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Structure of the Vanadium Nitrogenase
of Azotobacter vinelandii
and mechanistic insights into biological nitrogen
fixation
INAUGURALDISSERTATION
zur Erlangung des Doktorgrades
der Fakultät für Chemie und Pharmazie
der Albert-Ludwigs-Universität Freiburg im Breisgau
vorgelegt von
Daniel Sippel
aus Fulda
2017
I
Vositzender des Promotionsausschusses Prof. Dr. Stefan Weber
Referent: Prof. Dr. Oliver Einsle
Koreferentin: Prof. Dr. Susana Andrade
Datum der Promotion: 05. September 2017
II
III
Table of contents
Summary ......................................................................................................................................... 1
Zusammenfassung ......................................................................................................................... 3
1. Introduction ................................................................................................................................... 5
1.1 The biogeochemical nitrogen cycle .................................................................................. 5
1.2 Nitrogen fixation ............................................................................................................... 10
1.3 Nitrogenase from Azotobacter vinelandii ........................................................................... 13
1.3.1 Azotobacter vinelandii ..................................................................................................... 13
1.3.2 Nitrogenase genes (nif, vnf, anf) ................................................................................. 15
1.3.3 Introduction to nitrogenase ...................................................................................... 17
1.3.4 The nitrogenase MoFe-protein (NifDK) ................................................................ 19
1.3.4.1 The P-cluster .................................................................................................... 20
1.3.4.2 The FeMo-cofactor ......................................................................................... 22
1.3.5 The nitrogenase Fe-protein (NifH).......................................................................... 23
1.3.6 The N2ase complex .................................................................................................... 25
1.3.7 Nitrogenase cluster biosynthesis .............................................................................. 27
1.3.7.1 P-cluster biosynthesis ...................................................................................... 28
1.3.7.2 FeMo-cofactor biosynthesis ........................................................................... 29
1.3.8 Vanadium dependent nitrogenase (V-N2ase) ......................................................... 31
1.3.8.1 V-N2ase P-cluster and FeV-cofactor............................................................. 32
1.4 Mechanism of N2 reduction ............................................................................................ 33
1.4.1 N2ase turnover cycle and Lowe-Thorneley scheme .............................................. 33
1.4.1.1 N2ase turnover cycle ........................................................................................ 33
1.4.1.2 Fe-protein cycle ................................................................................................ 34
1.4.1.3 MoFe-protein cycle .......................................................................................... 34
1.4.1.4 Lowe-Thorneley kinetic model ...................................................................... 35
1.4.2 Substrate binding site ................................................................................................. 36
1.4.3 N2 reduction pathway ................................................................................................ 37
1.4.4 Molecular mechanism of N2 binding and reduction at the FeMo-cofactor ....... 39
1.4.4.1 Mechanistic model for N2 reduction via reductive elimination ................ 39
1.4.4.2 Computational mechanistic model for N2 reduction ................................. 42
1.4.5 Haber-Bosch process ................................................................................................. 44
1.5 Protein crystallography ..................................................................................................... 45
1.5.1 Protein crystallization ................................................................................................. 45
1.5.2 X-ray diffraction ......................................................................................................... 46
1.5.3 The electron density ................................................................................................... 48
1.5.4 Anomalous dispersion of heavy atoms and MAD ................................................ 50
IV
2. Scope of the study ....................................................................................................................... 53
3. Materials and Methods ................................................................................................................ 54
3.1 Materials ............................................................................................................................. 54
3.1.1 Chemicals and gases ................................................................................................... 54
3.1.2 Growth media ............................................................................................................. 54
3.1.3 Buffers and solutions ................................................................................................. 55
3.1.4 Chromatography ......................................................................................................... 56
3.1.5 Anoxic techniques ...................................................................................................... 57
3.1.6 Bacterial strains ........................................................................................................... 57
3.2 Microbiological methods ................................................................................................. 57
3.2.1 Permanent Mo glycerol culture ................................................................................ 57
3.2.2 Molybdenum depletion .............................................................................................. 57
3.2.3 Cultivation of A.vinelandii under Mo-limited conditions ...................................... 58
3.2.4 Whole cell nitrogenase acetylene reduction assay (ARA) .................................... 58
3.3 Protein biochemical methods .......................................................................................... 59
3.3.1 Purification of VFe-protein under low dithionite concentration (‘loDT’) ......... 59
3.3.2 Purification of VFe-protein under high dithionite concentration (‘hiDT’) ....... 60
3.4 Protein analytical methods ............................................................................................... 60
3.4.1 SDS-polyacrylamide-gel electrophoresis (SDS-PAGE) ........................................ 60
3.4.2 Determination of protein concentration (BCA-assay) .......................................... 61
3.4.3 Mass spectrometry ...................................................................................................... 61
3.4.4 Nitrogenase acetylene reduction assay .................................................................... 62
3.5 Crystallographic methods ................................................................................................ 62
3.5.1 Crystallization of VFe-protein .................................................................................. 62
3.5.2 X-ray data collection .................................................................................................. 63
3.5.3 Structure solution, model building and refinement ............................................... 63
3.5.4 Visualization of protein structures ........................................................................... 63
3.6 Spectroscopic methods .................................................................................................... 64
3.6.1 Electron paramagnetic resonance spectroscopy .................................................... 64
3.6.2 Differential scanning fluorimetry (DSF) ................................................................. 65
3.6.2.1 Optimal ratio of protein and SYPRO orange concentration .................... 65
3.6.2.2 Optimal pH and NaCl concentration ........................................................... 66
3.6.2.3 Influence of Na2S2O4 concentration ............................................................. 66
3.6.2.4 Influence of additives ...................................................................................... 67
4. Results ........................................................................................................................................... 68
4.1 Cultivation of A. vinelandii................................................................................................ 68
4.1.1 Mo depletion ............................................................................................................... 68
4.1.2 Cultivation of A. vinelandii under Mo-limited and V-containing conditions ...... 68
4.2 Isolation of VFe-protein .................................................................................................. 69
V
4.2.1 ‘loDT’ purification of VFe-protein .......................................................................... 69
4.2.2 ‘hiDT’ purification of VFe-protein .......................................................................... 70
4.3 Protein thermal stability ................................................................................................... 72
4.3.1 Optimal ratio of protein and SYPRO orange concentration ............................... 72
4.3.2 Optimal pH and NaCl concentration ...................................................................... 73
4.3.3 Influence of Na2S2O4 concentration ........................................................................ 74
4.3.4 Influence of additives ................................................................................................. 74
4.4 The structure of VFe-protein .......................................................................................... 75
4.4.1 Crystallization and data collection of VFe-protein ................................................ 75
4.4.2 The structure of ‘hiDT’ VFe-protein (‘resting’ state) ............................................ 77
4.4.2.1 Overall structural organization of VFe-protein ........................................... 77
4.4.2.2 Structure of the P-cluster ................................................................................ 80
4.4.2.3 Structure of the FeV-cofactor ........................................................................ 81
4.4.3 The structure of ‘loDT’ VFe-protein (‘active’ state).............................................. 84
4.4.3.1 Overall structural organization and P-cluster .............................................. 84
4.4.3.2 The FeV-cofactor in the ‘active’ state ........................................................... 85
4.4.3.3 The active site ................................................................................................... 86
4.5 Characterization of VFe-protein ..................................................................................... 90
4.5.1 Liquid chromatography mass spectrometry ........................................................... 90
4.5.2 Nitrogenase acetylene reduction assay .................................................................... 90
4.5.3 CW-EPR spectroscopy .............................................................................................. 91
4.5.3.1 ‘hiDT’/’resting’ state VFe-Protein ................................................................ 91
4.5.3.2 ‘loDT’/’active’ state VFe-protein .................................................................. 93
4.6 Comparison of EPR spectra and structures of VFe-protein ...................................... 94
5. Discussion ..................................................................................................................................... 96
5.1 Purification of VFe-protein in two states ...................................................................... 96
5.2 FeV-cofactor biosynthesis ............................................................................................... 98
5.2.1 The putative metallocluster carrier protein VnfY .................................................. 98
5.2.2 The unidentified hypothetical protein Avin_02580 .............................................. 99
5.3 Different redox properties of the V- and Mo-N2ase P-cluster and cofactor ........ 101
5.4 Mechanistic insights in to the active site of N2ase .................................................... 103
5.4.1 Mechanistic relevance of α-Gln176 ...................................................................... 103
5.4.2 Mechanistic outlook based on the structure of ‘active’ state VFe-protein...... 108
6. Appendix .................................................................................................................................... 113
6.1 Index of abbreviations .................................................................................................. 113
6.1.1 General abbreviations ............................................................................................. 113
6.1.2 Units .............................................................................................................. 114
6.1.3 Prefixes .............................................................................................................. 114
6.2 Thermofluor assay melting curves and temperatures ............................................... 115
VI
6.3 BLAST ............................................................................................................................. 117
6.4 Nitrogenase protein sequence alignment ................................................................... 118
6.5 Atom distances in N2ase structures ............................................................................. 123
6.6 Data collection statistics ................................................................................................ 125
7. Literature .................................................................................................................................... 128
Acknowledgement .................................................................................................................... 148
1
Summary
The nitrogen cycle describes the interconversion of the various chemical forms of nitrogen, an
essential element for all living organisms. The bulk amount of nitrogen is present as inert atmos-
pheric dinitrogen (N2) that cannot be metabolized by animals and most plants. Assimilation of
nitrogen into biomass occurs via ammonia (NH3). The reduction of molecular N2 into bioavaila-
ble NH3 is called N2 fixation and is of special importance and interest, as the N2 triple bond is the
most stable bond that any biological system has to break. In nature, nitrogenase is the only
known enzyme capable of overcoming the huge activation barrier of this exergonic reaction and
does so at ambient conditions. In the industrial Haber-Bosch process this reduction is performed
at ≈ 450 °C and ≈ 200 bar and is accordingly very energy and cost intensive. Nitrogenase is an
enzyme complex consisting of two metallo proteins, dinitrogenase (N2ase) and dintrogenase re-
ductase, and occurs in diazotrophic bacteria. N2ase contains two complex metal clusters and with
a composition of [7Fe-9S-C-Mo]-homocitrate the active site FeMo-cofactor even represents the
most complex metal cluster found in nature to date. Besides the common Mo-dependent N2ase,
the alternative and less wellstudied V- and Fe-only-dependent N2ases exist. Interestingly, next to
the unique ability to reduce N2, the V-N2ase is also capable of the catalytic CO reduction to pro-
duce CH4 and longer chain, C-C coupled hydrocarbons. Therefore, the V-N2ase combines the
two important industrial processes of Haber-Bosch and Fischer-Tropsch in one enzyme. As the
mechanism of N2 fixation is still unknown yet and so far only the structure of the Mo-N2ase has
been solved, structural information of the V-N2ase could give valuable insights into the mecha-
nism of N2 fixation and the differences in reactivity between these two N2ases.
In this work, the cultivation of the wild-type diazotroph Azotobacter vinalandii that is able to ex-
press all three nitrogenases, was carried out under Mo-limited and V-containing conditions, forc-
ing the bacteria to produce alternative V-N2ase. Using two different isolation strategies homoge-
neous V-N2ase could be isolated in two different states. One form (‘resting’ state) resembles in
EPR-spectra the previously studied V-N2ases. The second form (‘active’ state) shows new fea-
tures. By identification of an agent that increases the thermal stability of the protein via differential
scanning fluorimetry, successful protein crystallization was accomplished and protein structures at
atomic resolutions for both states could be solved via X-ray crystallography. The V-N2ase
(VnfDKG) core protein structure resembles the overall architecture of Mo-N2ase (NifDK), but
also shows also the additional, so far unknown small δ-subunit (VnfG), whose function is not yet
clear. Furthermore, besides the exchange of Mo to V at the active site FeV-cofactor, a new car-
2
bonate (CO32-) ligand that replaces a sulfide ([7Fe-8S-C-V]-homocitrate-CO3
2-) was identified and
rationalizes the distinctly different substrate reactivity between the Mo- and V-N2ase. In the ‘ac-
tive’ state structure at the cofactor, an N-species product intermediate was identified that displac-
es another sulfide ([7Fe-7S-C-V]-homocitrate-carbonate-N-ligand). Thus, the actual substrate
binding site on the FeV-cofactor has been determined the position of this sulfide (S2B), at the
Fe2-Fe6 edge of the cofactor. This N-ligand is coordinated by the conserved residue α-Gln176 of
the direct cofactor environment, which also underwent a conformational change. Concomitantly,
a storage position for the displaced sulfide was detected at the former position of the α-Gln176.
This arrangement likely represents a stable intermediate state during N2 reduction and thus pro-
vides valuable insights into the mechanism of N2 fixation by N2ase.
3
Zusammenfassung
Der Stickstoff-Kreislauf beschreibt die Umwandlung der verschiedenen chemischen Zustände
des für alle lebenden Organismen essentiellen Elementes Stickstoff. Fast die gesamte Menge an
Stickstoff liegt als inerter atmosphärischer Distickstoff (N2) vor, welcher von Tieren und den
meisten Pflanzen nicht metabolisiert werden kann. Die Assimilation von Stickstoff in Biomasse
verläuft über Ammonium (NH3 bzw. NH4+). Die Reduktion des molekularen N2
zubioverfügbarem NH3 heißt Stickstoff-Fixierung und ist von besonderer Bedeutung und
Interesse, weil die N2-Dreifachbindung die stabilste Bindung ist, die es in biologischen Systemen
zu brechen gilt. In der Natur ist die Nitrogenase das einzig bekannte Enzym, welches in der Lage
ist die immense Aktivierungsenergie für diese exergonische Reaktion aufzubringen, und macht
dies bei Normalbedingungen. Bei dem industriellen Haber-Bosch-Prozess wird diese Reduktion
bei ≈ 450 °C und ≈ 250 bar durchgeführt, und ist entsprechend energie- und kostenintensiv. Die
Nitrogenase ist ein Enzymkomplex, der aus zwei Metallo-Proteinen besteht, der Dinitrogenase
(N2ase) und der Dinitrogenase-Reduktase, und tritt in diazotrophen Bakterien auf. Die N2ase
enthält zwei Metallcluster, wobei der FeMo-Cofaktor des aktiven Zentrums, mit einer
Zusammensetzung von [7Fe-9S-C-Mo]-Homocitrat, der komplexeste bisher bekannte in der
Natur vorkommende Metallcluster ist. Außer der am meisten vorkommenden und untersuchten
Molybdän (Mo) abhängigen N2ase existieren auch die alternativen Vanadium (V) und nur Eisen
(Fe) abhängigen N2asen. Neben der einzigartigen Fähigkeit N2 zu reduzieren, ist die V-N2ase
ebenso zur katalytischen Kohlenmonoxid- (CO) Reduktion fähig, um Methan (CH4) oder
längerkettige, C-C gekoppelte Kohlenwasserstoffe zu produzieren. somit vereint die V-N2ase in
einem Enzym die zwei wichtigen industriellen Prozesse von Haber-Bosch und Fischer-Tropsch.
Aufgrund der Tatsache, dass der Mechanismus der N2-Fixierung immernoch nicht bekannt ist,
und bisher nur die Struktur der Mo-N2ase gelöst ist, könnten Informationen über die Struktur der
V-N2ase wertvolle Informationen über den Mechanismus der N2-Fixierung und über die
unterschiedliche Reaktivität der beiden Enzyme geben.
In dieser Arbeit wurde die Kultivierung des diazotrophen Bakteriums Azotobacter vinelandii,
welches in der Lage ist alle drei N2asen zu exprimieren, unter Mo-freien und V-beinhaltenden
Bedingungen etabliert, unter denen das Bakterium gezwungen ist die alternative V-N2ase zu
produzieren. Bei zwei unterschiedlichen Aufreinigungsmethoden wurden zwei Zustände von
jeweils reiner, homogener V-N2ase erhalten. Eine Form (‚resting‘ state) ähnelt in den EPR-Spektren
den bisher untersuchten V-N2asen und eine zweite Form (‚active‘ state) zeigt neue Charakteristika.
4
Via differential scanning fluorimetry konnte ein Reagenz bestimmt werden, welches die thermische
Stabilität des Proteins erhöht und eine erfolgreiche Proteinkristallisation ermöglicht. Durch
anschließende Röntgenbeugungs-Experimente konnten die Proteinstrukturen beider Zustände
bei atomarer Auflösung gelöst werden. Der V-N2ase (VnDKG) Proteinstrukturkern ähnelt sehr
stark der Gesamtstruktur der Mo-N2ase (NifDK), besitzt aber zusätzlich die bisher unbekannte
kleine δ-Untereinheit (VnfG), deren Funktion bisher noch unklar ist. Neben dem Austausch von
Mo gegen V am FeV-Cofaktor wurde ein neuer Carbonat- (CO32-) Ligand identifiziert, der ein
Sulfid ersetzt ([7Fe-8S-C-V]-Homocitrat-CO32-) und somit die deutlich unterschiedlichen
Substratreaktivitäten von Mo- und V-N2ase erklären könnte. In der Proteinstruktur des ‚active‘
state ist eine Produktzwischenstufe mit einer Stickstoff-Spezies am FeV-Cofaktor identifiziert
worden, welche den Platz eines weiteren Sulfids einnimmt ([7Fe-7S-C-V]-Homocitrat-CO32--N-
Ligand). Damit ist mit der Position dieses Schwefels (S2B) an der Fe2-Fe6-Kante die genaue
Substrat-Bindestelle am FeV-Cofactor gefunden worden. Der N-Ligand wird von der
konservierten Aminosäure α-Gln176 in der direkten Umgebung des aktiven Zentrums
koordiniert, die zudem einer konformativen Änderung unterliegt. Gleichzeitig ist an der
vorherigen Position von α-Gln176 die Zwischenlagerposition für das verdrängte Sulfid gefunden
worden. Diese Konstellation repräsentiert einen Zwischenzustand während der N2-Reduktion,
und liefert damit wertvolle Erkenntinisse über den Mechanismus der N2-Fixierung durch die
N2ase.
Introduction
5
1. Introduction
1.1 The biogeochemical nitrogen cycle
Nitrogen is one of the essential elements on earth. As mandatory component in nucleic acids and
proteins it is fundamental for every living organism. Nitrogen is the fifth most abundant element
in our solar system [1] and the fourth most abundant element in cellular biomass [2]. In our at-
mosphere and biosphere 99 % is present as inert dinitrogen gas (N2) and only 1 % is chemically
bound. [3], [4]. Therefore nitrogen can be divided into two classes: Unreactive nitrogen is the
atmospheric, gaseous N2 and reactive nitrogen represents any other form of this element. The
oxidation states range from +V like in the fully oxidized nitrate (NO3-) to -III in the fully reduced
ammonia (NH3) or ammonium (NH4+). Assimilation of nitrogen into organic biomass only takes
place via fixed nitrogen in form of NH4+. The inter-conversion of the three nitrogen compounds
N2, NO3- and NH4
+ via intermediate forms were the eponyms of the three major processes form-
ing the ‘classical’ nitrogen cycle. Nitrification is the oxidative formation of nitrate starting from
ammonium. Denitrification is the reduction of nitrate to gaseous dinitrogen. And the reduction
of inert dinitrogen to the fixed nitrogen in ammonium is called nitrogen fixation. This cycle was
fully controlled by microorganisms that performed these transformations [5]. Consequently bac-
teria participating in the pathways of the nitrogen cycle have been phylogenetically classified ac-
cording to their physiology as nitrifiers, denitrifiers or nitrogen fixers. With the invention of the
Haber-Bosch-process, the industrial nitrogen fixation of N2 with H2 to NH3, humanity started to
have a major influence on the nitrogen cycle [6]. This was the accepted view of the nitrogen cycle
for more than 100 years, until more recently, further nitrogen conversion processes, that play a
role in the interconversion of nitrogen forms, were discovered. These were the anaerobic ammo-
nium oxidation (anammox) [7], [8], [9], [10] and the anaerobic nitrite reduction [11], [12]. The
classical N-cycle thus had to be expanded and is not constrained by phylotype anymore, but ra-
ther seen in a more modular way [1], [13], [2]. A few more nitrogen conversion pathways, whose
relevance is unclear so far, such as a newly discovered NO dismutation into O2 and N2 by a pro-
posed NO-dismutase [14], [13], have not been considered here. For clarity, the five major pro-
cesses and the contemporary understanding of the biogeochemical nitrogen cycle are summarized
in Figure 1.
Introduction
6
Figure 1: The biogeochemical nitrogen cycle. The five major processes are labeled and highlighted by colour: 1, dark
blue) nitrification (respiratory; NH4+ → NO2
-/NO3-); 2, orange) denitrification (respiratory; NO3
-/NO2- → N2); 3,
green) anaerobic ammonium oxidation, anammox, (respiratory; NO3-/NO2
- + NH4+ → N2); 4, dark red) ammonifi-
cation, including nitrite-ammonification (respiratory and assimilatory, NO3-/NO2
- → NH4+) and N2-fixation (assimi-
latory, N2 → NH4+); 5, black) nitrite-nitrate interconversion (NO2
- ↔ NO3-), oxidation of NO2
- to NO3- represents
the second step of nitrification, reduction of NO3- to NO2
- represents first step of denitrification and is coupled to
anammox and nitrite ammonification. Aerobic processes are highlighted by light red arrows, anaerobic processes are
by light blue arrows. The oxidation states of nitrogen are shown on the left side in roman numerals. Abbreviations of
the enzyme genes performing the redox reactions are shown in the scheme and listed below for clarity. This scheme
was generated according to [15], [1], [13] and [2].
The different redox reactions and pathways are versatile and serve mainly either in assimilation or
in respiration. In assimilatory processes, reducing equivalents are used to generate a molecule,
NH4+ that is further incorporated into cell mass. Microbial respiration is designed for energy con-
servation. Therefore, in respiratory processes the electron flow is coupled to a membrane-bound
proton translocating complex that creates a proton motive force (pmf) to allow the synthesis of
adenosine triphosphate (ATP). The enzymes play mainly either a respiratory or an assimilatory
role in the biogeochemical cycle. But under stressful conditions for the bacteria, they can some-
NO3-
NO2-
NO
N2O
NH4+
N2
NH2OH
nirS, nirK
‚nas‘
narGHI
napAB
nosZ
+V
+IV
+III
+II
+I
-I
-II
-III
0
nxr
amo
Nit
rif.
hao
‚nas‘ nitrate reductase
cytoplasmic, assimilatory
narGHI nitrate reductase
membrane bound, respiratory
napAB nitrate reductase
periplasmic, dissimilatory and respiratory
norBC nitric oxide reductase
nosZ nitrous oxide reductase
nif nitrogenase
hzs hydrazine synthase
hdh hydrazine dehydrogenase
N2H4
NO
Nbiomass
Assim
ilatio
nM
inera
lizati
on
hzs
NO
3 -red
.
hdh
nirS, nirK nitrite reductase
periplasmic, respiratory
nir‘?‘ nitrite reductase
cytoplasmic, assimilatory
nrfA nitrite reductase
periplasmic, respiratory
amo ammonium monooxygenase
hao hydroxylamine oxidoreductase
nxr nitrite oxidoreductase
1
2
3
5
4
4
nirS, nirK
norBC
nir‘?‘
nrfA
nif
Introduction
7
times also perform dissimilatory reactions, for example to detoxify or to degrade excess reducing
power. But this function is then not anymore directly related to their function in the biogeochem-
ical nitrogen cycle [16]. In anaerobic respiratory pathways of the nitrogen cycle, nitrate or nitrite
is the terminal electron acceptor, and in the aerobic respiratory pathway, dioxygen is the terminal
electron acceptor.
The nitrogen cycle is divided into five common, major transformations of nitrogen: nitrification;
denitrification; anammox; nitrite-nitrate interconversion and ammonification, the creation of
ammonium, including anaerobic nitrite reduction to ammonium and nitrogen fixation (see Figure
1).
The oxidation of ammonium to nitrate (NH4+ → NH2OH → NO2
-/NO3-) is called nitrification.
Nitrification is an aerobic, respiratory pathway for chemolithoautotrophic bacterial microorgan-
isms using ammonium and nitrite as sole energy and reductant source and dioxygen (O2) as ter-
minal electron acceptor to fix carbon dioxide (CO2) without photosynthesis [2], [1]. It is the only
transformation, that is obligatory oxygen-dependent and therefore the only aerobic process. The
actual reactions of all remaining nitrogen transformations take place under locally anaerobic con-
ditions [1]. Nitrification can be divided into two steps [17]. The first step is the oxidation of am-
monium to nitrite, nitritation, and is carried out by ammonia-oxidizing bacteria. In this reaction,
ammonia monooxygenase (amo) oxidizes ammonium to hydroxylamine, which is subsequently
oxidized to nitrite by hydroxylamine oxidoreductase (hao). The second step is the oxidation of
nitrite to nitrate, nitratation, is carried out by nitrite-oxidizing bacteria and is catalyzed by the
nitrite oxidoreducase (nxr). In 2015 a bacterium, Nitrospira, was found to be able to perform the
complete ammonia oxidation (comammox) to nitrate [18].
The reduction of nitrate to gaseous dinitrogen (NO3-/NO2
- → N2O → NO → N2) is called deni-
trification [19]. Denitrification is an anaerobic respiratory pathway using nitrate, nitrite, nitrous
oxide (N2O) and nitric oxide (NO) as electron acceptors. The four-step reduction includes en-
zymes from the essential gene clusters nar, nir, nor and nos. The first two-electron redox reaction
from nitrate to nitrite is carried out in the cytoplasm by the membrane-bound, heterotrimeric
respiratory nitrate reductase NarGHI with NarG containing the active site molybdopterin cofac-
tor [15]. The product nitrite is subsequently transported to the periplasm by the nitrite trans-
porter NarK. The nitrate reductase NapAB, containing a molybdopterin cofactor as well, has
mainly dissimilatory functions under aerobic conditions as electron sink, but is also connected to
denitrification by nitrate reduction to nitrite in the periplasm [13]. The two respiratory nitrite re-
ductases nirS and nirK, a cytochrome cd1 nitrite reductase and a copper-containing nitrite reduc-
Introduction
8
tase, respectively, reduce nitrite to NO. Subsequently, the toxic NO that also causes nitrosative
stress [20] is reduced to nitrous oxide by nitric oxide reductase, NorBC [21]. The final reduction
of the chemically stable but potent greenhouse and ozone-depleting gas N2O [22] into the inert
atmospheric dinitrogen is achieved by nitrous oxide reductase NosZ. Denitrification from nitrate
to dinitrogen with three intermediate nitrogen compounds is a very complex pathway that in-
volves enzymes from several gene clusters. Studies of protein-protein interaction propose the
formation of a super- and a supra-complex that integrates all steps with a more efficient energy
conservation [23].
An alternative anaerobic respiratory pathway is the anaerobic ammonium oxidation (anammox)
(NO2- → NO + NH4
+ → N2H4 → N2). Chemolithoautotrophic anammox bacteria gain energy
for cellular growth from this exergonic reaction, with ammonia as reductant, nitrite as electron
acceptor and dinitrogen as product [24]. The reaction takes place in their vacuolar cell organelle,
the anammoxosome [25]. NO and hydrazine (N2H4) were identified as key intermediates [26].
Nitrite reduction to NO is probably carried out by the respiratory nitrite reductase NirS and
NirK, like in denitrification (see above), and/or by a yet unknown nitrite reductase among the
hao-like proteins, and maybe via hydroxylamine as intermediate. For the second step, the combi-
nation of NH4+ and NO to N2H4, the enzyme hydrazine synthase (hzs) is supposed to be in-
volved. A variant of hydroxylamine oxidoreductase (hao) is hypothesized to catalyze the final
oxidation to dinitrogen, the hydrazine dehydrogenase (hdh) [27]. Since the first discovery and
identification of anammox bacteria [9] several phylogenetically different bacterial species, all affil-
iated with the phylum Planctomycetes, have been identified in natural and synthetic ecosystems [28].
Anammox is responsible for major fixed nitrogen loss and dinitrogen production in marine envi-
ronments [29]. Due to its ability to simultaneously remove NO2- and NH4
+, it is beneficial for
waste water treatment and is used in large-scale bioreactors [30].
Ammonification, the formation of ammonium from either nitrite or dinitrogen (NO2- or N2 →
NH4+), plays a special role in the nitrogen cycle, as every assimilation of nitrogen into biomass by
living organisms takes place via nitrogen in the form of ammonium to start the biosynthesis of
the amino acid glutamine. There are two versions how nature realizes the creation of ammonium,
either by anaerobic nitrite ammonification or by nitrogen fixation.
The pathway of anaerobic nitrite ammonification (NO2- → NH4
+), can be divided into two pro-
cesses with either assimilatory or respiratory function [13]. Respiratory nitrite reduction, as real-
ized in denitrification and anammox, is the one-electron reduction by the respiratory, NO-
producing nitrite reductases NirS and NirK. Respiratory nitrite ammonification, the six-electron
Introduction
9
reduction of nitrite to ammonium in a single step, is carried out in the periplasm by cytochrome c
nitrite reductase, NrfA, which clearly differs from the NO-producing nitrite reductases [13]. As-
similatory nitrite ammonification takes place in the cytoplasm, catalysed by a siroheme containing
nitrite reductase, ‘nir?’ [31], [32], [12], [33]. A clear assignment, like nirS or nirK acting in denitrifi-
cation, cannot be done for assimilatory nitrite reductases yet. In Mycobacterium tuberculosis, nirB
encodes for the siroheme-containing subunit of the assimilatory nitrite reductase NirBD. But for
example in E. coli, NirBD has just a detoxifying function [34].
Nitrogen fixation is the reduction of dinitrogen to ammonium (N2 → NH4+). Nitrogen fixation is
an assimilatory process serving to create fixed nitrogen from the chemically inert gaseous dinitro-
gen. This process is carried out by the enzyme nitrogenase. Nitrogen fixation will be discussed in
detail in section 1.2.
Nitrite-nitrate interconversion (NO2- ↔ NO3
-) is not a nitrogen transformation pathway on its
own. Nitrate reductases or the nitrite oxidoreductase are rather involved in the other nitrogen
metabolisms. The oxidation of NO2- to NO3
- by nitrite oxidoreductase (nxr), containing a molyb-
dopterin cofactor [13], represents the second part of nitrification and is involved in anammox,
maybe by participation in replenishing the electron need for NAD(P)+ reduction for pmf creation
[35], [27]. In the reduction of NO3- to NO2
-, the nitrate reductases of the nar (respiratory NO3-
reduction), nap (periplasmic NO3- reduction), and nas (assimilatory NO3
- reduction) systems are
involved, all containing a molybdopterin cofactor as well [36], [15]. In the nar system, narGHI
encodes for the respiratory membrane-bound nitrate reductase that carries out the first step in
denitrification and generates the pmf. In the nap system, napAB encodes for the periplasmic ni-
trate reductase. Its definite role is not clear yet. The nap system is used under anoxic and/or (mi-
cro)oxic environment and might have a function in anaerobic respiration, or as an electron sink
during aerobic (photo)organoheterotrophic growth. There is no evidence for acting in a pro-
tonmotive fashion, which is why the main function could also be the nitrate-dependent quinone
regeneration [13]. The cytoplasmic, assimilatory nitrate reductase is supposed to be in the nas
gene cluster, but a final assignment is not yet possible. In Paracoccus denitrificans, a three-
component complex consisting of the nitrate reductase NasC, the nitrite reductase NasB and the
Rieske-Fe-S-protein NasG has been proposed [37]. However, there are also other genes, such as
nasA, which are hypothesized to encode for an assimilatory nitrate reductase [36]. How the nap,
nar and nas nitrate reductases are connected to assimilatory and respiratory nitrite ammonification
is not known for sure [33], [13].
Introduction
10
1.2 Nitrogen fixation
At 78 %, gaseous N2 comprises the vast amount of air in our atmosphere, but for animals and
most plants, nitrogen in form of N2 is not bioavailable. For assimilation, nitrogen needs to be in
the form of NH4+. Nitrogen fixation means the reductive dissociation of the nitrogen-nitrogen
triple bond of N2 into NH4+ (N2 → NH4
+). However, the term “fixed nitrogen” in general is of-
ten used for any metabolizable and thus bioavailable form of nitrogen. Therefore, the terms fixed
and reactive nitrogen are used analogously.
The ability for nitrogen fixation is restricted to organisms with a nif-gene cluster expressing the
enzyme nitrogenase (see section 1.3). They are able to live with N2 as only source of nitrogen and
this lifestyle is called diazotrophy. All known N2-fixing organisms are prokaryotes from selected
families of bacteria and archaea [38]. N2-fixation is a highly regulated mechanism, mainly depend-
ing on the presence or absence of oxygen and ammonia, leading to the induction or repression of
N2-fixation genes. Organisms capable of diazotrophy react accordingly to the environmental and
nutritional conditions and are therefore not restricted to a certain habitat, but can live in various
environments [39]. The rapid expansion of microbial genome sequencing in the last decade led
and leads to discovery of more and more N2-fixing and potentially N2-fixing organisms [40]. Dia-
zotrophs either live in symbiosis with plants or are free-living organsims. Common, known sym-
biotic diazotrophs are bacteria of genus Rhizobium associated with legumes. Nitrogen fixaton of
Rhizobia is fully restricted to the symbiosis [41]. Cyanobacteria can be either free-living or in sym-
biosis and are found in soil, fresh water and salt water. Very known are the free-living Trichodes-
mium cyanobacteria, living in marine systems and being responsible for major nitrogen fixation in
oceans, and the in symbiosis living cyanobacteria, Anabaena azollae associated with the azolla fern
and used in rice paddies [42]. Common free living soil bacteria are the facultative anaerobe
Klebsiella pneumoniae, the obligate aerobe Azotobacter vinelandii [43] and the obligate anaerobe Clos-
tridium pasteurianum [44]. These three bacteria are the best studied model organisms for nitrogen
fixation.
Although biological nitrogen fixation was first discovered in 1888 [45], the ability of N2-fixing
prokaryotes to create bioavailable nitrogen for food production was actively, maybe unknowingly,
used by humankind perhaps already ≈ 7000 years ago [46]. Legumes with the ability for self-
fertilizing via the symbiosis with N2-fixing organisms were used as well as rice cultivation tech-
niques that created an anaerobic environment for N2-fixing cyanobacteria. This way of creating
fixed nitrogen is called cultivation-induced biological nitrogen fixation (C-BNF) and represents
the first influence of humans on the creation of fixed nitrogen, anthropogenic nitrogen fixation.
Introduction
11
For C-BNF are used: Rhizobium-associated seed legumes, like peas and beans, and leguminous
forages, like alfalfa and clover; non-Rhizobium N2-fixing organisms associated with some crops,
e.g. cereals, and trees; cyanobacteria associated with rice paddies; and endo- as well as ectophytic
diazotrophs associated with sugar cane. Altogether, C-BNF accounted in 1860 for ≈ 15 Tg N yr-1
and in the early 1990’s for ≈ 31.5–33 Tg N yr-1 [47], [42].
Another natural source is the atmospheric nitrogen fixation via lightning. In lightning strikes,
nitric oxides (NOx) are produced from molecular N2 and O2, which is subsequently oxidized via
NO2 to HNO3 and then is deposited into ecosystems in form of nitrate (NO3-) [42].
Besides the natural sources, the industrial nitrogen fixation was invented. In 1913, Carl Bosch
and Fritz Haber developed the Haber-Bosch process that allows for the generation of NH3 from
the elements H2 and N2. Although the reaction is thermodynamically favored (see equation 1) it
takes place under extreme conditions with a temperature of 400-500°C and a pressure of
200-300 bar, using an α-Fe iron catalyst, consisting of magnetite (Fe3O4) promoted with irreduci-
ble oxides (K2O, Al2O3 and CaO) [48], [49]. The reaction, as displayed in equation 1, is exother-
mic and volume decreasing, favoring low temperatures and high pressures. In contrast, the cata-
lyst needs working temperatures of >400°C to efficiently bind N2 for decreasing the activation
barrier of 230 kJ mol-1 and catalyze the reaction with a conversion to NH3 of ≈ 18 vol % [50],
[51], [4]. These conditions and the low yield make industrial nitrogen fixation very energy inten-
sive.
(1)
The creation of fixed nitrogen by humans increased 10-fold from 1860 to 1990 from ≈ 15 to
≈ 156 Tg N yr-1 [42]. Besides the negligible amount of ≈ 0.3 Tg N yr-1 from salt mines in 1860 C-
BNF was with these ≈ 15 Tg N yr-1 the almost the only reactive nitrogen source. Although C-
BNF increased in early 1990’s to ≈ 31.5—33 Tg N yr-1 [42], [47] reactive nitrogen from the Ha-
ber-Bosch process was with the three-fold amount, 100 Tg N yr-1 [52], the main part. The re-
maining ≈ 24.5 Tg N yr-1 come from fossil fuel combustion for energy production [42]. Hence
reactive nitrogen from the Haber-Bosch process accounts for ≈ 64 % of the total amount of
anthropogenic reactive nitrogen. With ≈ 86 % (≈ 86 Tg N yr-1), the major portion of Haber-
Bosch reactive nitrogen was used for fertilizer. Nitrogen fertilizer and C-BNF represent ≈ 77 %
of the total anthropogenic reactive nitrogen and were used in agriculture for food production,
2 NH3 + 92.28 kJ
α-Fe-cat.
400-500°C
200-300 bar N2 + 3 H2
Introduction
12
displaying the immense demand for reactive nitrogen in agriculture. Fixed nitrogen from the Ha-
ber-Bosch process accounted for ≈ 74 % of all reactive nitrogen used for food production, but
Haber-Bosch ammonia was not only used for agriculture. The remaining ≈ 14 % (≈ 14 Tg N yr-1)
of Haber-Bosch fixed nitrogen, which is ≈ 9 % of total anthropogenic reactive nitrogen, were
used to produce for example synthetic fibers, nylon, refrigerants, explosives, plastics, rocket fuel,
nitroparaffins and some also was dispersed to the environment [47], [42], [53]
The enormous demand for fixed nitrogen correlated with a rapidly increasing human population
since beginning of the 20th century. Between 1908 and 2008 ≈ 27 % of the world’s population,
equivalent to ≈ 4 billion people born or 42 % of the estimated total births, were supported by
nitrogen fertilizer [6]. According to estimates for 2000, nitrogen fertilizer was responsible for
feeding 44 % of world’s population and for 2008 the number was 48 % [54], [55]. That means
that Haber-Bosch nitrogen allowed the existence of almost half of humankind. The rising de-
mand for reactive nitrogen, especially Haber-Bosch reactive nitrogen, can be seen in the further
increase from 156 Tg N yr-1 in 1995 to 187 Tg N yr-1 in 2005. Again, Haber-Bosch reactive nitro-
gen from 100 Tg N yr-1 to 121 Tg N yr-1 represents the major contribution to the increase [56],
[55].
Besides the use of fertilizer in agriculture for food supply and cotton, a new and growing role for
fertilizer has emerged for the production of biofuels. Around 2008, biofuels from corn in the
United States, covering 29 million ha, were fertilized with an average of 160 kg N ha-1 yr-1
(≈ 4.64 Tg N yr-1) and from sugar cane in Brazil, covering 7 million ha, were fertilized with an
average of 100 kg N ha-1 yr-1 (≈ 0.7 Tg N yr-1) [55].
In 2002, about 1 % of the world’s total annual energy supply was consumed only by the Haber-
Bosch process [57], showing how energy intensive this reaction is
A growing human population with increasing standard of living leads to a further rise in the need
for fixed nitrogen. Consequently, the requirement of reactive nitrogen especially Haber-Bosch
fixed nitrogen will increase as well. Cosidering that the Haber-Bosch process is very energy inten-
sive and that the demand for this process will increase in future, it points out the necessity and
importance for research in the field of N2-fixation to find a more efficient or improved way to fix
dinitrogen.
Introduction
13
1.3 Nitrogenase from Azotobacter vinelandii
1.3.1 Azotobacter vinelandii
Azotobacter vinelandii is a free-living, Gram-negative soil bacterium belonging to the group of
Gammaproteobacteria. The genus Azotobacter belongs to the family Pseudomonadaceae. Recent
phylogenetic comparisons between Azotobacter and Pseudomonas showed very high similarity be-
tween especially A. vinelandii and the Pseudomonas genus, raising the suggestion to reclassify Azoto-
bacter to Pseudomonas [58].
A. vinelandii is one of a few bacterial species being capable of cyst formation, which is the trans-
formation of the cell from a vegetative into a dormant stage. The encystment of A. vinelandii,
depending on the carbon source, its concentration and other growth conditions, leads to creation
of a capsule surrounding the cell to prevent desiccation and to be resistant to other chemical and
physical challenges [59].
The diazotroph A. vinelandii is an obligate aerobic bacterium [60]. To combine the energetically
most efficient respiration of O2 as terminal electron acceptor with the O2-sensitive process of
nitrogen fixation, A. vinelandii evolved several O2 protection mechanisms. This is necessary, as
under N2-fixing conditions nitrogenase enzymes (dinitrogenase and dinitrogenase reductase) can
account for up to ≈ 10 % of the total cellular cytosolic protein [61].
The first mechanism to keep the cytoplasm anaerobic deals with the prevention of oxygen enter-
ing the cell by creating an oxygen barrier. A. vinelandii can produce alginate to create a capsule
surrounding the cell which hinders oxygen entrance [62]. Alginate is a linear copolymer consisting
of 1→4-linked β-D-mannuronic acid and α-L-guluronic acid and is also major component of the
cysts capsule [60].
Further protection mechanisms cope with intracellular O2. A. vinelandii is supposed to be able to
adjust the oxygen consumption rates to the ambient oxygen concentration. That means that with
an increasing extracellular oxygen concentration, the oxygen consumption at the cell surface in-
creases as well, thereby maintaining low cytoplasmic oxygen levels. This is called ‘respiratory pro-
tection’ [63], [60]. The genus Azotobacter was cited to have the highest respiratory rates of all
known bacteria [64] and in fact, A. vinelandii has an extensive respiration system containing four
NADH-ubiquinone-oxidoreductases and five terminal oxidases [60]. Complementarily, it was
shown that respiratory rates of A. vinelandii increase proportional with an increasing ambient oxy-
gen concentration up to ≈ 70 μM O2 [65], [66]. Besides high respiratory rates, especially the
Introduction
14
NADH-ubiquinone-oxidoreductase Ndh and the terminal oxidase CydAB are apparently directly
involved in oxygen-tolerant nitrogen fixation [67]. Thereby respiration contributes maintaining a
low, non-harmful cytoplasmic O2-level, although at oxygen concentrations higher than 70 μM O2
up to a maximum of ≈ 230 μM O2, respiratory rates increase only slightly [65], [66].
Another O2 protection mechanism is related to the intracellular ATP-level. Protection is said to
depend on the supply of sufficient reducing equivalents together with a high intracellular ATP-
level to maintain N2ase at an adequately reduced redox state for its function. [68], [69], [66]. An
alternative way to explain the protective role of high ATP-concentrations is that they are in direct
correlation with high electron flux to nitrogenase, which has an influence on the dissociation rate
constant of the nitrogenase components [70] and thereby on the susceptibilty of the dinitrogen-
ase reductase to oxygen damage [71], [60].
A protection mechanism when O2 is present in high, harmful cytoplasmic concentrations for
N2ase is called ‘autoprotection’ [71], [72]. It proposes that the dinitrogenase reductase reduces O2
to a superoxide radical or hydrogen peroxide (H2O2) which are subsequently removed by the
superoxide dismutase (SOD) or catalase/peroxidase, without dinitrogenase reductase becoming
inactivated [73], [74], [66]. The hypothesis that dinitrogenase reductase does not get inactivated is
questionable, as evidently this protein with an oxygen sensitivity of t1/2 ≈ 45 s, compared to the
dinitrogenase with t1/2 ≈ 10 min, is very sensitive and gets rapidly deactivated by O2 [75], [76]. But
on the other side, as dinitrogenase reductase shows a higher oxygen sensitivity than dinitrogenase
and thus by reducing the intracellular O2 concentration, it could act as an O2 scavenger to prevent
oxygen damage on dinitrogenase, whose biosynthesis is way more complex and energy intensive
(see section 1.3.7) [77].
Another protection mechanism dealing with detrimental high cytoplasmic O2 concentrations is
the ‘conformational protection’. A ferredoxin-like iron-sulfur protein, called Shetna-protein II or
FeSII, forms a protective complex with both nitrogenase enzyme system components. This com-
plex is stable against oxygen but also inactive for N2-fixation [78], [76].
Lastly, if intracellular O2 concentration is too high and conditions are actually unsuitable for ni-
trogen fixation, nitrogenase is repressed by O2 [79].
A. vinelandii is one of the rare, N2-fixing bacteria encoding for three different nitrogenases. De-
pending on metal availability, either the molybdenum-dependent nitrogenase [80], or one of the
alternative nitrogenases [81], the vanadium-dependent nitrogenase or the Fe-only-dependent ni-
trogenase is expressed [82], [60]. Through the usage of a Mo storage protein, A. vinelandii can
store high levels of intracellular Mo to maintain nitrogen fixation via the most efficient Mo-
Introduction
15
dependent nitrogenase after environmental Mo exhaustion or to bridge a short-term Mo deficit
[83]. Nevertheless, under Mo starvation A. vinelandii expresses one of the alternative nitrogenases
for nitrogen fixation [84], [85], [81].
The amenability to genetic manipulation, the fact that its genome has been fully sequenced, the
ability of having an aerobic lifestyle while performing oxygen-sensitive N2-fixation, expressing
nitrogenase proteins in high yields and encoding for three different nitrogenases makes A. vine-
landii easy and practical to grow in laboratory scale and the ideal model organism for exploring
nitrogen fixation.
1.3.2 Nitrogenase genes (nif, vnf, anf)
The A. vinelandii genome encodes for three nitrogenases, the molybdenum-dependent nitrogen-
ase, encoded by the nif genes for nitrogen fixation, the vanadium-dependent nitrogenase, encod-
ed by the vnf genes for vanadium-dependent nitrogen fixation and the Fe-only-dependent nitro-
genase, encoded by the anf genes for alternative nitrogen fixation [60]. A total number of ≈ 82
genes are likely involved in N2 fixation by these three systems [86].
In the presence of Mo, the most common and in all diazotrophs present Mo dependent nitrogen-
ase (Mo-N2ase) is expressed. The genes for Mo-N2ase are located in two regions adjacent to and
equidistant from the origin of replication. The proximity to the origin might be responsible for
the high expression level of Mo-N2ase [60]. In total, twenty genes have been annotated as part of
the nif-system: nifA, nifB, nifD, nifE, nifF, nifH, nifK, nifL, nifM, nifN, nifO, nifQ, nifS, nifT, nifU,
nifV, nifW, nifX, nifY and nifZ. According to their function the genes can be divided into at least
five groups: nitrogenase structural proteins; cofactor biosynthesis and metal trafficking; regula-
tion; electron transfer and nitrogenase components maturation [87]. The major nif region encodes
for the structural subunits of nitrogenase, namely the dinitrogenase (nifDK) and the dinitrogenase
reductase (nifH), the major part of the cofactor assembly apparatus, protein maturation and elec-
tron transfer. The minor nif region includes regulatory genes, as well as genes necessary for Mo
trafficking and nitrogenase cofactor biosynthesis. An overview of the gene-cluster is given in
Figure 2.
Introduction
16
Figure 2: The nif, vnf anf genes encoding for the three different nitrogenases in A. vinelandii. This scheme was
adapted from [60]
The gene products of nifB, nifE, nifH, nifN, nifQ, nifS, nifU and nifV are directly involved in metal
trafficking and cofactor biosynthesis [77]. The gene products of nifX and nifY are proposed to be
involved in cofactor assembly [87].
The gene products of nifF [88] and nifH, in its role of nitrogenase reductase, are involved in elec-
tron transfer.
The gene products of nifA and nifL are responsible for regulation of nitrogen fixation. The nif, vnf
and anf genes are under control of a σ54-dependent promotor [60]. NifA is the σ54-dependent pos-
itive transcription regulatory element and acts as activator for nitrogenase gene expression under
NH4+-limiting conditions. The O2- and fixed-nitrogen-sensitive NifL, the negative transcription
regulator, interacts with NifA to form an inhibitory complex under harmful O2 concentrations
and sufficiently high levels of fixed nitrogen [79], [89].
The gene products of nifW and nifZ are involved in maturation of NifDK [90], [91], whereas nifM
plays a role in maturation of NifH [92].
The exact functions of the gene products of nifT and nifO have not been elucidated yet [43].
In the absence of Mo and presence of V, the expression of V dependent nitrogenase (V-N2ase)
system is induced. V-N2ase is encoded by vnfDKG, showing an additional δ-subunit, and vnfH.
Besides the structural genes, the vnf gene cluster contains an incomplete cofactor biosynthesis
apparatus (vnfE, vnfN, vnfU, vnfX, vnfY) and the supposed transcription regulator vnfA, whose
gene product carries a [3Fe-4S]-cluster and thus might also have another function [93]. The co-
factor assembly machinery is complemented by the corresponding nif genes nifB, nifM, nifQ, nifS,
Introduction
17
nifU, nifV, nifW and nifZ [94]. Interestingly, three genes are located on the vnf gene cluster that
show sequence similarity to genes participating in molybdopterin biosynthesis, which separate the
vnf genes in two sections [60].
In the absence of both Mo and V, the expression of the Fe-only nitrogenase (Fe-N2ase) system is
initiated. Fe-N2ase is encoded by anfDKG, showing the additional δ-subunit as well, and anfH.
The anf gene cluster is the smallest of the three gene clusters and carries, besides the structural
genes, only genes for the supposed transcriptional regulator anfA [95], the putative cofactor as-
sembly protein anfU and two proteins with unknown function (anfR and anfO) [60], [43]. There-
fore the Fe-N2ase system is complemented by the expression of the nif genes nifB, nifM, nifQ, nifS,
nifU, nifV, nifW and nifZ as well as the vnf genes vnfE, vnfH, vnfN, vnfX and vnfY [94].
The vnf and anf gene clusters are incomplete and the production of catalytically active V- or Fe-
N2ase requires the expression of the corresponding nif-genes, at least nifU, nifS, nifV, nifM, and
nifB [96], [97], [98], [43] and maybe also nifQ, nifW and nifZ [94]. This implies the general usage of
these basic proteins for maturation and cofactor assembly from all three nitrogenase systems.
And this hints towards a significant cross talk between the three systems during regulation and
assembly of the V- and Fe-N2ase [94].
1.3.3 Introduction to nitrogenase
N2 reduction by H2 to NH3 is an exergonic reaction and thus thermodynamically favorable (see
equation 1) but it is kinetically disfavored by a high activation barrier. Actually, with 945 kJ/mol
[4] N2 has the second highest dissociation energy after carbon monoxide (CO) with 1070 kJ/mol
[99], but due to its polarity, CO is more reactive than inert N2, making the N2 triple bond the
most stable bond that any biological system has to break. The enzyme nitrogenase reduces N2 to
NH3 at ambient conditions in a magnesium adenosine triphosphate (MgATP) dependent reaction
and is the only known enzyme capable of N2 reduction.
The conventional Mo-N2ase has been reviewed in e.g. [80], [100], [101]. Nitrogenase is an enzyme
complex consisting of two metallo-proteins, the dinitrogenase or molybdenum-iron protein (Mo-
Fe-protein) containing the active site, and dinitrogenase reductase or iron-protein (Fe-protein)
that is the electron donor for the MgATP-driven reaction. Besides the Mo-dependent nitrogen-
ase, two alternative nitrogenases exist with an analogous protein component system. They differ
in the active site cluster heterometal composition and contain vanadium (see section 1.3.8) or
only Fe [81].
Introduction
18
For N2 fixation Fe- and MoFe-protein form a complex under turnover, and the MgATP-
dependent electron transfer takes place (see Figure 3). This process has to be repeated until suffi-
cient electrons have been accumulated to reduce N2 to NH3. Besides N2, also protons (H+) are
reduced and H2 evolves. For the ‘standard model’, a single electron transfer per two MgATP hy-
drolysed [102] and production of one obligatory H2 molecule per reduced N2 [103], [104] is as-
sumed, leading to the common stoichiometry (see equation 2) for biological N2 fixation by N2ase
of
(2)
Figure 3: Nitrogenase MoFe-Fe-protein complex under turnover conditions. The protein complex was trapped using
MgADP·AlF4- as MgATP-analogue. The MoFe-protein is shown in green (NifD) and red (NifK) colours and the
two γ-subunits of the Fe-protein in dark and light ochre (NifH). On the right side, positions of the the nitrogenase
clusters and the nucleotide as well as the electron pathway for N2 reduction are highlighted. The metal clusters are
shown in ball-and-stick representation with sulfur in yellow, iron in orange, molybdenum in cyan, nitrogen in blue,
aluminium in grey, fluor in light cyan, magnesium in neon green and carbon in black. The figure was generated using
the PDB-ID 1M34 [105].
In addition to the physiological substrate N2, nitrogenase can reduce some small compounds
containing double or triple bonds [80]. The reduction of the non-physiological substrate acety-
lene (C2H2) to ethylene (C2H4) is usually used to determine N2ase activity (see section 3.2.4) [106],
[107] [108], [109], [110].
1 N2 + 8 H+ + 8 e- + 16 MgATP 2 NH3 + 1 H2 + 16 Pi + 16 MgADP
NifD
(α)
NifK
(β)
NifD‘
(α‘)NifK‘
(β‘)
Mo
Fe-p
rote
inF
e-p
rote
in
NifH
(γ)
NifH‘
(γ‘)
NifH
(γ)
NifH‘
(γ‘)
Fe-p
rote
in
2 MgADP·AlF4
[4Fe-4S]
P-cluster
FeMo-
cofactor
e-
N2
2 NH3
e-
e-
Introduction
19
Nitrogenase is a relatively slow-acting enzyme, with a turnover time of ≈ 5 e- s-1 [111]. Depending
on the organism, additional posttranslational regulation exists next to genetic regulation via NifA
and NifL (see section 1.3.2). When the nitrogenase product NH3 becomes available, the ADP-
ribosyltransferase (DraT) covalently links an ADP-ribose moiety to a specific arginine residue on
the Fe-protein, switching off nitrogenase activity. After ammonium exhaustion, the ADP-
ribosylhydrolase (DraG) removes the modifying group, restoring nitrogenase activity [112].
In the following, N2ase will be used for the dinitrogenase component. In the same way, Mo-
N2ase or MoFe-protein or NifDK will be used for the molybdenum-dependent nitrogenases.
Also, Fe-protein or NifH will be used for the dinitrogenase reductase.
1.3.4 The nitrogenase MoFe-protein (NifDK)
MoFe-protein is a α2β2-heterotetramer of ≈ 240 kDa, representing two catalytically active αβ-
subunits, each containing two complex metallo-clusters. The [8Fe-7S]-cluster (P-cluster) serves as
intermediate electron transfer center, transmitting electrons from Fe-protein to the [7Fe-9S-Mo-
C]-homocitrate iron-molybdenum cofactor (FeMo-cofactor or M-cluster), the active site for sub-
strate reduction and the most complex metal cluster found in nature [113], [114], [115], [116],
[117], [118].
The homologous α- and β-subunits each contain three domains of the α/β-type (see Figure 4).
Each domain shows the common structural element of a central four-stranded parallel β-sheet
flanked by α-helices, called the Rossman fold. The P-cluster (see section 1.3.4.1) is located at the
interface of the α- and β-subunit ≈ 10 Å below the protein surface and the FeMo-cofactor (see
section 1.3.4.2) is buried in the α-subunit, also ≈ 10 Å below the protein surface. The α- and β-
subunits within one αβ-dimer are roughly related by a two-fold rotation axis going through the P-
cluster. The two catalytic αβ-subunits are related by the molecular two-fold rotation axis. In each
subunit a wide, shallow cleft is found between the three domains. While in the α-subunit in this
position the FeMo-cofactor is located, this place is occupied by the residues β-His193, β-Gln294,
β-His297 und β-Asp372 in the β-subunit. The edge-to-edge distance of FeMo-cofactor to P-
cluster within one αβ-dimer is ≈ 14 Å. The N-terminus of the β-subunit (≈ 50 residues) wraps
around the α-subunit, possibly for stabilization of each αβ –dimer and/or the whole tetramer.
The tetramer interface between two catalytic αβ-dimers consists mainly of interactions between
the β-subunits and seems to be stabilized by a cation binding at the interface of both β-subunits.
Based on the octahedral coordinational environment by the carboxyl oxygen of β-Glu109, β’ -
Asp353, β’-Asp357, the carbonyl oxygen of β-Arg108 and two water molecules and depending on
Introduction
20
the crystallization condition either a Ca2+ or Fe2+/3+ is present at the cation site [113], [119]. This
metal binding site has a distance of ≈ 25 Å and ≈ 21 Å to the P-cluster and to the FeMo-
cofactor, respectively, and thus doesn’t play a mechanistic role but rather serves for stabilization.
Figure 4: Structural architecture of the α- and β-subunits of the MoFe-protein. In NifD (A) domains I, II, and III are
shown in dark, medium and light green, respectively. In NifK (B) domains I, II, III are shown in dark, medium and
light red, respectively.The FeMo-cofactor in NifD is situated between the three domains. The P-cluster is located at
the outside of domain I in both subunits at the NifDK interface. Additional loops connecting the domains, α-helices
interacting with the corresponding α’-and β’-subunits and the N-terminus of NifK wrapping around NifD are shown
in grey. Metal clusters are displayed in ball-and-stick representation, with sulfur in yellow, iron in orange, oxygen in
red, nitrogen in blue and carbon in grey. The figure was generated from the PDB-ID 3U7Q [116].
1.3.4.1 The P-cluster
The overall structure of the [8Fe-7S] P-cluster in the reduced (presence of sodium dithionite),
resting state, termed the PN-state, can be considered as two linked [4Fe-4S]-clusters that share
one μ6-sulfur atom (S1) (see Figure 5 A) [114], [120]. The symmetry of the P-cluster in the resting
state is roughly C2v, with a twofold axis passing through the S1 sulfur at the intersection of two
perpendicular mirror planes. The four-fold coordination spheres of the eight iron atoms are
completed by the cysteinyl sulfur of six cysteines, with four cysteines coordinating single iron
atoms (α-Cys62, α-Cys154, β-Cys70, β-Cys153) and two cysteines (α-Cys88, β-Cys95) each bridg-
ing two iron atoms from the separate cluster sub-fragments. Upon a two-electron oxidation from
the PN state to the Pox state, the P-cluster undergoes a structural rearrangement concomitant with
a loss of symmetry. In this conformation, one cubic unit opens up by release of two Fe-S bonds
(Fe5-S1 and Fe6-S1) and formation of two novel metal-protein ligand bonds, Fe6-O, to a serin-O
(β-Ser188), and Fe5-N, to a backbone amide (α-Cys88) [120] (see Figure 5 B). The P-cluster and
the [4Fe-3S] cluster of O2-tolerant [NiFe] hydrogenase [121] are the only known naturally occur-
P-cluster
I
II
III
FeMo-
cofactor
P-cluster
I
II
III
N-terminus
A B
Introduction
21
ring [Fe-S] clusters that contain serin-O and amide-N ligands, in addition to typical cysteinate-S
ligands. All iron atoms remain four-fold-coordinated in the Pox-state.
Figure 5: Metal clusters of the nitrogenase MoFe-protein. The P-cluster is displayed in the reduced PN (A) and the
oxidized Pox (B) state. The structural rearrangement of the P-cluster upon two electron oxidation is displayed. The
FeMo-cofactor is illustrated in C. Sulfur is yellow, iron is orange, molybdenum in cyan, oxygen is red, nitrogen is blue
and carbon is medium green. The figure was generated from PDB-ID 3U7Q [116].
The P-cluster, relative to the PN-state, can be reversibly oxidized by up to three electrons (P0 =
PN, P1+, P2+ = Pox, and P3+) [122], [123], [124] (see equation 3). Mössbauer spectroscopy revealed
that all iron atoms in the PN-state are in the ferrous oxidation state [8Fe2+-7S]2+ [125]. The mid-
point potentials for the three reversible P-cluster redox couples have been determined at pH 7.5
to be Em(P0/P1+) = -307 mV, Em(P1+/P2+) = -307 mV and Em(P2+/P3+) = +90 mV (see equation
3) [122]. More reduced states have not been observed yet [126] and further oxidation beyond
> + 250 mV is not reversible anymore and seems to degrade the P-cluster [122]. While the PN
state is diamagnetic, both P1+ and P2+ states are paramagnetic. The P1+ state shows a mixed spin
system (S = ½ and 5/2) with perpendicular mode EPR signals in the g = 2 and 5 regions [123],
and the P2+ state shows an integer spin system (S = 3) with parallel-mode EPR signals at g = 11.8
[122]. The P3+ state has a S = ½ and 7/2 mixed spin system. A proposed ‚electron deficit spend-
ing’ mechanism describes the kinetics of a one electron transfer event between Fe-protein and
MoFe-protein [127] (see section 1.4.1.3). It suggests upon complex formation, at first an electron
transfer from the P-cluster to the FeMo-cofactor, which is then followed by the electron transfer
from the [4Fe-4S]-cluster reducing the P-cluster. This process supports a working PN/P1+ redox
couple. In another study, a two electron transfer event from the Fe-protein to the MoFe-protein
homocitrateα-His442
α-Cys275
FeMo-co
α-Cys62
α-Cys154
β-Cys95
α-Cys88
β-Cys143
β-Ser188
β-Cys70
Fe6
Fe5
Pox
α-Cys62
α-Cys154
β-Cys95
β-Cys143
β-Ser188
β-Cys70
Fe6 Fe5
PN
α-Cys88
A B C
Introduction
22
could be observed [128], which would hint to a possibly operating PN/P2+ redox couple as well.
There is no evidence for a mechanistic relevance of the P3+ state.
(3)
1.3.4.2 The FeMo-cofactor
The structure has been solved by crystallographic and spectroscopic methods and consists of an
inorganic framework, with seven iron atoms, nine sulfur atoms, one molybdenum atom and one
carbon atom, and additionally one organic homocitrate molecule ([7Fe-9S-Mo-C]-HC) [113],
[114], [115], [116], [117]. The carbon atom was shown to be a carbide (C4-) [129]. The cluster is
ligated to the protein environment through one cysteine ligand (α-Cys275) bound to the Fe atom
Fe1 at one end and through one histidine ligand (α-His442) bound to the Mo atom at the oppo-
site end (see Figure 5 C). The inorganic components exhibit very closely a C3v (threefold) sym-
metry, which becomes particularly apparent in the trigonal prismatic arrangement of the six cen-
tral iron atoms. Each iron atom is coordinated by three cluster sulfide atoms and either by the
interstitial C4- (Fe2 – Fe7) or by the sulfur atom of α-Cys275 (Fe1). Intermetal (Fe-Fe and Mo-Fe)
and Fe-S distances show typical values of ≈ 2.6-2.7 Å and ≈ 2.2-2.3 Å, respectively [115], [116].
The octahedral Mo coordination environment by three sulfide atoms and residue α-His442 is
completed through bidentate coordination by homocitrate through its 2-hydroxy and 2-carboxyl
groups. Thus, the overall structure of the FeMo-cofactor can be regarded as one [4Fe-3S-C] cub-
ane and one [Mo-3Fe-3S-C] cubane that are connected by three bridging sulfides with one shared
μ6-C4- at the center.
Although the atomic structure of FeMo-co has been solved, its electronic structure and reactivity
are still under debate. In the as-isolated MoFe protein (reduced with sodium dithionite) FeMo-co
is present in its resting state, MN. This state is paramagnetic, with a S = 3/2 spin system and a
rhombic EPR signal with apparent g-values of g = 4.3, 3.7 and 2.0 [130]. The FeMo-co can be
reversibly oxidized by one electron from the resting state MN to a M1+ state, called Mox [131],
[132]. This state is diamagnetic with S = 0 and is EPR-silent. The redox potential for the redox
couple MN/Mox is Em(MN/Mox) = -42 mV at pH 7.5 [122]. Reduction of FeMo-co beyond the MN
state has only been observed either under physiological turnover to a one electron reduced MR
state [133] or via radiolytically reduction also by one electron but into another state, called MI
[134]. Both MR and MI states have an integer spin system (S ≥ 1) but differ in their spin state,
P0 = PN
S = 0
(diamagnetic)
1 e-
-307 mV P1+
S = ½, 5/2
1 e-
-307 mV P2+ = Pox
S = 3
1 e-
+90 mV P3+
S = ½, 7/2
Introduction
23
probably with MR having a S = 2 and MI showing a S = 1 spin state. Furthermore, Mössbauer
spectroscopy revealed that Fe sites in MR are as reduced as in MN, whereas in MI the Fe sites are
further reduced than in MN [134], [135]. EXAFS showed that metal-metal distances in FeMo-co
contract by about 0.04 Å upon one-electron reduction under turnover, indicating that electrons
are not stored in the Fe atoms [136], [80]. As the MR state is observed under physiological turno-
ver, the redox couple MN/MR is likely mechanistically relevant, while the MI state is probably not.
The relevance of oxidized Mox state and the corresponding redox couple Mox/MN during N2 re-
duction is not clear yet.
With the recent determination of molybdenum being Mo3+ [137] and identification of the intersti-
tial C4- [116], [117], [129], current computational analysis and the comparison with experimental
Mössbauer isomer shifts [134] suggest a negative charge of [7Fe-9S-Mo-C]1- [138]. The most re-
cent assignment of metal oxidation states in the FeMo-cofactor proposes a Mo3+-3 Fe2+-4 Fe3+
distribution [138] with Fe1, Fe3, Fe7 having a Fe2+ and Fe2, Fe4, Fe6, Fe8 having an Fe3+ oxida-
tion state [139]. It could also be interpreted as Mo3+-1 Fe2+-4 Fe2.5+-2 Fe3+ [138].
Recently, a MoFe-protein structure with carbon monoxide (CO) bound to the FeMo-co was
solved. The CO replaces one of the bridging belt sulfurs (S2B) and is coordinated by the two Fe
atoms Fe2 and Fe6 [140]. Furthermore, it was shown that an incorporated selenium atom (Se) at
the S2B site of the FeMo-co diffuses under turnover and after ≈ 500 cycles the Se is equally dis-
tributed over all three bridging sulfurs [141]. Both findings hint to a certain degree of flexibility at
the FeMo-co, at least concerning the belt sulfurs, which was not expected before.
1.3.5 The nitrogenase Fe-protein (NifH)
Fe-protein is a γ2-homodimer ≈ 64 kDa with two identical subunits that coordinate a single [4Fe-
4S]-cluster and has two MgATP binding sites (see Figure 6 A, B) [142]. Each subunit symmetri-
cally ligates the surface-exposed and subunit-bridging [4Fe-4S]-cluster and has the MgATP bind-
ing site at the subunit’s interface. The Fe-protein mediates MgATP hydrolysis with electron trans-
fer from the [4Fe-4S]-cluster to the MoFe-protein. It was the only known electron donor for the
MoFe-protein that supports N2 reduction until recently light driven N2 reduction by CdS:N2ase
MoFe-protein biohybrid nanorods was discovered [143].
Each monomer adopts the general polypeptide fold present in P-loop-NTP-ases like ras p21 or
G-proteins [144], [145], that belong to the nucleotide-switch protein family (see Figure 6 A) [146].
Their common structural core elements are a central β-sheet flanked by several α-helices (Rossman
Introduction
24
fold), and three conserved loops, called the phosphate-binding loop (P-loop), the switch I and the
switch II region, representing the nucleotide binding site. The P-loop consists of the Walker A
motif G-X-X-X-X-G-K-S/T [147], which can be found in residues γ-Gly9 to γ-Ser16, and inter-
acts with the α- and β-phosphate of the MgATP, thus being sensitive for absence or presence of
a nucleotide. The switch I region includes the sequence γ-Cys38 to γ-Asp43 and primarily inter-
acts with Mg2+. The switch II region, including residues γ-Asp125 to γ-Phe135 [111], starts with
the conserved motif D-X-X-G, corresponding to residues 125 and 128, and interacts mainly with
the γ-phosphate, thus being sensitive to the form of bound nucleotide (diphosphate or triphos-
phate).
Figure 6: Structure of the nitrogenase Fe-protein. A monomer of the Fe-protein in the MgADP·AlF4- bound nitro-
genase complex conformation is displayed in A. The nucleotide binding site represented by the P-loop in dark green,
switch I in blue and switch II in red also ligating the [4Fe-4S] cluster. In B, the structure of the nucleotide-free Fe-
protein dimer is displayed in light and dark grey with the bridging [4Fe-4S] cluster and the nucleotide binding sites at
the subunits interface. The conformational change including the subunit rotation and the [4Fe-4S] cluster shift are
illustrated in C by a superposition of the Fe protein in the nitrogenase complex (light and dark ochre) and nucleo-
tide-free. Sulfur is yellow, iron is orange, molybdenum is cyan, aluminium is grey, fluor is light cyan, magnesium in
neon green, oxygen is red, nitrogen is blue and carbon is medium green. The figure was generated from the PDB-
IDs 1G5P [148] and 1M34 [105].
P-loop
MgADP·AlF4
Switch II
Switch I
[4Fe-4S]
A B
C
[4Fe-4S]
nucleotide binding sites
≈ 4 - 5 Å
Introduction
25
The [4Fe-4S]-cluster is ligated by γ-Cys97 and γ-Cys132 from both subunits, which are located
within the switch II region. Thereby the switch II region, which interacts with the γ-phosphate
being sensitive to the nucleotide phosphorylation state, is connected to the cluster. Nucleotide-
switch proteins share the common feature to adopt different distinct conformations depending
on the form of the bound nucleotide. The function of these proteins is a signaling or energy
transduction to a second protein that is coupled with nucleotide hydrolysis. In case of nitrogen-
ase, it is the electron transfer from the Fe-protein to the MoFe-protein which is coupled with
MgATP hydrolysis. The Fe-protein alone shows no MgATP hydrolyzing reactivity [102], high-
lighting that it cannot hydrolyse MgATP on its own and requires the interaction with the Mo-
Fe-protein. In the reduced state with two bound MgATPs, the Fe-protein adopts a conformation
not capable of MgATP hydrolysis, but able to form a complex with the MoFe-protein, the ‘on-
state’ [145]. Subsequent complex formation triggers a conformational change that leads to the
second conformation (‘complex conformation’) [146] of the Fe-protein, that enables hydrolysis
of MgATP. A direct chemical coupling of nucleotide hydrolysis to electron transfer can be ex-
cluded, as a distance of ≈ 20 Å from the nucleotide binding site to the cluster is too far [142].
However a concomitant conformational change of the switch II region, which is connected to
the cluster via γ-Cys129 and to the nucleotide binding site via γ-Asp125, leads to a shift of the
cluster by 4-5 Å towards the protein surface (see Figure 6 C) and section 1.3.6) [149] and thus
influences and enables the coupled electron transfer. After MgATP-hydrolysis, having MgADP
bound, the Fe-protein adopts a third conformation, the ‘off-state’ [145], leading to complex dis-
sociation and release of Pi and MgADP. The rate of nucleotide turnover, the combination of
hydrolysis and nucleotide exchange, is the ultimate timing event in such signal or energy trans-
duction processes [100].
The Fe-protein shows a nucleotide-dependent redox-potential behavior with -300 mV, -430 mV
and -490 mV depending on whether no nucleotide, MgADP or MgATP is bound, respectively
[102].
1.3.6 The N2ase complex
A key step in the N2ase mechanism with Fe-protein as a P-loop-NTPase is the formation of the
Fe-protein–MoFe-protein complex, where MgATP hydrolysis is coupled with electron transfer
[100]. The transition state analogue ADP-aluminium fluoride (AlF4-) was used to trap the N2ase
component proteins in a stable complex [150], [151]. ADP-AlF4- is considered to mimic the tran-
sition state during nucleotide hydrolysis, and AlF4- is supposed to represents the trigonal bi-
Introduction
26
pyramidal geometry of the terminal phosphate that undergoes nucleophilic attack by a water mol-
ecule. The structure of the MgADP-AlF4--stabilized complex [149], [105] shows substantial struc-
tural changes for the Fe-protein, while no large structural changes are detected in the MoFe-
protein.
The overall quaternary structural change in Fe-protein can be seen as a ≈ 13 ° rotation of each
monomer towards the interface leading to a subunit approach including inter-subunit interactions
and a concomitant shift of the [4Fe-4S]-cluster of ≈ 4-5 Å to the protein surface closer to the
MoFe-protein (see Figure 6 C). This cluster shift results in a distance decrease of the [4Fe-4S]-
cluster to the P-cluster of ≈ 18 Å to ≈ 14 Å. Therewith in the nitrogenase complex the [4Fe-4S]-
cluster, the P-cluster and the FeMo-cofactor are ≈ 14 Å center-to-center distant to each other
(see Figure 7). Furthermore by decrease to ≈ 14 Å, the maximum distance for rapid electron tun-
neling between protein redox centers is warranted [152].
Figure 7: Metal cluster distances and positions in nitrogenase complex compared with nucleotide free Fe-protein.
Metal clusters in nitrogenase complex are displayed in ball and coloured line representation. The [4Fe-4S] cluster of
nucleotide free Fe-protein is highlighted by grey sticks. Distance decrease of [4Fe-4S] cluster to P-cluster upon com-
plex formation is visible. Sulfur is yellow, iron is orange and molybdenum is cyan. The figure was generated with the
PDB-IDs 1G5P [148] and 1M34 [105].
The conformational changes within the Fe-protein in the complex-structure (‘complex confor-
mation’ see section 1.3.5) are the mediator that connects the mechanism of ATP-hydrolysis with
electron transfer. An efficient ATP-hydrolysis needs the proper positioning of two residues. One
residue, from the switch II region, needs to be located close to the γ-phosphate to assist in ori-
enting the attacking water or acting as a general base. The other residue, from the P-loop, needs
to be a positively charged residue that stabilizes the negatively charged leaving β-phosphate [146].
The crucial conformational rearrangements occur in the P-loop and the switch II region. The
positively charged residue γ-Lys10 from the P-loop coordinates the oxygen of the β-phosphate in
the second subunit across the interface, thus stabilizing the leaving group. The basic γ-Asp129
from the switch II region also interacts across the interface with AlF4- in the nucleotide binding
14 Å 14 Å
shift of 4-5 Å
Introduction
27
site of the second monomer through a water molecule, thereby acting as activator of the attack-
ing water. The switch II region (residues γ-125 to γ-135) in the complexed Fe-protein contains
residues, that are involved in nucleotide binding (conserved motif D-X-X-G with γ-Asp125 and
γ-Gly128), in cluster ligation (γ-Cys132) and that are essential for ATP-hydrolysis (γ-Asp129). It
is crucial, that the conformation of the switch II region, that is decisive for ATP hydrolysis, with
γ-Asp129 from one subunit orienting the water in the nucleotide binding site of the other subunit
in the proper position for ATP hydrolysis, is simultaneously connected to a repositioning of γ-
Cys132, that leads to the [4Fe-4S]-cluster shift of ≈ 4-5 Å towards the P-cluster.
Crucial is that the for ATP-hydrolysis decisive conformation of the switch II region, where γ-
Asp129 from one subunit orients the water molecule in the nucleotide binding site of the other
subunit in the proper position for ATP-hydrolysis, is simultaneously connected with a reposition-
ing of γ-Cys132 and thus the [4Fe 4S]-cluster shift of ≈ 4-5 Å towards the P-cluster. Conforma-
tional changes in the loop (residues γ-91 to γ-97) containing the second cluster-coordinating lig-
and, γ-Cys97, also impact the location and environment of the [4Fe-4S]-cluster, resulting in the
cluster placement closer to the surface. Thereby the carbonyl-oxygens of MoFe-protein residues,
α-Val158 and β-Leu157, are in van-der-Waals distance (≈ 3.2 - 3.5 Å) to the [4Fe-4S]-cluster.
The key point of the mechanism of coupling ATP-hydrolysis to electron transfer is that the con-
formation of the switch II region, which is needed for ATP-hydrolysis, is also needed to properly
position the [4Fe-4S]-cluster for efficient electron transfer. Fe-protein, MoFe-protein and
MgATP are necessary to attain the proper conformation and position of the switch II region
[146]. In this case, the MoFe-protein serves to stabilize the short-lived transition state confor-
mation for the activation of MgATP hydrolysis in Fe-protein.
1.3.7 Nitrogenase cluster biosynthesis
The complexity of the nitrogenase metallo-clusters, the FeMo-cofactor, also called M-cluster, and
the P-cluster, also requires a complex assembly machinery. In the biosynthesis of both clusters,
several nif-gene products, like NifB, NifEN, NifH, NifQ, NifS, NifU, NifV, NifZ, are involved
[153], [87]. Whereas the P-cluster is assembled ‘in situ’ on NifDK, the maturation of the M-
cluster takes place ‘ex situ’, on the specified cluster assembly proteins NifB and NifEN, and is
after fully biosynthesis transferred to NifDK [154], [77].
Introduction
28
1.3.7.1 P-cluster biosynthesis
The maturation of the P-cluster is initiated by the proteins NifS and NifU generating [4Fe-4S]-
building blocks, that are then used for the assembly of the P-cluster. NifS is a pyroxydal-
dependent cysteine desulfurase delivering sulfide, and NifU is a scaffold protein that builds the
[2Fe-2S]- and [4Fe-4S]-clusters [97], [155], [156], [157], [158], [159].
A pair of [4Fe-4S]-clusters is transferred to the interface of each of the two αβ-halves of NifDK
where the proposed stepwise biosynthesis of the P-clusters takes place ‘in situ’ [154]. This un-
bridged [4Fe-4S]-cluster pair, also called P*-cluster, is thought to consist of one normal [4Fe-4S]-
cluster and one atypical [4Fe-4S]-like cluster [160], [161], [162]. The two P-clusters of NifDK are
not synthesized simultaneously, but one after the other one. For maturation of the first P-cluster,
NifH is supposed to interact with NifDK at the αβ-subunit interface and via a reductive coupling
of both [4Fe-4S]-clusters one [8Fe-7S]-cluster is generated (see Figure 8 ‘P1’). This reaction takes
place under MgATP and electron consumption [163] and these dependencies specify a second
function of NifH, as it is involved in the reductive coupling of two [4Fe-4S]-clusters into one
[8Fe-7S]-cluster. So far it is not clear how the eighth sulfur is eliminated. For the assembly of the
second P-cluster the protein NifZ in addition to NifH is necessary (see Figure 8 ‘P2’). The exact
role of NifZ is not known, but it might act in a chaperon-like function by modifying some key
residues at the second αβ-interface enabling the second coupling [91], [164]. The maturation of
the P*- to the P-cluster is connected with a conformational change in the αβ-subunit. On the one
site, a 6 Å gap at the αβ-interface from the [4Fe-4S]-cluster-pair is closed, maybe induced by
complexation with NifH, thereby protecting the P-cluster from solvent exposure and stabilizing
the protein structure [165]. On the other site, the conformational change leads to an opening of
the α-subunit, presenting the positively charged M-cluster insertion path. Thus after P-cluster
maturation the subsequent insertion of the M-cluster on NifDK is allowed [166].
Introduction
29
Figure 8: Schematic illustration of the biosynthesis of nitrogenase clusters. Proteins are displayed as cubes with
NifDK in light and medium red, NifB in blue and NifEN in light and medium green. Dashed arrows represent inter-
protein cluster transfer and straight arrows intra-protein cluster assembly steps. NifS and NifU generate [4Fe-4S]
clusters and deliver them to NifDK and NifB. The in situ P-cluster maturation is highlighted in light blue, the ex situ
FeMo-co biosynthesis in orange. P1: reductive coupling of two [4Fe-4S] clusters from one P*-cluster results in a
MoFe-protein with one P-cluster and one P*-cluster; the reaction is mediated by NifH under MgATP and electron
consumption. P2: assembly of the second P-cluster where additionally NifZ is needed; P-cluster maturation leads to
a conformational change of NifDK, enabling M-cluster insertion. M1a: The radical-SAM-dependent coupling of a
[4Fe-4S] cluster pair (K-cluster) and the simultaneous central carbon and 9th sulfur insertion results in the [8Fe-9S-S]
L-cluster on NifB. M1b: L-cluster transfer from NifB to NifEN. M2: Mo and homocitrate incorporation results in
the matured M-cluster; this step is mediated by NifH under MgATP and electron consumption and leads to a con-
formational change of NifEN, enabling interaction with NifDK. M3: The transfer of matured M-cluster to NifDK
results in MoFe-protein with fully assembled P-clusters and M-clusters. The scheme is generated according to [154].
1.3.7.2 FeMo-cofactor biosynthesis
The biosynthesis of the M-cluster is initiated by the proteins NifS and NifU generating [4Fe-4S]-
building blocks like as for the P-cluster.
The proposed ‘ex situ’ assembly of the M-cluster or FeMo-cofactor starts with a pair of [4Fe-4S]-
clusters delivered from NifU to NifB and can then be divided into three major steps: 1a) the
formation of a [4Fe-4S]-pair, called K-cluster, into the [8Fe-9S-C]-core cluster, called L-cluster,
on NifB and 1b) subsequent transfer to NifEN; 2) the incorporation of Mo and homocitrate to
yield the [7Fe-9S-Mo-C]-HC FeMo-cofactor, or M-cluster, on NifEN and; 3) the transfer of the
fully assembled M-cluster to NifDK [77] (see Figure 8 ‘M1 – M3’).
NifB is a radical S-adenosyl-L-methionine (SAM) dependent enzyme [167], [168] and carries one
[4Fe-4S]-SAM-cluster [169] in close proximity to the [4Fe-4S]-cluster pair (K-cluster). In a novel
NifS
+
NifU
NifB
K-cluster
„SAM“
[4Fe-4S]
NifB
L-cluster
SAM:
C
9th S
2x[4Fe-4S] [8Fe-9S-C]
[8Fe-9S-C]
[8Fe-9S-C]
NifH
MgATP
e-
NifEN
L-cluster
[4Fe-4S]
FeMoco
FeMoco
NifEN
M-cluster
FeMoco
FeMoco
NifDK
M-cluster
Mo
HC
Fe
2x[4Fe-4S]
2x[4Fe-4S]
NifDK
P*-cluster
([4Fe-4S] + [4Fe-4S]-like)
[8Fe-7S]
2x[4Fe-4S]
NifDK
P-cluster
P*-cluster
NifH
MgATP
e-
[8Fe-7S]
[8Fe-7S]
NifDK
P-cluster
NifZ
NifH
MgATP
e-
[8Fe-7S]
[8Fe-7S]
[4Fe-4S]
[4Fe-4S] [4Fe-4S]
[4Fe-4S]
6 A
P1 P2
M1a M1b
M2
M3
in situ
P-cluster
ex situ
M-cluster/
FeMo-cofactor
„SAM“
[4Fe-4S]
Introduction
30
synthetic route, NifB is supposed to couple and rearrange the two [4Fe-4S] clusters into an [8Fe-
9S-C]-cluster (L-cluster), concomitant with the radical SAM-dependent insertion of carbide and
the addition a ‘9th’ sulfur [169], [170] (see Figure 8 ‘M1a’). The pattern of the SAM cleavage gave
hints for a radical-SAM-dependent chemistry in this process [171]. While SAM is the origin of the
carbon [171] and a direct transfer from SAM to the cluster is suggested, the origin of the ‘9th’
sulfur is not clear yet. Though SAM has been shown to act as sulfur donor [172], [173] and could
act in this role here as well. Yet, the mechanism for incorporation of both atoms is unclear. Nev-
ertheless, the carbon species has to undergo some additional deprotonation and/or dehydration
steps until the necessary carbide atom is generated in the L-cluster center [129]. Subsequently, the
L-cluster is to be transferred to NifEN (see Figure 8 ‘M1b’).
NifEN has a high sequence homology to NifDK, is structurally homologous and was hypothe-
sized to carry analogous cluster sites serving as scaffold protein for M-cluster assembly [174]. In
the NifDK P-cluster site, NifEN shows a [4Fe-4S]-cluster [175] and in the active FeMo-cofactor
site NifEN presumably binds the L-cluster [176], [177]. The conversion of the [8Fe-7S-C] L-
cluster to the fully matured [7Fe-9S-Mo-C]-HC FeMo-cofactor or M-cluster, the attachement of
Mo and homocitrate while one Fe atom will be distracted, is supposed to take place on a NifEN-
NifH complex and is MgATP- and redox-dependent [178], [179], [180] (see Figure 8 ‘M2’). While
NifQ probably serves as the physiological Mo donor [181], NifV is the homocitrate synthase
[182]. Upon maturation of L- to M-cluster, NifEN undergoes a conformational change with two
effects [183]. At first, while the L-cluster is located at the surface, probably to ease the access for
Mo and homocitrate incorporation, the matured M-cluster is hypothesized to be relocated within
the protein [177]. And secondly, this maturation step enables interaction with NifDK, as only
fully assembled M-cluster-containing NifEN can form a complex with NifDK [183]. Mo and
homocitrate are mandatory comobilized and are not stepwise inserted [183], but it is unknown,
how their incorporation and the simultaneous distraction of one Fe takes place. Even the deliv-
ered form of Mo is not clear yet, as on one hand in in-vitro assays, containing Mo in form of
molybdate (MoO4-), NifEN carrying the L-cluster, NifH, MgATP, reducing agent and homo-
citrate, M-cluster assembly takes place [179]. And furthermore, MoO4- can bind to NifH in a po-
sition corresponding to the γ-phosphate of MgATP in an ADP-bound structure [142]. Interest-
ingly, on the other hand in NifQ, the putative physiological Mo donor, Mo is proposed to be
bound in a [3Fe-Mo-4S] cluster [181], [184]. The MgATP and redox dependence of this reaction
specifies a third function of NifH, and a key role as MgATP-dependent Mo/homocitrate in-
sertase [179], [180].
Introduction
31
The final step of M-cluster maturation is the cluster transfer from NifEN to NifDK (see Figure 8
‘M3’). M-cluster is proposed to be delivered upon NifEN-NifDK complex formation via direct
protein-protein interactions [178], initiating a suggested migration of the M-cluster from the tran-
sient binding site on NifEN, the ‘low affinity’ site, to the final active binding site on NifDK, the
‘high affinity’ site [185]. The hypothesized, differential affinities remain to be proven. Anyway, a
positively charged insertion path, in the α-subunit of matured P-cluster containing NifDK, may
facilitate insertion of the negatively charged M-cluster through ionic interactions and some con-
served, positively charged key residues [166]. Finally, after M-cluster insertion a conformational
rearrangement of NifDK closes the insertion path and buries the M-cluster in the α-subunit
≈ 10 Å below the protein surface [113], [166].
1.3.8 Vanadium dependent nitrogenase (V-N2ase)
The alternative, vanadium-dependent N2ase (V-N2ase) [81], [186] is also a two-component en-
zyme complex consisting of an iron protein (Fe-protein or VnfH) and the vanadium iron protein
(VFe-protein or VnfDKG) and is supposed to be an analogue to the Mo-N2ase. VnfH has a se-
quence identity of ≈ 91 % with NifH. The α- and β-subunits (encoded by vnfD and vnfK) of
VnfDKG share a sequence identitiy of ≈ 33 % and ≈ 32 % with NifDK [187], but V-N2ase has
an additional δ subunit (encoded by vnfG) (see section 1.3.2). The subunits have a size of
VnfD ≈ 53.9 kDa, VnfK ≈ 52.8 kDa and VnfG ≈ 13.4 kDa (Mr based on gene size from Uni-
ProtKB entries: vnfD = P16855; vnfK = P16856; vnfG = P16857). V-N2ase is primarily found in
Azotobacter, cyanobacteria and methanogens, groups of organisms without a DraT posttransla-
tional mechanism [188].
Although a vanadium-dependent alternative nitrogenase is known for more than 30 years [189],
[84], [190], [191], so far any structural information of V-N2ase remained elusive due to problems
to isolate homogeneous and stable VFe-protein [192], [186], although recently a homogeneous
and stable His-tagged VFe-protein could be isolated [193]. Thus the exact subunit composition of
the VFe-protein is not known yet. Several V-N2ases have been isolated, like from A. chroococcum as
α2β2δ2 [186] and from A. vinelandii as either a mixture of a αβ2(δ)-trimer and α2β2(δ)-tetramer each
with minor amounts of the δ-subunit [192] or as recently in the His-tagged variant as a α2β2δ4-
octamer [193]. The presence of conserved cluster ligands for P-cluster and FeMo-cofactor of the
MoFe-protein in the sequence of the VFe-protein suggests the existence similar of clusters in the
V-N2ase, like the P-cluster and in that case an iron-vanadium cofactor (FeV-cofactor or FeV-co)
with a different heterometal composition (see section 1.3.8.1).
Introduction
32
Both N2ases in general show reduction of the same substrates, although they also show distinct
differences in their substrate range and catalytic activities. Mo- and V-N2ase reduce their physio-
logical substrate N2 and the most common non-physiological substrate C2H2 [194], [195]. Where-
as at 30 °C V-N2ase shows ≈ 60 % decreased specific N2 reduction activity compared to Mo-
N2ase, at 5 °C, with overall lower activities, V-N2ase is more active [196]. Acetylene appears to be
a poorer substrate for V-N2ase with a specific C2H2 reduction activity of only ≈ 28 % compared
to Mo-N2ase [193]. In vivo, V-N2ase reduces three molecules of H2 per reduced N2 [81, 197] im-
plementing a lower efficiency compared to Mo-N2ase. A significant reactivity difference is the
ability of V-N2ase to reduce C2H2 not solely to C2H4, like Mo-N2ase, but also to form ≈ 5 %
ethane (C2H6) [198], [199].
The most striking difference in reactivity between V-N2ase and Mo-N2ase is their catalytic activity
towards CO reduction and hydrocarbon formation [200], [201]. Both N2ases are able to reduce
CO, but V-N2ase is ≈ 800 fold more active in CO reduction, with 16.5 nmol reduced car-
bon/nmol protein/min compared to 0.02 nmol reduced carbon/nmol protein/min of Mo-N2ase,
which is below catalytic turnover for the Mo-N2ase [201], [202]. Depending on the assay condi-
tion both N2ases produce ethylene (C2H4), ethane (C2H6), propane (C3H8), propylene (C3H6), α-
butylene (C4H8) and n-butane (C4H10). Additionally, V-N2ase can produce methane (CH4). The
predominant product for both proteins is C2H4 [200], [201]. Single mutated α-Val70→Ala/Gly
MoFe variants show the same decreased CO reduction activity than wild-type Mo-N2ase [203].
Thereby V-N2ase is able to perform two important industrial processes, the Haber–Bosch- and
Fischer-Tropsch-process as one enzyme at ambient conditions.
This definite and distinct discrepancy in catalytic reactivity suggests differences in redox poten-
tials of FeV- and FeMo-cofactor, which could arise from e.g. the different heterometal composi-
tion (V vs Mo) and/or different electronic properties and/or differing direct environments of
cofactors within the proteins (VFe vs MoFe).
1.3.8.1 V-N2ase P-cluster and FeV-cofactor
Spectroscopic studies with Mössbauer [204], magnetic circular dichroism (MCD) [205] and EPR
[206], [207] of VFe-protein from Hales and collegues and Eady and collegues (non-His-tagged
VFe-protein) [186] indicate the presence of a homologous P-cluster in V- and Mo-N2ase. In con-
trast, recent X-ray absorption spectroscopy (XAS) and extended X-ray absorption fine structure
(EXAFS) spectroscopy studies of a His-tagged VFe-protein from Hodgson, Hedman and Ribbe
suggested the P-cluster may consist of paired [4Fe-4S]-like clusters [208], [187].
Introduction
33
While EXAFS studies of non-His-tagged VFe-protein and its solvent-extracted FeV-cofactor
[209], [210], [211], [212] and Mössbauer spectroscopy [204] suggested largely homologuos cofac-
tors in VFe- and MoFe-proteins, XAS and EXAFS studies with His-tagged VFe-protein also
generally show close resemblance, but the extracted cofactors show distinct differences in the
electronic properties and structural topology [213]. MCD studies of the non-His-tagged V-N2ase
indicated some differences in the electronic and magnetic properties of both FeV-co and FeMo-
co as well [205]. This is consistent with observations in EPR spectroscopy, where both reduced
and, in case of the His-tagged V-N2ase, oxidized protein spectra of VFe- and MoFe-protein differ
in their intensity and line-shape [206], [193].
However, in EPR the P-cluster in its P1+ state is associated with an S = ½ spin state, with EPR
signals at g = 2.03 and 1.92 and the P-cluster is also associated with an S = 5/2 spin state with an
EPR signal at g = 6.68 [208], [187]. The P2+-specific S = 3 integer spin state shows an EPR sig-
nal at g ≈ 12 [206]. The FeV-co in the resting state gives rise to an S = 3/2 spin state, with EPR
signals at g = 5.50, 4.32 and 3.77 [193], [187]. Interestingly, the signal at g ≈ 5.50 of non-His-
tagged VFe-protein shows a redox-dependent splitting [206].
1.4 Mechanism of N2 reduction
1.4.1 N2ase turnover cycle and Lowe-Thorneley scheme
1.4.1.1 N2ase turnover cycle
The basic mechanism for reduction of one N2 molecule at the protein level is the following: 1)
The MoFe-protein forms a complex with the reduced, MgATP-bound Fe-protein; 2) MgATP
hydrolysis and electron transfer take place, with subsequent Pi release and complex dissociation;
3) after Fe-protein reduction and MgATP exchange the cycle restarts and 4) is repeated until suf-
ficient electrons have been transferred for N2 binding and reduction. This process can be divided
in the Fe-protein cycle and the MoFe-protein cycle (see Figure 9) [214], [80], [100].
Introduction
34
Figure 9: Schematic illustration of the N2ase turnover cycle consisting of Fe-protein cycle (left) and MoFe protein
cycle (right). The electron flow from a reduced ferredoxin (Fd) or flavodoxin (Fld) to the Fe-protein, the electron
transfer from Fe-protein to MoFe-protein coupled to MgATP hydrolysis and the final substrate reduction leaving the
MoFe protein in the resting state is displayed. For reduction of N2 the Fe-protein cycle has to be repeated (repre-
sented by dashed arrow) eight times. The figure was adapted from [214], [100].
1.4.1.2 Fe-protein cycle
The Fe-protein cycle describes an individual electron transfer event (see Figure 9). The Fe-
protein, in its role as nucleotide dependent switch-protein (see section 1.3.5 and 1.3.6), partici-
pates in the N2ase turnover cycle in a three-state process. The reduced Fe-protein ([4Fe-4S]1+)
with two bound MgATP molecules represents the first state (‘on-state’), where it is able to associ-
ate with the MoFe-protein. Upon complex formation, the Fe-protein adopts the second state
(‘complex conformation’) in which hydrolysis of the two MgATP and the concomitant single-
electron transfer to MoFe-protein takes place. After hydrolysis of the two bound MgATP, the
oxidized Fe-protein ([4Fe-4S]2+) adopts the third state (‘off-state’) triggering complex dissocia-
tion, ADP and Pi release, which is the overall rate-limiting step in nitrogenase catalytic cycle
[215]. After reduction of the Fe-protein from the most common physiological reductants ferre-
doxin or flavodoxin [216], [217], [88], and exchange of MgADP against MgATP, the Fe-protein
cycle restarts. The order of reduction and nucleotide exchange is not established yet, but it is as-
sumed to be reduction first, followed by nucleotide exchange [215].
1.4.1.3 MoFe-protein cycle
The MoFe-protein cycle represents the accumulation of electrons, as well as substrate binding
and reduction at FeMo-cofactor (see Figure 9). Eight electrons are required for N2-reduction (see
equation 2), and only one electron is transferred per Fe-protein cycle. Therefore the Fe-protein
cycle has to occur eight times for reduction of a single N2 molecule. The different states of accu-
Fe-proteinred
2 ATP
Fe-protein-
MoFe-protein-complex
2 ATP
Fe-proteinox
2 ADP
Fd/Fldred
Fd/Fldox
2 ADP
2 ATP
MoFe-proteinred (E1-E8)
MoFe-proteinresting (E0)
substrateox substratered
Introduction
35
mulated electrons on MoFe-protein are denoted En, where n represents the number of accumu-
lated electrons on the FeMo-co. E0 represents the resting state, E1 to E7 represent the intermedi-
ate states during N2 catalysis after which E0 is restored [214]. N2 binds reversibly to states E3 and
E4 and concomitant with N2 binding a H2 molecule is released (see Figure 10) [218], [103], [219].
This reversible interaction is the basis for the ability of H2 to competitively inhibit N2 reduction,
as H2 can in turn displace N2. N2 is the only known substrate whose reduction is inhibited by H2,
making H2 a specific, competitive inhibitor for N2 reduction [220], [221], [222], [223]. The prop-
erty of N2/H2 to reversibly bind and exchange, leads to HD formation in the presence of N2 and
D2 at the E4 reduction level without scrambling with the solvent and only under N2 turnover
conditions with high electron flux [214], [80], [224], [104]. In the absence of N2, only protons are
reduced, resulting in H2 production, and under these conditions no states beyond E4 can be
reached. While N2 binds to E3 and E4, non-physiological substrates, such as C2H2 bind reversibly
to E1 and E2, and inhibitor CO binds also already to E2 [225], [214]. Thus, although binding oc-
curs at the same substrate binding site at FeMo-co, C2H2 and CO are non-competitive inhibitors
to N2 [226], [101]. The electron flow within the three metal clusters of the N2ase complex for a
single electron transfer event is divided into two electron transfer substeps, from the [4Fe-4S]-
cluster to P-cluster and from the P-cluster to FeMo-cofactor. Recently, the order and kinetics of
these two steps were determined via stopped-flow spectrophotometry. At first, the slow and con-
formationally gated electron transfer from the P-cluster to the FeMo-co takes place, followed by
the second, fast and backfilling electron transfer from the [4Fe-4S]-cluster to the oxidized P-
cluster. This process was nicknamed an ‘electron deficit spending’ mechanism [127].
1.4.1.4 Lowe-Thorneley kinetic model
The Lowe-Thorneley kinetic model (LT-scheme) describes the nitrogenase catalysis in terms of
rate constants for the transition of intermediates (E0, E1 to E7) within one nitrogenase turnover
cycle [70], [227], [219], [228], [229], [214], [230] (see Figure 10).
Introduction
36
Figure 10: The simplified Lowe-Thorneley kinetic scheme for N2ase catalysis. E0 is the resting state, E1 to E7 repre-
sent the catalytic intermediate states and the number of occurred e-/H+ transfer steps is indicated by subscript. N2
binds reversibly with concomittant H2 release at E3 or E4. H2 can be released by states E2, E3 and E4. Acidic (pH 0)
or basic (pH 14) treatment gives product N2H2. NH3 is produced and released between the fifth and eigth e-/H+
transfer step. This scheme was adapted from [219].
Proton supply is accompanied with electron transfer to FeMo-co, resulting in a proton coupled
electron transfer mechanism (PCET). Thus. n displays the number of e-/H+ delivery steps, result-
ing in a description of En in the LT-scheme by En(nH). N2 binding accompanied to H2 exchange
as in E4(4H) yielding E4(2N2H), is a reversible process. Compliance with the ‘key constraints’ on
N2ase mechanism (see Table 1) is a pivotal part for LT-scheme [214], [80], [224], [104], [231].
Table 1: ‚Key constraints‘ for nitrogenase mechanism.
(i) State when N2 is reduced:
N2 is reduced at the E4 stage of e-/H+ accumulation
(ii) D2 or T2 only react during N2 turnover, during which
(a) 2 HD form stoichiometrically: M-N2 + D2 + 2 H+ → 2 HD + M + N2
(b) No scrambling with solvent: ‘No’ T+ released to solvent under T2
(c) Reduction level of this reaction: D2/T2 reacts at E4(2N2H) level
1.4.2 Substrate binding site
Site-directed mutations of α-Val70 to either decrease (α-Val70→Ala) or increase (α-Val70-Ile) the
residue size either led to access of larger compounds such as propyne (HC≡C-CH3) and propar-
gyl alcohol (HC≡C-CH2-OH) [101], or limited the turnover rates for C2H2, N2 or N2H4, while
keeping normal H2 production rates [232]. These findings led to two conclusions. First, the sub-
E0 E1H E22H E33H E32NH
E44H E42N2H N2H2
E5E6E7
H2N2
2 NH3
pH 0/14
e-/H+
- H2- H2
- H2
e-/H+ e-/H+
e-/H+e-/H+
e-/H+ e-/H+
e-/H+
e-/H+
H2N2
H2N2
H2N2
Introduction
37
strate range can be controlled by altering α-Val70, while electron flow and catalytic integrity of
the active site are maintained. And secondly, the side chain of α-Val70 is situated above one site
of the FeMo-co including Fe atoms Fe2, Fe3, Fe6, Fe7 indicating that these Fe sites, rather than
Mo, constitute to the substrate binding site.
The MoFe-protein structure with CO bound to Fe2 and Fe6 of FeMo-co [140] (see section
1.3.4.2) and the fact that MoFe-protein can reduce the non-competitive inhibitor CO [201], [203]
reinforces the Fe-atoms, especially Fe2 and Fe6, to be the substrate binding site of the FeMo-co.
Whether Fe3 and Fe7 also belong via H or hydride bridges to the substrate binding site is not
clear yet.
In direct protein vicinity to the now assumed substrate binding site, Fe2 and Fe6, residues α-
His195 and α-Gln191 showed significant influence on N2ase reactivity. Alteration of α-
His195→Gln reduces the N2 reduction activity below 1 %, while C2H2 and proton reduction
rates are comparable to wild type [233], [234]. A α-His195→Asn MoFe-protein is no longer ca-
pable to reduce N2 [224]. α-His195 is in H-bond distance to S2B with ≈ 3.2 Å [116] and is hy-
pothesized to be involved in proton delivery [235], [104] or in general stabilization of H-species
bound to FeMo-co [236].
The site mutation α-Gln191→Ala allowed reduction of the bigger substrate 2-butyne (CH3-
HC≡CH-CH3) pointing to a sterical role for α-Gln191 [237], [101]. Another alteration to α-
Gln191→Lys diminished N2 reduction and even binding completely [224].
1.4.3 N2 reduction pathway
There are two pathways proposed for the reduction of N2 (states E4 to E8/E0) that differ in the
site of reduction and protonation on bound N-N fragments. In the ‘distal’ pathway, the distal N
is hydrogenated at first, followed by reduction of the second N. In the ‘alternate’ pathway the N
atoms are hydrogenated in alternate steps (see Figure 11) [101].
The distal pathway is based on the Chatt [238], [239] or Chatt-Schrock cycles [240] for N2 reduc-
tion by organometallic, mononuclear Mo-metal-complexes, that cleave N2 [241], [242] and reduce
it catalytically [243], [244], [245], [246]. In this mechanism, Mo is assumed to be the substrate
binding site [247]. The bound N2 is successively hydrogenated at first at the distal N. After three
added e-/H+, the N-N bond is cleaved and the first NH3 is released. Upon addition of three fur-
ther e-/H+ to the remaining nitrido-N species the second NH3 is released [248], [249].
Introduction
38
An alternative type of reaction with an alternate pathway was proposed for N2ase by several theo-
retical studies [250], [248]. In this route, Fe atoms are supposed to be the catalytic binding site
[251], [252]. With an alternately hydrogenation of both N atoms the first two e-/H+ steps lead to
a diazene-level intermediate (N2H2), the next two steps yield hydrazine (N2H4) and only upon the
fifth addition the first NH3 is generated and released [248].
Figure 11: Possible distal (left) or alternating (right) N2 reduction pathways. After N2 binding to a structurally unde-
fined binding site (M) on the cofactor the first reduction/protonation to break the dinitrogen triple bond occurs. In
the following reduction and protonation takes place either first at the distal N releasing the first NH3 after three e-
/H+ transfers to the substrate or alternately at both N atoms with first NH3 production after more e-/H+ transfer
steps. The types of bound N-ligands are named. Possible entry points for diazene and hydrazine are shown. This
scheme was adapted from [253], [101].
The distal pathway was supported by Mo-model complexes, that until recently were solely able to
catalyze N2 reduction, and in fact these react via the distal pathway [245] that is computationally
favored for Mo [247]. Invalidating this reason, small W clusters were identified, that reduce N2
via the alternate route [254]. Moreover, several Fe-model complexes were discovered, that are
able to cleave [255] and also catalytically reduce N2 [256], [257], [253].
The alternate pathway is supported by the fact that hydrazine (N2H4) is both a substrate [258] and
a minor product upon acid or base treatment of N2ase (see Figure 10) [259], [214], [80]. The al-
ternate route is computationally favored on Fe sites [252]. Due to economical considerations it is
most reasonable that nature created one reaction pathway for all three types of N2ase. Based on
N≡N−M
HN=N−M
HN=NH ⇌ H2N−N=M HN=NH−M ⇌ HN=NH
H2N=NH−MN≡M
HN=M H2N−NH2−M ⇌ H2N−NH2
H2N−M
M
NH3
NH3
NH3
diazenediazene
diazenido
hydrazidonitrido
dinitrogen
hydrazineimido
amido
distal alternating
Introduction
39
this assumption, the fact that V-N2ase produces traces of N2H4 under N2 turnover is a strong
argument for the alternate route as reaction pathway of N2ases [260].
Recapitulating these arguments and the findings concerning the substrate binding site (see section
1.4.2), e.g. that CO is bound to Fe2 and Fe6, as well as the studies with α-Gln191-, α-His195- and
α-Val70-variant Mo-N2ases indicating Fe2, Fe3, Fe6 and Fe7 as binding site, Mo can almost be
excluded and Fe sites seem to be most likely the active binding site. At current state, the alternate
pathway seems likely and is assumed as the reaction pathway for N2 reduction by N2ase [261],
although it is not proven yet. Assuming the alternate route, the reduction of the substrates N2,
N2H2 and N2H4 can be easily explained by postulating that they just enter the reaction pathway at
their appropriate reduction stage to the reduced and thus sufficiently activated FeMo-co [262],
[104].
1.4.4 Molecular mechanism of N2 binding and reduction at the FeMo-cofactor
For defining a molecular mechanism of N2 reduction by N2ase, the intermediate states of catalysis
E1 to E8/E0 need to be known. While the active site FeMo-co in the resting state E0 is well-
characterized, there was no characterization of FeMo-co in the intermediate states. Substrate
binding requires FeMo-co to be more reduced than the ‘as-isolated‘ state E0, and the major prob-
lem was to trap any FeMo-co intermediate for analysis and characterization. Hence, there was no
possibility to integrate a reaction pathway into the LT-kinetic scheme to develop a molecular
mechanism for N2 reduction.
This problem could be overcome within the last ≈ 10 years by studies of nitrogenase with indi-
vidual amino acid substitutions, where especially α-Val70 played a crucial role. By sterical hin-
drance it controls the access of substrates to the active site FeMo-co, while maintaining catalytic
integrity [232], [101] (see section 1.4.2). In subsequent studies, intermediates E4(4H) [232], [263],
[264], E4(2N2H) [265], E7 [266] and E8 [262] could be trapped, or at least populated in a higher
concentration, investigated and characterized. And based on these genetic, biochemical and spec-
troscopic studies, a molecular mechanism for N2 binding and reduction was proposed [267],
[268], [104], [231].
1.4.4.1 Mechanistic model for N2 reduction via reductive elimination
Analysis of the four-electron and proton-accumulated state E4(4H) showed the presence of two
bridging hydrides and two protons [232], [263], [264]. This lead to the concept of a central role of
hydride chemistry [267]. Based on this idea it was proposed that upon delivery of the second e-
Introduction
40
/H+ to reach E2 state, both electrons are shuttled from the metal-cluster onto one proton to
form a [Fe-H-Fe] hydride, leaving the remaining proton bound to sulfur for electrostatic stabiliza-
tion and the FeMo-co formally in the resting state redox level, M0 or MN. In analogy, the e-/H+
delivery to reach E4 state would result in the observed two hydrides and two protons (see Figure
12 ‘I’). Via such a process, by storing two electrons as hydrides, the metal-ion core of the FeMo-
cofactor would only cycle through the two oxidation states of one redox couple throughout the
whole MoFe cycle. This is consistent with 57Fe ENDOR studies and ‘Electron inventory analysis’
[269], [270]. The process of hydride formation could be realized in two ways. Either hydrides are
created after each second electron transfer. This would mean after the first e-/H+ transfer the
FeMo-co would be in a one electron reduced M1- oxidation state and upon the second redox
event hydride formation and restoring of M0 would occur. Or in the second way, hydride for-
mation occurs after the first e-/H+ delivery, leaving the FeMo-co in a one-electron-oxidized M1+
that is restored to M0 upon the second redox event. This electron storage process would also
explain how such reducing power is generated and kept at the constant redox potential of the Fe-
Protein [102], (see section 1.3.5). E2, E3 and E4 can relax by release of H2 via hydride protona-
tion [263], [271], which is a side reaction and represents normal H2 evolution in case of substrate
absence.
N2 binding takes place at E3 and E4, but E4 is the key stage in the process of N2 reduction [214],
[230], called the ‘Janus’ state [267], as it uniquely enables N2 hydrogenation. There are no absolute
hydride locations known, rather a fluxionality between the Fe2, Fe3, Fe6 and Fe7 face is as-
sumed, maybe with a vertex on Fe6 [104]. The obligatory production of one H2 molecule upon
binding and reduction of one N2 [103], [214] is explained in this mechanism via a reductive elimi-
nation (re) [272], [273], [104]. Both hydrides on E4 undergo a transient terminalization (probably
on Fe6) and subsequently the reversible reductive elimination of H2 with binding of N2 takes
place (see Figure 12 ‘re’) [268], [104], [265], [231]. The crucial point in this step is that the evolv-
ing H2 takes only two electrons, while the remaining two electrons from both hydrides stay on
Fe. Thereby, this Fe atom (Fe6) has transiently two additional reducing equivalents, is thus highly
activated and hence solely able to bind and reduce N2. The Fe atom can be viewed e.g. as a Fe2+
which is for a short time reduced to a Fe0. The N2 form in the E4(2N2H) state of the FeMo-co is
not known and could be a bound N2 with still two electrons and two protons present, or a re-
duced [N2]2- form and two protons or a diazene (N2H2) itself, which has to be created at some
point on E4.
For the subsequent states E5 to E8, the N-intermediates are bound to Fe6. The e-/H+ delivery is
proposed to form hydrides, as explained above for E1 to E4, that reduce the bound intermediates
Introduction
41
by migratory insertion [104] and protons bind accordingly (see Figure 12 ‘II’). In the E6 state hy-
drazine (N2H4) is bound. Upon the seventh e-/H+ delivery N-N bond cleavage occurs, releasing
one NH3 molecule and leaving the characterized [NH2]- bound to FeMo-co [266]. The final step
to the characterized state E8 results in bound NH3 to FeMo-co with redox level M0 [262]. After
release of the second NH3 molecule FeMo-co restores to its resting state E0.
Figure 12: Illustration of the proposed mechanistic model for N2 reduction via reductive elimination. Depicted is the
Fe2, Fe3, Fe6, Fe7 face of the FeMo-cofactor with Fe2 down left and Fe6 down right. The four e-/H+ transfer steps
from E0 to E4 are represented in I yielding a Fe site with two bridging hydrides (representative Fe2-Fe6, Fe6-Fe7)
and two protonated sulfurs. Reversible binding of N2 with by reductive elimination of H2 at E4 on Fe6 and the re-
duction to diazene at E4 is shown in re. Subsequent reduction/protonation steps from E5 to finally the resting state
E0 is shown in II. The one electron oxidized FeMo-co compared to resting state is labeled with a ‘+’. re: reductive
elimination; mi: migratory insertion; bc: bond cleavage. This figure is from [104].
The mechanism of reductive elimination of H2 by N2 is built on the characterized intermediate
states E4(4H) and E4(2N2H) and the implication that hydride chemistry is central to N2 reduction
by N2ase. It justifies its correctness by satisfying the ‘key constraints’ for the N2ase mechanism
[214], [80], [224], [104], [231] (see section 1.4.1.4). The proposed reaction pathway and mechanis-
tic model for N2 reduction are based on the trapped and characterized states E4(4H), E7 with a
bound [NH2]- [266], E8 with bound [NH3] [262] and the assumption of an alternate reaction
pathway (see section 1.4.3). However, the reductive elimination and the mechanistic model are
not commonly accepted yet [274]. Furthermore they do not take it into account the structure of
Introduction
42
the MoFe protein with substrate/inhibitor CO bound to Fe2 and Fe6 replacing a bridging sulfide
[140] (see section 1.4.2).
1.4.4.2 Computational mechanistic model for N2 reduction
This computational model is based on Density Functional Theory (DFT) calculations with
known cluster models, spin states and corresponding constraints [275]. The recent structure of
the MoFe-protein with a CO bound to Fe2 and Fe6 replacing S2B [140] led to the proposal of a
second protonation of S2B to form and remove a dihydrogen sulfide (H2S), revealing a very reac-
tive μ2 Fe-Fe edge for N2 reduction at the FeMo-co. The model describes the full pathway of
eight e-/H+ transfer steps of N2 reduction by N2ase and suggests approximate pictures of the
according intermediate states (E0, E1 to E7) within the Lowe-Thorneley kinetic scheme. The in-
termediate states are not described in detail concerning the electronic structure of the FeMo-co
or the oxidation states of the bound N-species during turnover.
The pathway starts with the resting state of FeMo-co (E0) and can be divided into three regions
(see Figure 13). The first region (‘unsealing’; E0 to E4) describes the first four e-/H+ transfers. It
contains formation of a hydride (H*) on an Fe atom adjacent to the one-fold protonated S2B
(E2), followed by the second protonation of S2B to form and remove a H2S, thus revealing the μ2
site where a hydride is still bound (E3). After creation and release of H2 to fully expose the Fe-Fe
edge, ‘unsealing’ ends with the binding of N2 (E4). The second area (‘activation’; E5 to E7) de-
scribes the reduction of N2 until production of the first NH3. It starts with the initial first e-/H+
transfer to N2 to form the activated N2H* (E5) and is followed by further e-/H+ delivery steps
according to a distal route until the first NH3 is released (E7). The last region (‘resealing’, E7 to
E0) describes the readsorption of H2S, the formation of the second NH3 and the regeneration of
the resting state. It starts after dissociation of NH3, leaving a reduced N-species (N*), with the
readsorption of H2S to the μ2 site (E7) and is followed by proton shuttling from H2S to N*. Upon
the final e-/H+ transfer, the second NH3 is produced. After product desorption the resting state
of FeMo-co is restored (E0), ending the turnover cycle.
With an intact FeMo-co the initial e-/H+ transfer to N2 to form N2H* is the critical step in N2
reduction with ≈ 2 eV versus Computational Hydrogen Electrode (CHE) [276], [275]. Consider-
ing the energy gain from hydrolysis of two MgATP of ≈ 0.7 eV (vs CHE), this step seems to be
very implausible. Assuming a model with the possibility of an H2S removal and a highly reactive,
free, μ2 site other reactions and reaction barriers occur.
Introduction
43
Figure 13: Computational mechanistic model for N2 reduction by N2ase. Proposed FeMo-co structures for the rest-
ing (E0) and intermediate states (E1 – E7) during one N2 turnover cycle are illustrated based on computational DFT
calculations. Atoms are displayed as balls; hydrogen in light grey, carbon in grey, sulfur in yellow, iron in orange,
oxygen in red, nitrogen in blue and molybdenum in cyan. This scheme was adapted from [275].
The energy barrier for the hydride formation on a Fe atom adjacent to the singly protonated S2B
(E2) is calculated to be ≈ 0.8 eV (vs CHE). The presence of this hydride reduces the energy barri-
er for the second protonation of S2B to form a removable H2S (E3) from ≈ 1.4 eV to only
≈ 0.6 eV (vs CHE). The dissociation of H2S exposes the much more reactive μ2 site, although it
is still partially blocked by the remaining hydride. The subsequent e-/H+ transfer (E4) leads at
ambient temperatures to evolution of the obligatory H2 molecule per reduced N2 [214], [103] and
eventually fully exposes the highly reactive, free and reduced μ2 Fe-Fe edge (see Figure 13 ‘*’)
which then binds N2. At very low temperatures, also an intermediate with two bound hydrides
could be stabilized, that represents the in freeze-quench experiments observed ‘Janus intermedi-
ate’ [104]. The energy for formation of both species on E4 is equal. The initial e-/H+ transfer to
N2, resulting in the ‘activated’ N2H* (E5), is calculated to be only ≈ 0.4 eV (vs CHE). All subse-
quent reduction and protonation steps beyond the N2 activation proceed with low energy barri-
ers. Therefore, compared to a model with intact FeMo-co, where the critical step is the first re-
duction of N2 to N2H* with ≈ 2 eV (vs CHE), in a model with a free Fe-Fe edge, the critical step
is hydride formation at a Fe atom, which is necessary for the stabilization of H2S and accounts
for just ≈ 0.8 eV (vs CHE). Thereby, the critical step in the μ2 site model becomes compatible
with the energy gain from MgATP hydrolysis. And additionally a supporting ligand or an appro-
priate docking site for stabilization of the H2S would further decrease the energy barrier for hy-
dride formation.
S* SH* SH-H* H* * N2H* N2H2* N* NH-SH* NH2-S* S*
Unsealing Activation Resealing
0 1 2 3 4 5 6 7 8
e- / H+ transfers
Introduction
44
The readsorption of H2S to the Fe-Fe edge, with subsequent proton shuttle from H2S to N*, is
considered to be most thermodynamically favorable after release of the first NH3 leaving a re-
duced, unprotonated N-species (N*). It would be energetically preferable if H2S stays in vicinity
during turnover. While the binding of NH3 to a free Fe-Fe edge is stabilized by ≈ 0.7 eV, binding
of NH3 to the intact FeMo-co with readsorbed S2B is unstable and leads to dissociation of NH3.
Thus the readsorption of H2S to the μ2 site in E7 is a vital step to initiate product desorption after
the last e-/H+ delivery step yielding the second NH3 and regenerating the resting state E0.
In ≈ 90 % of the ≈ 2000 statistically independent pathways calculated for this model, the hydride
formation (≈ 0.8 eV vs CHE), prior to H2S dissociation and revealing the reactive μ2 site, is the
limiting step. Assuming the μ2 site model, in ≈ 9 % of the corresponding calculations the initial
activation of N2 to N2H* (≈ 0.4 eV vs CHE) is limiting. In comparison, assuming intact FeMo-
co, the activation step with ≈ 2 eV (vs CHE) is limiting in 100 % of all calculations. Furthermore,
a model including H2S removal explains the MoFe structure with S2B exchanged to CO. But
according to this model apparently all substrates and inhibitors bind and thus block the highly
reactive μ2 site. This would imply that all these substrates are competitive inhibitors, although
they are non-competitive. The removal and readsorption of H2S to the Fe-Fe edge to enable de-
sorption of the product NH3 from FeMo-co is a process that can be observed at the Fe-catalyst
in the Haber-Bosch process in a different way, but concerning a similar problem, as there tem-
perature and pressure control the initial adsorption and final desorption in order not to poison
the catalyst with bound NH3 [275], [277].
1.4.5 Haber-Bosch process
In industry, N2-fixation is carried out in the Haber-Bosch-process. N2-reduction actually takes
place at the Fe-atoms of a promoted α-Fe catalyst [48], [49] (see section 1.2). In this process N2
and H2 molecules are split in two atoms upon adsorption into the Fe atoms of the catalyst, called
‘dissociative adsorption’ [278], [279], [280], [281], and subsequently bound H-atoms hydrogenate
N-atoms. In this mechanism of N2-reduction and NH3-synthesis (see Figure 14) single electrons,
protons or charged ions or molecules occur at no point [277].
Introduction
45
Figure 14: Haber-Bosch mechanism. Hydrogen and nitrogen atoms are readicals and labeled as adsorbed hydrogen
(Had) and as single nitrogen (Ns). Adsorbed dinitrogen molecule is labeled as N2,ad.
In comparison, in the mechanism of N2-fixation by N2ase the nitrogen binds as a N2 molecule to
the FeMo-co (‘associative adsorption’) [278] and subsequently sequential hydrogenation and NH3
production takes place, independent of the proposed mechanism and pathway [214], [104], [275].
Therefore, especially the crucial first reduction ‘step’ is essentially different. Whereas in the Ha-
ber-Bosch process the N2 molecule is directly separated into two radical atoms, a single electron
reduction to the complete N2 molecule takes place in N2ase. Due to this fundamental mechanistic
difference between N2-reduction via Haber-Bosch with at α-Fe and N2ase at the FeMo-co, the
mechanisms of both are hardly comparable on a molecular level.
1.5 Protein crystallography
The goal of protein crystallography is the determination of a protein structure. Protein Crystals
are produced, investigated in an X-ray diffraction experiment and the subsequent computational
data evaluation and result interpretation yields the protein structure.
1.5.1 Protein crystallization
The most common method for protein crystallization is by vapor diffusion. The underlying prin-
ciple is the competition between protein and a precipitation agent for solvent molecules. Reduc-
tion of solvent molecule amount by vapor diffusion leads to a decrease in protein solubility until
supersaturation of the protein solution is reached and results in protein crystallization (see Figure
15).
H2 ⇋ 2 Had
N2 ⇋ N2,ad ⇋ 2 Ns
Ns + Had ⇋ NHad
NHad + Had ⇋ NH2,ad
NH2,ad + Had ⇋ NH3,ad ⇋ NH3
Introduction
46
Figure 15: Phase diagram of protein crystallization. A crystallization process is displayed by blue arrows: vapor diffu-
sion leads to protein and precipitant concentration increase until protein nucleation takes place what only occurs in
the nucleation zone; subsequently crystals grow in nucleation and metastable zone until the solubility curve is
reached.
1.5.2 X-ray diffraction
A protein crystal is a repetitive assembly of protein molecules in a highly ordered, three-
dimensional system forming a protein lattice. The smallest building block is the unit cell, whose
dimensions are defined by the axes a, b, c and the angles α, β, γ. Within the unit cell, further sym-
metry elements can exist, dividing it into several asymmetric units that represent the basic structural
element of the crystal. The unit cell geometry, in combination with the possible symmetry opera-
tions defines the space group of the crystal. This knowledge is sufficient to describe each mole-
cule and atom in the crystal. Due to amino acid chirality, only 65 out of 230 possible space
groups occur in protein crystallography.
The maximum resolution a given object can be investigated at is determined by the applied wave-
length. In protein crystallography, atomic distances of e.g. C-C-bonds with ≈ 1.54 Å (0.154 nm)
require the usage of X-rays which offer a range of 0.25-0.01 nm. Photons interact with the elec-
trons of the atom’s electron shell. This interaction leads to the emission of random scattered ra-
diation with a phase shift of 180° and with the same frequency as the photons (elastic or coherent
scattering; Thomson scattering). While destructive interference leads to deletion of scattered radi-
ation, constructive interference results in X-ray diffraction with discrete intensity maxima.
The Laue equations describe the relation of X-ray reflections in crystals between real atomic lat-
tice planes and virtual three-dimensional lattice planes in the real space of the crystal. For a con-
precipitant concentration
pro
tein
co
nce
ntr
atio
nmetastable
zone
precipitation
zonenucleation
zone
solubility curve
undersaturation
supersaturation
Introduction
47
structive interference of X-rays at such virtual lattice planes in the real space, Bragg’s law (see
equation 4) [282] has to be fulfilled:
2 𝑑ℎ𝑘𝑙 ∙ sin 𝜃 = 𝑛 ∙ 𝜆 (4)
This equation describes diffraction of incoming X-rays with wavelength λ on virtual lattice planes
specified by the Miller indices h, k, l and with the distance d. Constructive interference of both
X-rays reflected at the angle θ from parallel lattice planes only takes place when the path differ-
ence is a integer multiple of the wavelength (see Figure 16).
Figure 16: Virtual lattice planes and Bragg’s law. A) Representation of virtual lattice planes in the unit cell; the Miller
indices h, k, l denote in how many equal parts the cell axis is divided; in the given example, the lattice plane would be
specified as (3 1 2). B) Representation of X-ray reflection on virtual lattice planes; θ is the glacing angle and dhkl is the
distance between two adjacent lattice planes; the path distance of 2 dhkl sinθ is highlighted by the shadowed line area.
X-ray diffraction at the virtual lattice in real space according to Bragg’s law creates a second
three-dimensional lattice with diffraction maxima. The geometric properties of this lattice are
inverse to the virtual lattice in real space, and thus it is called the reciprocal lattice. Each reflection
at a lattice plane, that is in agreement with Bragg’s law yields a lattice point in the reciprocal space
described by the Miller indices as (h, k, l). The corresponding lattice plane in the unit cell of the
crystal is accordingly labeled as (h, k, l), with the distance dhkl.
The Ewald sphere visualizes the spots of the reciprocal lattice, that are in agreement with Bragg’s
law (see Figure 17). The Ewald sphere can be displayed in two dimensions as a circle with radius
1/λ and the crystal as origin (C; x, y, z = 0, 0, 0) in the middle in real space. The incoming X-ray
radiation and the origin of the reciprocal lattice (O; h, k, l, = 0, 0, 0) are on opposite ends of this
circle. Every reflection, that is in agreement with Bragg’s law, intersects at a given crystal rotation
with the Ewald sphere and can be measured by a detector. The experimental result of an X-ray
diffraction experiment is the detection of the spots of the reciprocal lattice. The primary result of
x
y
zl = 2
h = 3
k = 1
dhkl
Θ
Θ
dhkl · sin Θ
A B
Introduction
48
evaluation of the reciprocal lattice is the space group and the unit cell dimensions (a, b, c, α, β, γ).
While the geometry of the unit cell determines the spot positions of the reciprocal lattice, the unit
cell content determines the intensity of the spots.
Figure 17: Ewald sphere. In real space, the origin is denoted with the crystal C and virtual lattice planes are displayed
as red lines with distance dhkl. In reciprocal space, the origin is labeled with O representing the reflection (0 0 0); the
reciprocal lattice is represented by small black circles; highlighted red dots of the reciprocal lattice are spots that at
the given crystal rotation are in agreement with Bragg’s law and can be detected; S is the vector in reciprocal space of
a reflection (h, k, l)
1.5.3 The electron density
The aim of an X-ray crystallography experiment is the determination of the electron density dis-
tribution of the crystal that is then interpreted by finding a protein model matching the electron
density.
All reflections in reciprocal space and the electron density map, the distribution of electrons in
real space, are equal representations of the protein crystal structure. Mathematically, both can be
converted into each other by a Fourier transformation (see equation 5). The maxima of the recip-
rocal lattice are the representation of the crystal in the Fourier space. Thus, the Fourier transform
of all reflections F(hkl) in the reciprocal lattice delivers the electron density.
(5)
Experimentally measured and regarded, each reflection of the incident beam on the lattice plane
dhkl yielding a spot (h, k, l) in the reciprocal lattice can be described by the structure factor F(hkl)
as a complex vector consisting of an amplitude and a phase angle:
dhkl
Θ
S
1/λ
CO
Fourier transform
Introduction
49
ℎ𝑘𝑙 = | ℎ𝑘𝑙| ∙ 𝑒𝑖𝜑ℎ𝑘𝑙. (6)
Mathematically, a structure factor F(hkl) of a reflection can be described via atomic scattering
factors fj, representing the diffraction contribution of each individual atom j with position (x, y, z)
in the unit cell. The structure factor F(hkl) of a single reflection (h, k, l) is described by a summa-
tion of these:
ℎ𝑘𝑙 = ∑𝑓𝑗 𝑒2𝜋𝑖(ℎ𝑥𝑗+𝑘𝑦𝑗+𝑙𝑧𝑗)
𝑗
. (7)
A Fourier summation of all structure factors F(hkl) in reciprocal space leads to the electron den-
sity for every point in real space in the unit cell:
( ) = 1
𝑉 ∑ ℎ𝑘𝑙 ∙ 𝑒
−2𝜋𝑖(ℎ𝑥+𝑘𝑦+𝑙𝑧)
ℎ 𝑘 𝑙
. (8)
As individual atom positions xj, yj, zj are not known yet, electron density cannot be calculated.
Insertion of the experimental structure factor (equation 6) in the Fourier summation of structure
factors for electron density (equation 8) yields:
( ) = 1
𝑉 ∑| ℎ𝑘𝑙| ∙ 𝑒
−2𝜋𝑖(ℎ𝑥+𝑘𝑦+𝑙𝑧) ∙ 𝑒𝑖𝜑ℎ𝑘𝑙
ℎ 𝑘 𝑙
. (9)
The experimentally measured intensity is proportional to the square of the structure factor ampli-
tude (see equation 10) and thus the structure factor amplitude can be determined.
𝐼ℎ𝑘𝑙 ~ | ℎ𝑘𝑙|2 (10)
The phase angle is a complex number and squaring it consequently results in a real number.
Therefore, the phase angle is lost during the X-ray diffraction experiment and cannot be deter-
mined. Consequently electron density cannot be calculated. This phenomenon is called the phase
problem.
The phase problem in protein crystallography can be solved by different methods. If a sufficient-
ly similar protein structure is available, phases can be adopted from this protein and the structure
can be solved by molecular replacement [283]. Otherwise, the phase problem has to be overcome
by analysis of a sufficiently easy substructure. This include the methods of single-wavelength
anomalous dispersion (SAD) [284], multiple-wavelength anomalous dispersion (MAD) [285],
multiple isomorphous replacement (MIR) [286], single isomorphous replacement with anomalous
dispersion (SIRAS) and multiple isomorphous replacement with anomalous dispersion (MIRAS).
Introduction
50
In all cases, the Patterson-Function is used (see equation 11). The Patterson function is no electron
density function, but has the same cell unit dimensions. It shows intensity maxima for atom posi-
tions depending on the inter-atom distance vectors, but it is phase-angle-independent. A number
of n atoms in the unit cell of real space yield n(n-1) maxima in Patterson space.
𝑃(𝑢 𝑣 𝑤) = 1
𝑉 ∑| ℎ𝑘𝑙|
2 ∙ 𝑒−2𝜋𝑖(ℎ𝑢+𝑘𝑣+𝑙𝑤)
ℎ 𝑘 𝑙
(11)
For molecular replacement Patterson maps are calculated and compared, leading to initial usable
phases. In case of a sufficiently simple substructure analysis, real space atom positions can be
determined in the Patterson map. These atoms can be heavy atom derivatives (MIR) or anoma-
lous scatterers (SAD, MAD). In a second step, the phase angle for the entire protein structure
can be recalculated.
1.5.4 Anomalous dispersion of heavy atoms and MAD
A plot of the X-ray absorption coefficient of an element versus the X-ray wavelength shows sev-
eral sharp edge-like features. Especially heavy atoms show these edges within the spectral range
of applied X-rays for protein crystallography. At these edges, the photon energy is exactly as high
as the energy to eject a core electron from the atom. While the incoming X-ray energy is ab-
sorbed from the ejected electron, the re-emitted X-rays arise from a valence shell electron, filling
up the hole in the atom core shell and are of lower energy than the absorbed.
Normal X-ray scattering is far from those X-ray absorption edges and is regarded as elastic and
coherent scattering. At X-ray absorption edges, the phase shift deviates from the normal 180°
and is called anomalous scattering.
While for normal X-ray diffraction the structure factor is wavelength-independent, the structure
factor (FHA,ano) becomes wavelength-dependent for heavy atoms at an X-ray absorption edge and
additionally shows a real or dispersive (Δf’) and an imaginary (Δf’’) contribution (see equation 12
and Figure 18 B). The phase of Δf’’ is always 90° ahead to Δf’.
𝐹𝐻𝐴 𝑎𝑛𝑜(𝜆) = 𝐹𝐻𝐴0 + Δ𝑓′(𝜆) + Δ𝑓′′(𝜆) (12)
For normal X-ray diffraction far from X-ray absorption edges without anomalous dispersion, the
structure factor amplitudes of a Friedel pair F+(h,k,l) and F-(-h,-k,-l) are equal. This property is
called Friedel’s law (see equation 13 and Figure 18 A).
Introduction
51
| +ℎ𝑘𝑙| = | −ℎ𝑘𝑙| (13)
At an X-ray absorption edge, the anomalous dispersion of heavy atoms leads to a breakdown of
Friedel’s law. With the difference between the Friedel pairs (Δano) (see equation 14 and Figure 18
B) an anomalous difference map only consisting of heavy atoms can be calculated with the Pat-
terson function. In case, this substructure is sufficiently simple enough, the phases and positions
of the heavy atoms can be determined.
Δ𝑎𝑛𝑜 = | +ℎ𝑘𝑙| − |
−ℎ𝑘𝑙| (14)
Figure 18: Friedel pairs for normal and anomalous diffraction. A) Representation of the structure factors of a Friedel
pair far from an absorption edge without anomalous dispersion according to Friedel’s law. B) Breakdown of Friedel’s
law; structure factor amplitudes of Fano+ and and Fano
- differ (Δano) due to anomalous dispersion of heavy atoms
highlighted by the anomalous contribution of Δf’HA,ano and Δf’’HA,ano to FHA,ano.
With the phases of the heavy atoms and according to
𝑃 = − 𝐻𝐴 (15)
and analogously with anomalous dispersion
𝑃 = 𝑎𝑛𝑜 − 𝐻𝐴 𝑎𝑛𝑜 (16)
every single structure factor of the protein (FP) can be re-calculated with the help of the Harker
construction [287]. By drawing a circle with radius |FP| (|FP+| = |FP
-|) and two circles with the
radius of |Fano+| and |Fano
-| with the offsets of the determined FHA,ano+ and FHA,ano
-, respectively,
the intersection of these three circles yields the structure factor FP+ (see Figure 19).
Im
Re
F+(h,k,l)
F-(-h,-k,-l)
FP+(h,k,l)
FP-(-h,-k,-l)
FHA+(h,k,l)
FHA-(-h,-k,-l)
A Im
Re
Fano+(h,k,l)
FHA, ano+
Δf‘HA, ano
Δf‘‘,+HA, ano
FHA, ano- Fano
-(-h,-k,-l)
Δano Δf‘‘,-HA, ano
Δf‘,-HA, ano
Fp+
Fp-
FHA+
FHA, ano+
B
Introduction
52
Figure 19: Harker construction for phase phase determination by anomalous scattering. The determined heavy atom
structure factors FHA,ano+ and FHA,ano
– are set as offset at the circle origin; FHA,ano- has been mirrored on the horizon-
tal line; the vector of intersection point of the three circles to the origin yields the structure factor FP+
Im
Re
|Fano+| circle
|FP| circle
|Fano-| circle
FP+
FHA, ano+
FHA, ano-
Scope of the study
53
2. Scope of the study
Both Mo- and V-N2ases reduce N2 to NH3. While CO is an inhibitor for Mo-N2ase V-N2ase is
additionally capable of reduction of CO to CH4 and to perform C-C coupling for the formation
of longer chain hydrocarbons. The previously studied V-N2ases from A. vinelandii and A. chroococ-
cum from nifHDK deletion strains either could not be isolated to homogeneity in a stable form, or
are genetically modified. In any case crystallization did not succeed so far and thus no three-
dimensional structure of V-N2ase is available.
The primemodel organism to investigate N2 fixation and N2ase is the obligatly aerobic diazotroph
A. vinelandii, whose genome encodes for all three types of N2ases. It is easy to grow at high yields
of N2ase proteins in a laboratory scale. Although A. vinelandii has a Mo-storage protein to main-
tain N2 fixation by the most efficient Mo-N2ase under short term Mo deficit, this bacterium can
also grow diazotrophically under Mo depletion, by expressing either V- or Fe-only N2ase genes,
depending on the available metal source. The ability of wild-type A. vinelandii to produce V-N2ase
can thus be capitalized to avoid using deletion strains or genetically modified protein constructs.
The task in this work was to grow wild-type A. vinelandii via Mo-depletion under Mo-limited and
V-containing conditions diazotrophically for producing V-N2ase, establishing a purification pro-
cedure for homogeneous and stable protein, subsequent protein crystallization and finally struc-
ture solution.
Materials and Methods
54
3. Materials and Methods
3.1 Materials
3.1.1 Chemicals and gases
All chemicals and reagents were of analytical grade and obtained from SERVA (Heidelberg,
Germany), Carl Roth (Karlsruhe, Germany), Applichem (Darmstadt, Germany), VWR (Darm-
stadt, Germany), Hampton Research (Aliso Viejo, USA) und Merck (Darmstadt, Germany). All
gases were obtained from SWF (Friedrichshafen, Germany) and Air Liquide (Ludwigshafen,
Germany) and had a purity of at least 99.6 % for C2H2, 99.8 % for O2, 99.97 % for CO and
99.9999 % for N2, H2 and Ar.
3.1.2 Growth media
Growth media used for cultivation of A. vinelandii are listed in Table 2.
Table 2: Composition of Burk’s media which were used for cultivation of A. vinelandii [288], [220], [289], [290],
[291], [292], [293], [294]. a) for preparation of permanent Mo glyceol culture (see section 3.2.1) 0 mM NH4Cl and
0.01 mM Na2MoO4*2H2O was used; b) for Mo depletion (see section 3.2.2) 0.01 mM Na2MoO4*2H2O was used.
Medium Reagent Concentration
Burk’s medium (pH 7.5; pre culture) sucrose 58 mM
CaCl2 0.9 mM
MgSO4 1.67 mM
FeSO4*7H2O 0.04 mM
citric acid 0.20 mM
Na3VO4 (Na2MoO4*2H2O a), b)) 0.01 mM
KH2PO4 2.5 mM
K2HPO4 10 mM
NH4Cl 10 mM (0 mM a))
Burk’s medium (main culture) see above, except:
NH4Cl 1.36 mM
Materials and Methods
55
3.1.3 Buffers and solutions
All buffers and solutions used for SDS-PAGE, chromatography, BCA-assay, acetylene reduction
assay are summarized in Table 3 to Table 5.
Table 3: Buffers and solutions for SDS-PAGE [295], [296].
Buffer / solution Reagent Concentration
Stacking gel (5 %) Acrylamide-bisacrylamide (37.5:1) 5 %
Tris/HCl, pH 6.8 0.125 M
SDS 0.1 % (w/v)
TEMED 0.1 % (v/v)
APS 0.05 % (w/v)
Resolving gel (12.5 %) Acrylamide-bisacrylamide (37.5:1) 12.5 %
Tris/HCl, pH 8.8 0.375 M
SDS 0.1 % (w/v)
TEMED 0.1 % (v/v)
APS 0.05 % (w/v)
5x Sample buffer Tris/HCl, pH 6.8 0.5 M
glycerol 40 % (v/v)
SDS 8 % (w/v)
Bromphenol Blue 0.004 % (w/v)
β-Mercaptoethanol 0.25 M
Electrophoresis buffer Tris 0.025 M
Glycin 0.192 M
SDS 0.1 % (w/v)
Staining solution ethanol 10 % (v/v)
acetic acid 5 % (v/v)
Coomassie Brilliant Blue R250:G250 (4:1) 0.002 % (w/v)
Destaining solution ethanol 10 % (v/v)
Materials and Methods
56
Table 4: Buffers used for chromatography [297]; abbreviation: size exclusion chromatography (SEC).
Buffer Reagent Concentration
‘loDT’ purification ‘hiDT’ purification
lysis buffer Tris/HCl, pH 7.4 50 mM 50 mM
NaCl 100 mM 100 mM
Na2S2O4 2 mM 10 mM
loading buffer Tris/HCl, pH 7.4 50 mM 50 mM
NaCl 100 mM 100 mM
Na2S2O4 2 mM 2.5 mM or 5 mM
elution buffer Tris/HCl, pH 7.4 50 mM 50 mM
NaCl 500 mM 500 mM
Na2S2O4 2 mM 2.5 mM or 5 mM
SEC buffer Tris/HCl, pH 7.4 20 mM 20 mM
NaCl 100 mM 100 mM
Na2S2O4 2 mM 2.5 mM
Table 5: Reaction mixture for the nitrogenase acetylene reduction assay [297].
Reagent Concentration
Tris/HCl pH 7.4 20 mM
MgCl2 5 mM
phosphocreatine kinase 0.125 mg/ml
phosphocreatine 15 mM
ATP 2.5 mM
Na2S2O4 12.5 mM
3.1.4 Chromatography
Table 6: Column, column material and manufacturer.
Column and column material Manufacturer
HiLoad 26/600 Superdex 200 prep grade GE Healthcare
HiTrap Q HP (5 ml), Q Sepharose High Performance GE Healthcare
Resource Q (6 ml), Source 15Q GE Healthcare
Materials and Methods
57
3.1.5 Anoxic techniques
Due to the proteins oxygen sensitivity all procedures handling VnfDKG and VnfH were carried
out under strict exclusion of dioxygen, either by using modified Schlenck techniques or by work-
ing with in an anaerobic chamber having an atmosphere of 95 % N2 / 5 % H2.
Buffers and solutions were anoxified by applying eight cycles of nitrogen overpressure (0.1 bar;
1 min) and vacuum (1–10*10-5 bar; 10 min). The oxygen level was monitored by an oxygen elec-
trode (Oxyview Systems, model:OXYV1, Hansatech instruments, Norfolk, England). Finally, 2
mM Na2S2O4 were added to all buffers and solutions.
3.1.6 Bacterial strains
Azotobacter vinelandii Type strain Lipman 1903 [298] (DSM-No. 2289) from Leibniz-Institute
DSMZ-German Collection of Microorganisms and Cell Cultures was used for cultivation.
3.2 Microbiological methods
3.2.1 Permanent Mo glycerol culture
Freeze-dried A. vinelandii cells were resuspended in Molybdenum containing and NH4Cl free
growth media (modified after Burk; see Table 2). Cells were cultured by inoculation of Mo con-
taining growth medium supplemented 10 mM NH4Cl (pre culture; see Table 2) with the cell re-
suspension and incubated at 30 °C under 180 rpm agitation. Bacterial growth was monitored by
measuring the optical density at 600 nm (OD600). At an OD600 = 2.5 500 μl culture medium was
mixed with 500 μl 90 % (v/v) sterile glycerol, subsequently shock-frozen in liquid nitrogen and
stored at -80 °C.
3.2.2 Molybdenum depletion
Mo containing growth medium supplemented with 10 mM NH4Cl (pre culture; see Table 2) was
inoculated with a permanent Mo glycerol culture (see section 3.2.1) and cells were grown at
30 °C, agitated at 180 rpm and bacterial growth was monitored by measuring OD600. At an OD600
= 3.0 culture medium was streaked on Agar plates consisting of Mo containing, NH4Cl free
growth medium complemented with 5 % (w/v) Agar-Agar and incubated at 30 °C. Bacterial
Materials and Methods
58
growth was successful when A. vinelandii cells actively fixed nitrogen, which resulted in brownish
colonies. Mo depletion was initiated by inoculation of Vanadium containing growth medium
[291], [292], [293], [294], [83], [84] supplemented with 10 mM NH4Cl (see Table 2) with nitrogen
fixing colonies. The molar amount of Na2MoO4 in the medium was replaced by the same amount
of Na3VO4. Cells were grown at 30 °C, agitated at 180 rpm and bacterial growth was monitored
by measuring OD600. At an OD600 = 3.0 culture medium was streaked on Agar plates consisting
of V containing, NH4Cl free growth medium complemented with 5 % (w/v) Agar-Agar and in-
cubated at 30 °C until A. vinelandii cells fixed nitrogen. This cycle of growing A. vinelandii cells in
liquid V containing growth medium supplemented with 10 mM NH4Cl followed by growing the
bacteria on Agar plates with V containing, nitrogen-free growth medium supplemented with 5 %
(w/v) Agar-Agar was performed five times to ensure having Mo depleted A. vinelandii cells. For
permanent V glycerol cultures subsequently cells were once more grown in V containing growth
medium supplemented with 10 mM NH4Cl and at OD600 = 3.8 750 μl culture medium was mixed
with 250 μl 90 % (v/v) sterile glycerol, subsequently shock-frozen in liquid nitrogen and stored at
-80 °C.
3.2.3 Cultivation of A.vinelandii under Mo-limited conditions
A. vinelandii was grown aerobically in V containing growth medium (see Table 2): Pre culture me-
dium (100 ml) was supplemented with 10 mM NH4Cl as sole nitrogen source; main cultures
(500 ml) were complemented with 1.36 mM NH4Cl resulting in a de-repression of nitrogenase
gene expression upon ammonium exhaustion [299]. Cultures were grown at 30 °C and agitated at
180 rpm. Bacterial growth and nitrogenase production was monitored by measuring OD600 and
the whole cell nitrogenase acetylene reduction assay (see section 3.2.4), respectively. At an
OD600 = 2–4 and at a nitrogenase activity maximum cells were harvested by centrifugation at
5.000×g and 4 °C for 20 min, subsequently shock-frozen and stored in liquid N2.
3.2.4 Whole cell nitrogenase acetylene reduction assay (ARA)
2 ml culture medium was filled in an air-tight glass vial (13.2 ml; Wheaton), air-tight sealed with a
rubber septum, headspace was replaced by Argon, 1 ml Acetylene as well as 0.2 ml (≈ 0.02 atm)
or 1.0 ml (≈ 0.09 atm) oxygen were added and the reaction vial was incubated for 30 min at
25 °C under 200 rpm agitation. The conversion of acetylene to ethylene by nitrogenase was quan-
tified from the headspace via gas chromatography (SRI 8610C Gas Chromatograph, SRI instru-
ments) [106], [108], [300], [109].
Materials and Methods
59
3.3 Protein biochemical methods
VFe-protein from A. vinelandii was purified under a low dithionite (‘loDT’) and a high dithionite
(‘hiDT’) concentration according to the protocol shown in Figure 20. All purification steps until
loading the crude extract onto the anion exchange chromatography were performed at 4 °C. If
not highlighted all subsequent steps were performed at RT.
Figure 20: Schematic illustration of the purification procedure of VnfDKG in ‘active’ and in ‘resting’ state.
3.3.1 Purification of VFe-protein under low dithionite concentration (‘loDT’)
Cells were resuspended with the threefold amount of lysis buffer (see Table 4; 2 mM Na2S2O4)
and homogenized by pottering. Cells were ruptured with an Emulsiflex C5 homogenizer (Aves-
tin) at 15.000 psi under a nitrogen atmosphere. The cell lysate was centrifuged 100.000 ×g for 1 h
and the supernatant was sterile filtered and loaded onto a HiTrapQ HP anion exchange column
(GE Healthcare) with flowrate of 2 ml min-1, pre-equilibrated in loading buffer (see Table 4;
2 mM Na2S2O4). After a wash step at 150 mM NaCl, VFe-protein was eluted at 350 mM NaCl.
The VnfDKG containing solution was diluted and loaded onto a 6 ml ResourceQ anion ex-
change column (GE Healthcare) with a flowrate of 2 ml min-1, pre-equilibrated in loading buffer.
cell suspension
crude extract
cells
resuspension 1:3 (w/v) with lysis buffer
cell disruption: 3 x Emulsiflex @ 15.000 psi
centrifugation: 30 min @ 100.000 ×g
‚loDT‘ VFe-protein
VFe_QSeph
VFe_ResourceQ
VFe_SEC
anion exchange chromatography:
HiTrapQ HP
size exclusion chromatography::
26/600 Superdex 200 pg
anion exchange chromatography:
ResourceQ
1. day
2. day
cell suspension
crude extract
cells
resuspension 1:3 (w/v) with lysis buffer
cell disruption: 3 x Emulsiflex @ 15.000 psi
centrifugation: 30 min @ 100.000 ×g
‚hiDT‘ VFe-protein
VFe_QSeph
VFe_ResourceQ_2
VFe_SEC
anion exchange chromatography:
HiTrapQ HP
size exclusion chromatography::
26/600 Superdex 200 pg
anion exchange chromatography:
ResourceQ No.1
VFe_ResourceQ_1
anion exchange chromatography:
ResourceQ No.2
1. day
2. day
3. day
Materials and Methods
60
After a wash step at 150 mM NaCl, VFe-protein was eluted with a linear gradient from 150 –
350 mM NaCl within 20 column volumes (CV) and stored overnight at 4 °C. Subsequently
VnfDKG was loaded onto a HiLoad 26/600 Superdex 200 pg size exclusion column (GE
Healthcare) with a flowrate of 1 ml min-1, pre-equilibrated in SEC buffer (see Table 4; 2 mM
Na2S2O4). Pure VFe-protein was concentrated to 30 mg/ml using a Vivaspin 20 MWCO 100.000
PES concentrator (Sartorius) under 4 bar N2 overpressure, shock-frozen and stored in liquid ni-
trogen.
3.3.2 Purification of VFe-protein under high dithionite concentration (‘hiDT’)
Cells were resuspended with the threefold amount of lysis buffer with 10 mM Na2S2O4 (see Table
4) and homogenized by pottering. Cells were ruptured with an Emulsiflex C5 homogenizer
(Avestin) at 15.000 psi under a nitrogen atmosphere. The cell lysate was centrifuged 100.000 ×g
for 1 h and the supernatant was sterile filtered loaded onto a HiTrapQ HP anion exchange col-
umn (GE Healthcare) with flowrate of 2 ml min-1, pre-equilibrated in loading buffer with 5 mM
Na2S2O4 (see Table 4). After a wash step at 150 mM NaCl, VFe-protein was eluted at 400 mM
NaCl. The VnfDKG containing solution was diluted and loaded onto a 6 ml ResourceQ anion
exchange column (GE Healthcare) with a flowrate of 2 ml min-1, pre-equilibrated in loading buff-
er with 5 mM Na2S2O4. The VFe-protein containing flow through was stored overnight at 4 °C
and then loaded a second time onto a 6 ml ResourceQ anion exchange column (GE Healthcare)
with a flowrate of 2 ml min-1, pre-equilibrated in loading buffer with 2.5 mM Na2S2O4. After a
wash step at 150 mM NaCl, VnfDKG was eluted with a linear gradient from 150 – 350 mM
NaCl within 25 CV and stored overnight at 4 °C. Subsequently VnfDKG was loaded onto a Hi-
Load 26/600 Superdex 200 pg size exclusion column (GE Healthcare) with a flowrate of
1 ml min-1, pre-equilibrated with SEC buffer (see Table 4). Pure VFe-protein was concentrated to
30 mg/ml using a Vivaspin 20 MWCO 100.000 PES concentrator (Sartorius) under 4 bar N2
overpressure, shock-frozen and stored in liquid nitrogen.
3.4 Protein analytical methods
3.4.1 SDS-polyacrylamide-gel electrophoresis (SDS-PAGE)
Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) was used to analyze
protein presence and purity. The method for conducting a discontinuous SDS-PAGE has been
Materials and Methods
61
described elsewhere [295], [296]. Used buffers, gels and solutions are listed in Table 3. 1-30 μg
protein was treated with 5x sample buffer, incubated for 5 min at 95 °C prior to loading on gel.
Pierce Unstained Protein MW Marker (Thermo Fisher) was used as molecular weight standard.
Separation was performed at a current of 45 mA for 45 min. For protein band visualization gels
were treated with staining and destaining solution. Developed gels were scanned.
3.4.2 Determination of protein concentration (BCA-assay)
The 2,2’-Bichinoline-4,4’-dicarboxylic acid-assay (BCA-assay) was used to determine the protein
concentration [301], [302]. The BCA-reagent contains Pierce BCA Protein Assay Reagent A
(Thermo Fisher) and 4 % (w/v) CuSO4 in a 49:1 ratio. The assay principle consists of the reduc-
tion of Cu2+ to Cu1+ by protein and a subsequently complexing of Cu1+ with two molecules BCA.
This complex can be detected photometrical at 562 nm. The amount of reduction is proportional
to the protein present.
50 ul protein sample were mixed with 1 ml BCA reagent, incubated 30 min at 60 °C, subsequent-
ly incubated on ice for 3 min and then 3 min at RT. The sample absorption was detected photo-
metrical at 562 nm using a GeneQuant 1300 (GE Healthcare) spectrometer and a triple determi-
nation. Comparison of the absorbance with a calibration curve with Bovine serum albumin (BSA)
of known protein concentrations (50, 100, 150, 200, 250 μg/ml) delivered the protein sample
concentration.
3.4.3 Mass spectrometry
Liquid chromatography-mass spectrometry (LC-MS) was used to identify the protein VnfDKG.
LC-MS was performed at the Core Facility Proteomics, Center for Biological Systems Analysis
(ZBSA), Freiburg University, Freiburg, Germany [303].
Gel slices containing proteins to be analyzed by mass spectrometry, were in-gel digested using
trypsin (Promega, Mannheim, Germany). Digests were performed overnight at 37 °C in 0.05 M
NH4HCO3 (pH 8). About 0.1 µg of protease was used for each gel band. Peptides were extracted
from the gel slices with ethanol.
All LC-MS/MS analyses were performed with the 1200 Agilent Chip-HPLC system (Agilent
Technologies, Böblingen, Germany), either coupled to a quadrupole time-of-flight (Q-TOF; Ag-
ilent 6520) mass spectrometer. Peptides were separated on the HPLC-Chip with an analytical
column of 75 µm inner diameter and 150 mm length and a 40 nl trap column, both packed with
Materials and Methods
62
Zorbax 300SB C-18 (5 µm particle size; Agilent). Peptides were eluted with a linear acetonitrile
gradient with 1%/min at a flow rate of 300 nl/min (starting with 3% acetonitrile). The Q-TOF
spectrometer was operated in the 2 GHz extended dynamic range mode. MS/MS analyses were
performed using data-dependent acquisition mode. After a MS scan (2 spectra/s), a maximum of
3 peptides were selected for MS/MS. Singly charged precursor ions were excluded from selec-
tion. Internal calibration was applied using one reference mass. Software MassHunter (Agilent)
and Mascot Deamon version 2.4.0 (Matrix Science, London, UK) were used for data acquisition
and processing (These information were kindly provided from Dr. Veronica Dumit, Center for
Biological System Analysis Freibug).
3.4.4 Nitrogenase acetylene reduction assay
Nitrogenase acetylene reduction assays with isolated nitrogenase proteins (NifDK and NifH,
VnfDKG and VnfH) was used to determine the specific nitrogenase acetylene reduction activity
[106], [107], [108], [109], [110].
In a 10 ml reaction vial (Wheaton), 1 ml reaction mixture was sealed under an atmosphere of
95 % N2 and 5 % H2. The mixture contained 0.435 nmol Mo- or V-N2ase and 26.1 nmol corre-
sponding Fe-protein (1:60), 12.5 mM Na2S2O4, 5 mM MgCl2, 20 mM Tris/HCl buffer at pH 7.4
and an ATP-regenrating system of 0.125 mg/ml phosphocreatine kinase and 15 mM phospho-
creatine. The headspace was exchanged for Argon and 1 ml of acetylene was added via a gastight
syringe. The mixture was incubated at 30 °C for 3 min. The reaction was started by addition of
ATP to a final concentration of 2.5 mM and further incubated at 30 °C for 3 min. The reaction
was terminated by the addition of 200 µl glacial acetic acid and 1 ml of headspace was subse-
quently analyzed via gas chromatography for the conversion of acetylene to ethylene by nitrogen-
ase (SRI 8610C Gas Chromatograph, SRI instruments).
3.5 Crystallographic methods
3.5.1 Crystallization of VFe-protein
VnfDKG was crystallized using the sitting drop vapor diffusion method [304]. Crystallization
plates were set up in an anaerobic chamber containing an atmosphere of 95 % N2 / 5 % H2. Ini-
tial hits were obtained by using Morpheus screen HT-96 (Molecular Dimensions, Suffolk, UK)
[305]. 50 ul of reservoir solution was filled in Intelli-Plate 96-3 low-profile crystallization plates
Materials and Methods
63
(Hampton research, Aliso Viejo, USA). The drop size was set to 1.4 μl with a protein to reservoir
ratio of 1:1. The protein solution contained a ≈ 5.5 mg/ml VnfDKG concentration and
5 mM Na2S2O4 and 0.1 mM ZnCl2 as additives. Immediately after pipetting plates were sealed
with VIEWseal, advanced adhesive sealer (Greiner Bio One, Frickenhausen, Germany) and incu-
bated at 20–23 °C. Crystal optimization was performed by fine screening in 24-well crystallization
Cryschem M Plate (Hampton research, Aliso Viejo, USA) and microseeding [306], [307]. Best
crystallization conditions contained 10–13 % (w/v) PEG 8.000, 15-20 % ethylene glycol,
0.03 M MgCl2, 0.1 M MOPS/HEPES-Na or Tris/HCl pH 7.5, ≈ 5.5 mg/ml VnfDKG and the
additives 5 mM Na2S2O4, 0.1 mM ZnCl2. PEG, ethylene glycol, MgCl2, buffer and ZnCl2 solu-
tions were sterile filtered. Buffer solution MOPS/HEPES-Na pH 7.5 was used up to 14 days.
Crystals grew within ≈ 7 days. Crystals were flash-cooled and stored in liquid nitrogen
3.5.2 X-ray data collection
Diffraction data were collected on beamline X06SA using an EIGER 16M detector (Dectris) or
on X06DA using a PILATUS 2M-F detector at the Swiss Light Source (Paul-Scherrer-Institute,
Villigen, CH). For structure solution, datasets were collected to high multiplicity at and above the
Fe K-edge, at a wavelength of 1.7413 Å (7120 eV, inflection) and 1.7357 Å (7143 eV, peak), to-
gether with a high-resolution remote datasets at 1.0000 Å (12398 eV), 0.9000 Å (13776 eV) or
0.8000 Å (15498 eV). For an enhancement of anomalous scattering of S relative to Fe datasets
were collected to high multiplicity below the Fe K-edge at wavelength 1.7463 Å (7100 eV).
3.5.3 Structure solution, model building and refinement
For structure solution peak data set was indexed, integrated, scaled and merged with autoSHARP
[308]. Experimental phasing and model building have been performed using programs AutoSol
and AutoBuild, respectively, from the PHENIX suite [309]. Structure was refined using COOT
[310] and REFMAC5 [311].
All remaining datasets were indexed and integrated with XDS [312] and scaled and merged with
AIMLESS [313] from the CCP4 suite [314]. Structures were refined using COOT [310] and
REFMAC5 [311].
3.5.4 Visualization of protein structures
Protein structures were visualized using the program PyMOL [315].
Materials and Methods
64
3.6 Spectroscopic methods
3.6.1 Electron paramagnetic resonance spectroscopy
Buch: Electron paramagnetic resonance spectroscopy (EPR) is a resonance spectroscopy of mo-
lecular systems with unpaired electrons. EPR spectroscopy of biomolecules is used e.g. for inves-
tigation of metal centers (which often represent the enzyme’s active site for the biochemical reac-
tions). It is a method to investigate and determine the electronic structure of metal sites and by
interpretation of the EPR experiments you can draw conclusions about e.g. the chemical identity,
the redox state, the structural conformation and composition and the intra- or intermolecular
interactions of the metal center. EPR is based on the interplay of electromagnetic radiation with
the magnetic momentums of the electrons. Two degenerated energy levels can be split by the
presence of an external magnetic field and the energy difference is proportional to the magnetic
field strength (Zeeman splitting). Electron transition between the two different energy levels is
induced by electromagnetic radiation at resonance condition [316] (see equation 15).
∙ 𝜈 = Δ𝐸 = 𝑔 ∙ 𝜇𝐵 ∙ 𝐵 (15)
ħ = Planck constant (6.6262 x 10-34 Js)
ν = microwave frequency in GHz
ΔE = energy difference between the two levels
g = Landé factor (free electron in vacuum: ge = 2.00231930)
μB = Bohr magneton (9.274096 x 10-24 JT-1)
B = external magnetic field in Tesla (T)
EPR spectra were recorded using a continuous-wave (cw) X-band EPR-spectrometer (Elexsys E-
500, 10″ ER073 electromagnet, super high Q resonator cavity, Bruker) at a microwave frequency
of 9.339 GHz, equipped with a continuous- flow liquid helium cryostat (ER 41112HV, Oxford
Instruments). 300 μl of VFe-protein were filled into an X-band EPR tube and frozen in liquid
nitrogen. The ‘hiDT’ VFe-protein sample in the as isolated state had a protein concentration of
≈ 20 mg/ml with ≈ 2 mM Na2S2O4 in 50 mM Tris/HCl pH 7.4 and 100 mM NaCl. For the re-
duced ‘hiDT’ VFe-protein the as isolated sample was thawed, complemented with Na2S2O4 to a
final concentration of 20 mM and frozen in liquid nitrogen. For the ‘loDT‘ VFe-protein in the as
isolated state the Na2S2O4 concentration was decreased below 0.2 mM by dilution and concentra-
tion with 50 mM Tris/HCl buffer pH 7.4 and 100 mM NaCl. The EPR sample had a protein
Materials and Methods
65
concentration of ≈ 17 mg/ml. For the reduced ‘loDT’ VFe-protein the as isolated sample was
thawed and complemented with Na2S2O4 to a final concentration of 8 mM and frozen in liquid
nitrogen. The measurement was carried out at a temperature of 10 K and a power of 2 mW with
a modulation amplitude of 6 G and a receiver gain of 60 dB.
3.6.2 Differential scanning fluorimetry (DSF)
Differential scanning fluorimetry (DSF) was used to determine and optimize the protein thermal
stability, also called Thermofluor assay [317], [318], [319], [320]. DSF is benefits from a fluores-
cence increase of a fluorescent dye that is quenched in aqueous solutions but regains flourescence
in a hydrophobic environment. The method is based on detectable changes of protein folding
upon heat treatment. The protein is exposed to a temperature gradient which leads to thermal
denaturation and eventually unfolding of the protein. Upon unfolding the dye can interact with
the hydrophobic protein core resulting in the fluorescence increase. The temperature of protein
unfolding is regarded as the melting point (Tm) and thus protein thermal stability can be analyzed.
Samples were set up in the anaerobic chamber on ice in Hard-Shell Low-Profile 96-Well Skirted
PCR Plates (Bio-Rad Laboratories) and closed with Microseal ‘B’ PCR Plate Sealing Films. The
assay was performed outside the anaerobic chamber in a CFX96 Touch Real-Time PCR Detec-
tion System (Bio-Rad Laboratories). Prior to the assay sample solutions were spinned down by
centrifuging PCR plates for 10 s at 1000 ×g. The analysed temperature range was from 20 –
80 °C with 1 °C temperature steps, holding the temperature for 10 s. SYPRO orange (5.000x
concentrate in DMSO) was used freshly and diluted with H2O for a one-time usage. Sample assay
volume was 25 μl in section 3.6.2.1, 3.6.2.2, 3.6.2.3 and 26 ul in section 3.6.2.4.
3.6.2.1 Optimal ratio of protein and SYPRO orange concentration
Various VnfDKG (0.25; 0.5; 1.0 mg/ml) and SYPRO orange (5x; 10x; 20x) assay concentrations
have been tested in ≈ 50 mM HEPES/NaOH pH 7.0. H2O for VnfDKG solution was used as
reference. The assay was performed under reducing (2 mM Na2S2O4) and non-reducing
(0 mM Na2S2O4) conditions to check dioxygen tightness of the sealed plate for assay perfor-
mance. Experiment was performed once.
Materials and Methods
66
Table 7: Composition of Thermofluor assay for optimal ration of protein and SYPRO orange concentration.
reducing non-reducing
VnfDKG (6.25; 12.5; 25.0 mg/ml) 1 μl 1 μl
SYPRO orange (125x; 250x; 500x) 1 μl 1 μl
Na2S2O4 (100 mM in 100 mM Tris base) 0.5 μl
50 mM HEPES/NaOH pH 7.0 22.5 μl 23.0 μl
3.6.2.2 Optimal pH and NaCl concentration
Various pH values (pH 6.0; 6.5; 7.0; 7.5; 8.0; 8.5; 9.0) and NaCl (0; 100; 200; 300; 400; 500 mM)
assay concentrations have been tested with 1 mg/ml VnfDKG and 20x SYPRO orange concen-
tration in 50 mM HEPES/NaOH under reducing conditions (2 mM Na2S2O4).
Table 8: Composition of Thermofluor assay for opimal pH and NaCl concentration.
volume
VnfDKG (25 mg/ml) 1 μl
SYPRO orange (500x) 1 μl
Na2S2O4 (100 mM in 100 mM Tris base) 0.5 μl
mixed pH and NaCl solution 22.5 μl
3.6.2.3 Influence of Na2S2O4 concentration
The influence of Na2S2O4 was tested by using 0, 1, 2, 4, 10 and 20 mM Na2S2O4 with 1 mg/ml
VnfDKG and 20x SYPRO orange in 50 mM Tris/HCl pH 7.0. Experiment was performed in
quadruplet measurements.
Table 9: Composition of Thermofluor assay for influence of the Na2S2O4 concentration.
volume
VnfDKG (25 mg/ml) 1 μl
SYPRO orange (500x) 1 μl
50 mM Tris/HCl pH 7.0 with 0, 1, 2, 4, 10, 20 mM Na2S2O4
23 μl
Materials and Methods
67
3.6.2.4 Influence of additives
Various chaotropic/dissociation reagents, salts, reducing agents, polyamines, chelating agents,
imidazole, multivalent and monovalent ions have been tested with 1 mg/ml VnfDKG and 20x
SYPRO orange concentration under reducing conditions (2 mM Na2S2O4) in ≈ 50 mM Tris/HCl
pH 7.0 (see Table 10, Table 11). Experiment was performed in quadruplet measurements.
Table 10: Composition of Thermofluor assay for additive screen.
volume
VnfDKG (12.5 mg/ml) 2 μl
SYPRO orange (500x) 1 μl
Na2S2O4 (50 mM in 50 mM Tris base) 1 μl
buffer-additive solution 22 μl
Table 11: Layout of Thermofluor additive screen with additive concentrations in the buffer-additive solutions; cha-
otropic/dissociation reagents [A1:A6], reducing agents [B1:B6], salts [C1:C4; D1:D5; [E4:F3], polyamines [C5:C6],
chelating agent [D6], imidazole [E1:E3], multivalent ions [G1:H1], monovalent ions [F4:F5; [H2:H6]; Abbreviations:
β-mercaptoethanol (β-ME), tris(2-carboxyethyl)phosphine (TCEP), dithiothreitol (DTT), hydrochloride (HCl).
1 2 3 4 5 6
A 0.1 M urea 0.5 M urea 1 M urea 6 M urea 0.1 M guani-dine-HCl
0.5 M guani-dine-HCl
B 0.1 % β-ME 0.01 M TCEP HCl
0.001 M DTT 0.005 M DTT 0.01 M DTT 0.02 M DTT
C 0.1 M Na-acetate
0.1 M Ca-acetate
0.1 M K-acetate
0.1 M NH4-acetate
0.001 M sper-midine
0.001 M sperm-ine-HCl
D 0.1 M Na2SO4 0.1 M MgSO4 0.1 M (NH4)2SO4
0.1 M NaH2PO4
0.1 M KH2PO4 0.005 M EDTA
E 0.05 M imidaz-ole
0.1 M imidaz-ole
0.3 M imidaz-ole
0.1 M NaHCO2 0.1 M KHCO2 0.1 M NH4HCO2
F 0.1 M Na-citrate
0.1 M Na-malonate
0.1 M NaNO3 0.1 M NaBr 0.1 M NaCl H2O (refer-ence)
G 0.01 M MgCl2 0.01 M CaCl2 0.001 M MnCl2 0.001 M NiCl2 0.001 FeCl3 0.001 M ZnCl2
H 0.001 M CoCl2 0.1 M LiCl 0.1 M KCl 0.1 M NH4Cl 0.1 M NaI 0.1 M KI
Results
68
4. Results
4.1 Cultivation of A. vinelandii
4.1.1 Mo depletion
Bacteria grown in Mo-limited Burk’s medium containing 10 mM NH4Cl and V were streaked out
on Mo- and N-limited agar plates supplemented with V (see section 3.2.2 and Table 2). After 3 to
four days, brownish colonies indicated a successful diazotrophic growth and the presence of ni-
trogenase (see Figure 21). Single colonies were picked to re-inoculate Mo-limited Burk’s medium
containing 10 mM NH4Cl and V for another round of liquid growth with subsequent streaking
out on Mo- and N-limited agar plates supplemented with V. This cycle was repeated five times.
Afterwards, cultivation of A. vinelandii under Mo-limited and V-containing conditions for the
production of V-N2ase could be performed.
Figure 21: Bacterial growth of A. vinelandii on Mo-limited and NH4Cl-free agar plates. Brownish colonies indicate
diazotrophic growth via nitrogen fixation and thus the presence of nitrogenase.
4.1.2 Cultivation of A. vinelandii under Mo-limited conditions
The diazotrophic growth and nitrogenase activity of A. vinelandii under Mo-limited conditions
under provision of vanadate was monitored by measuring OD600 and whole-cell nitrogenase ac-
tivity. At an OD600 ≈ 2.5 and at maximum activity (see Figure 22) after ≈ 18 h, cells were harvest-
ed with a cell yield of 2.5-5 g/l medium (wet weight).
Results
69
Figure 22: Cell growth and whole cell nitrogenase acetylene reduction activity.
4.2 Isolation of VFe-protein
4.2.1 ‘loDT’ purification of VFe-protein
The isolation of VFe-protein at low dithionite concentration (‘loDT’ VFe-protein) was per-
formed in a three-step purification procedure (see section 3.3.1) and is shown exemplary for a
VFe-protein isolation from 20 g of cells (wet weight).
After cell disruption and centrifugation, the cytosolic crude extract was loaded onto a 5 ml
HiTrapQ HP anion exchange column. After a wash step at 150 mM NaCl, the step elution frac-
tion at 350 mM NaCl contained VFe-protein (VFe_QSeph) (see Figure 23 A). After dilution, it
was loaded onto a 6 ml ResourceQ anion exchange column. After a wash step step at 150 mM
NaCl, the VFe-protein (VFe_ResourceQ) eluted in the linear gradient at ≈ 200 mM NaCl and
≈ 22 mS/cm (see Figure 23 C). After protein fraction concentration, it was loaded onto a HiLoad
26/600 Superdex 200 pg size exclusion column. The pure ‘loDT’ VFe-protein (VFe_SEC) eluted
at a retention volume of ≈ 160 ml (see Figure 23 D). The protein purity of each step is shown in
Figure 23 B. A comparison of MoFe- and the VFe-protein is shown in Figure 23 B as well and
was kindly provided by Christian Trncik.
The yield of pure ‘loDT’ VFe-protein was 0.52 mg protein per 1 g cells (wet weight).
9 10 11 12 13 14 15 16 17 18 19 20 21
0,0
0,5
1,0
1,5
2,0
2,5
3,0
incubation [h]
OD
60
0 [A
bs]
0
100
200
300
400
500
600
700
800
900
Activity [n
mo
l*h
-1*m
l-1]
Results
70
Figure 23: Exemplary isolation of VFe-protein under low-dithionite conditions. The elution profiles of the anion
exchange chromatographies are shown for the HiTrap Q HP in A and for the Resource Q in C; VFe-protein con-
taining fractions are coloured in brown. The chromatogram for the size exclusion chromatography is shown in D.
The corresponding SDS-PAGEs are shown in B. A comparison of the isolated MoFe- and VFe-protein on SDS-
PAGE is also shown in B
4.2.2 ‘hiDT’ purification of VFe-protein
The VFe-protein under high dithionite conditions (‘hiDT’ VFe) was isolated in a four step purifi-
cation procedure (see 3.3.2) and is described for a VFe-protein isolation from 48 g cells (wet
weight).
After cell disruption and preparation of cytosolic crude extract preparation, it was loaded onto a
15 ml HiTrapQ HP anion exchange column. Upon a 150 mM NaCl wash step, the step elution
fraction at 400 mM NaCl contained the VFe-protein (VFe_QSeph) (see Figure 24). The protein
solution was diluted and loaded onto a 6 ml ResourceQ anion exchange column. The VFe-
protein did not bind to the column, but rather eluted in the flow through (VFe_ResourceQ_1)
(see Figure 24 C). This happened probably due to a high degree of dithionite binding or interac-
tion with the ResourceQ anion exchange column and thus leading to a protein binding blockage.
The usage of 100 mM NaCl in the loading buffer should avoid vast binding of dithionite to the
ResourceQ functional quarternary amine groups and does so at lower dithionite concentrations
(≈ 2.5 mM). The absence of a massive protein elution, neither during gradient elution nor at
0 20 40 60 80 100 120 140 160 180 200
0
500
1000
1500
2000
2500
3000U
V/v
is-a
bso
rptio
n [m
Au
]
retention volume [ml]
VFe_QSeph
10
15
20
25
30
35
40
45
50
55
co
nd
uctivity [m
S/c
m]
100
150
200
250
300
350
400
450
500
550
Na
Cl [m
M]
A
0 20 40 60 80 100 120 140 160 180 200
-200
-100
0
100
200
300
400
500
600
700
800
900
1000
UV
/vis
-ab
so
rptio
n [m
Au
]
retention volume [ml]
VFe_ResourceQ
10
15
20
25
30
35
40
45
50
55
co
nd
uctivity [m
S/c
m]
100
150
200
250
300
350
400
450
500
550
Na
Cl [m
M]
C
0 20 40 60 80 100 120 140 160 180 200 220 240 260 280 300
-50
0
50
100
150
200
250
300
350
400
450
500
UV
/vis
-ab
so
rptio
n [m
Au
]
retention volume [ml]
0
1
2
3
4
5
6
7
8
9
10
11
12
co
nd
uctivity [m
S/c
m]
VFe_SEC
D
kDa
116.0
66.2
45.0
35.0
25.0
18.4
14.4
VnfD
VnfK
VnfG
NifK
NifD
B
Results
71
wash step with 500 mM NaCl, argues against a protein overload. After reequilibration with load-
ing buffer, containing a lower dithionite concentration (2.5 mM), the flow through protein frac-
tion was loaded a second time onto the ResourceQ column. The elution of VFe-protein
(VFe_ResourceQ_2) in the linear gradient occurred at ≈ 200 mM NaCl and ≈ 21 mS/cm (see
Figure 24 D). The concentrated protein fraction was loaded onto a HiLoad 26/600 Superdex 200
pg size exclusion column. The pure ‘hiDT’ VFe-protein (VFe_SEC) eluted at a retention volume
of ≈ 164 ml (see Figure 24 E). The protein purity of each step is shown in Figure 24 B.
The yield of pure ‘hiDT’ VFe-protein was 0.55 mg protein per 1 g cells (wet weight).
Figure 24: Isolation of VFe-protein under high dithionite conditions. The elution profiles of the anion exchange
chromatographies are shown for the HiTrap Q HP in A, for the first Resource Q in C and for the second Resource
Q in D; VFe-protein containing fractions are coloured in brown. The chromatogram for the size exclusion chroma-
tography is shown in D. The corresponding SDS-PAGEs are shown in B.
kDa
116.0
66.2
45.0
35.0
25.0
18.4
14.4
VnfD
VnfK
VnfG
B
0 50 100 150 200 250 300 350
1200
1400
1600
1800
2000
2200
2400
2600
2800
3000
3200
3400
3600
3800
UV
/vis
-ab
so
rption
[m
Au
]
retention volume [ml]
VFe_QSeph
10
15
20
25
30
35
40
45
50
55
co
nd
uctivity [m
S/c
m]
100
150
200
250
300
350
400
450
500
550
Na
Cl [m
M]
A
0 20 40 60 80 100 120 140 160 180 200 220 240 260
-400
-200
0
200
400
600
800
1000
1200
1400
1600
1800
UV
/vis
-ab
so
rption
[m
Au
]
retention volume [ml]
VFe_ResourceQ_1
10
15
20
25
30
35
40
45
50
55
co
nd
uctivity [m
S/c
m]
100
150
200
250
300
350
400
450
500
550
Na
Cl [m
M]
C
0 20 40 60 80 100 120 140 160 180
-400
-200
0
200
400
600
800
1000
1200
1400
1600
1800
2000
UV
/vis
-ab
so
rption
[m
Au
]
retention volume [ml]
VFe_ResourceQ_2
10
15
20
25
30
35
40
45
50
55
co
nd
uctivity [m
S/c
m]
100
150
200
250
300
350
400
450
500
550
Na
Cl [m
M]
D
0 20 40 60 80 100 120 140 160 180 200 220 240 260 280 300
-100
0
100
200
300
400
500
600
700
800
900
UV
/vis
-ab
so
rption
[m
Au
]
retention volume [ml]
0
1
2
3
4
5
6
7
8
9
10
11
12
co
nd
uctivity [m
S/c
m]
VFe_SEC
E
Results
72
4.3 Thermal stability of VFe-protein
4.3.1 Optimal protein and SYPRO orange concentrations
The results of the thermofluor assay to figure out the optimal protein and SYPRO orange con-
centration specifically for the VFe-protein in the assay condition is shown in Figure 25. Addition-
ally, the impact of the presence or absence of oxygen to eliminat Na2S2O4 in the assay condition
was tested, as the assay is actually performed in a Thermocycler outside the anaerobic chamber
(see section 3.6.2).
Figure 25: Optimal protein and SYPRO orange concentrations. The melting curves of the Na2S2O4 reducing and
non-reducing conditions are shown in A and B, respectively. A typical shape of the alternative representation of a
melting curve using the first derivative - (dRFU)/dT is shown in C and adapted from [320].
Reference experiments without protein showed no fluorescence change during the temperature
gradient, evidencing that the fluorescence increase arises from interactions of the dye with the
protein.
The shapes of the melting curve derivatives with and without Na2S2O4 differ clearly. In condi-
tions ‘+DT’ distinct minima are visible representing a definite point of unfolding of the protein
20 25 30 35 40 45 50 55 60 65 70 75 80
-1200
-1000
-800
-600
-400
-200
0
200
400
600
800
-d(R
FU
)/d
T
Temperature [°C]
0,25 mg/ml 5x
0,5 mg/ml 5x
1,0 mg/ml 5x
0,25 mg/ml 10x
0,5 mg/ml 10x
1,0 mg/ml 10x
0,25 mg/ml 20x
0,5 mg/ml 20x
1,0 mg/ml 20x
0 mg/ml 5x
0 mg/ml 10x
0 mg/ml 20x
with Na2S
2O
4
20 25 30 35 40 45 50 55 60 65 70 75 80
-1200
-1000
-800
-600
-400
-200
0
200
400
600
800-d
(RF
U)/
dT
Temperature [°C]
0,25 mg/ml 5x
0,5 mg/ml 5x
1,0 mg/ml 5x
0,25 mg/ml 10x
0,5 mg/ml 10x
1,0 mg/ml 10x
0,25 mg/ml 20x
0,5 mg/ml 20x
1,0 mg/ml 20x
0 mg/ml 5x
0 mg/ml 10x
0 mg/ml 20x
without Na2S
2O
4
A B
C
Results
73
and thereby a certain melting temperature (Tm) for the protein. In conditions ‘-DT’ the protein
seems to start to unfold at higher temperatures, but not in the manner of typical melting curve
shapes (see Figure 25) and thus probably not representing a characteristic protein unfolding nec-
essary to determine the melting temperature. Therefore, the presence of Na2S2O4 in assay condi-
tions was required for the determination of proper protein Tm. Whether these differences be-
tween ‘+DT’ and ‘-DT’ arise from possible O2 leakage of the sealed assay plate, or have other
reasons, is not known.
The comparison of the various protein and SYPRO orange concentrations show that the most
distinct curves occur with 20x SYPRO orange and 0.5 and 1 mg/ml VFe-protein. Lower concen-
trations yield lower fluorescence response and consequently less distinct melting curves and re-
sults. As the best condition for further assays, 1 mg/ml protein concentration and 20x SYPRO
orange was chosen.
FeCl2, CoCl2 and CuCl2 appear to quench the fluoroprobe at high concentrations (≈ mM) [321].
Although the VFe-protein has a high Fe content with 30 Fe-atoms per molecule, at 1 mg/ml
protein concentration and an entirely denatured protein with released Fe-atoms a Fe-
concentration of only ≈ 0.125 mM would be present in solution. Anyway, this correlation specifi-
cally for VnfDKG rationalizes a ratio with high protein and SYPRO orange concentrations com-
pared to the standard conditions of ≈ 0.05 mg/ml protein and 5x SYPRO orange.
4.3.2 Optimal pH and NaCl concentration
The results (Tm) of the thermofluor assay to test optimal pH and NaCl concentration for VFe-
protein are shown in Table 12. Corresponding melting curves are in section 6.2 in Figure 43.
Table 12: Optimal pH and NaCl concentrations. Tm decrease is indicated by colour change from green over yellow
to red.
pH 6.0 pH 6.5 pH 7.0 pH 7.5 pH 8.0 pH 8.5 pH 9.0
0 mM 48 °C 49 °C 50 °C 49 °C 49 °C 49 °C 47 °C
100 mM 46 °C 49 °C 48 °C 50 °C 49 °C 47 °C 46 °C
200 mM 45 °C 47 °C 48 °C 48 °C 46 °C 46 °C 46 °C
300 mM 44 °C 47 °C 47 °C 48 °C 46 °C 46 °C 45 °C
400 mM 45 °C 47 °C 47 °C 47 °C 46 °C 45 °C 45 °C
500 mM 44 °C 46 °C 47 °C 47 °C 46 °C 45 °C 44 °C
Results
74
Highest melting temperatures of Tm ≈ 50 °C occur in conditions with pH 7.0 and 7.5 as well as 0
or 100 mM NaCl. Tm is decreasing with higher or lower pH values and increasing salt concentra-
tions. As best condition for further assays pH 7.0 and 0 mM NaCl was chosen.
4.3.3 Influence of Na2S2O4 concentration
In Table 13, the average Tm of the quadruplet measurements are shown. Corresponding melting
curves and all melting temperatures are in section 6.2 in Figure 44 and in Table 16.
Table 13: Influence of Na2S2O4 concentration.
0 mM DT 1 mM DT 2 mM DT 4 mM DT 10 mM DT 20 mM DT
ØTm 52 °C 51 °C 51 °C 51 °C 50 °C 48 °C
Melting curves with and without Na2S2O4 differ in shape, as explained in section 3.6.2.1. There-
fore the Tm for ‘0 mM DT’ is not taken into comparison for various DT concentrations. The
melting temperatures, with 51 °C for the 1 mM DT and 48 °C for 20 mM DT, generally change
just slightly, but definetly a Tm decrease with an increasing Na2S2O4 concentration is observable.
This indicates an influence of DT concentration on VFe-protein. At low concentrations of 1, 2
and 4 mM DT, the effect seems to be similar and starts to increase with increasing Na2S2O4 con-
centrations. A 2 mM DT concentration was chosen to use in further assays.
4.3.4 Influence of additives
The influence of various additives and their concentration has been been tested. In Table 14, the
average Tm of quadruple measurements are shown. Corresponding melting curves and all melting
temperatures are listed in section 6.2 in Figure 45 and in Table 17.
The reference Tm is 51 °C (grey). Most additives show no or only a slight impact, with melting
temperatures between 47-52 °C (yellow). A few additives lead to a clear Tm decrease with
37-43 °C (red). For 6 M urea, 1 mM NiCl2, 1 mM FeCl3 and 1 mM CoCl2, no melting tempera-
ture could be determined from the melting curve derivatives. Only 1 mM ZnCl2 showed a distinct
melting temperature increase by ≈ 9 °C to Tm = 60 °C (green), resulting in a higher thermal sta-
bility of VFe-protein.
Results
75
Table 14: Additive screen. Reference is highlighted in grey; slight or no effects in yellow; distinct Tm decrease in red
and distinct Tm increase in green. a) For 6 M urea, 1 mM NiCl2, 1 mM FeCl3 and 1 mM CoCl2 no Tm could be deter-
mined from the melting curves.
1 2 3 4 5 6
A 0.1 M urea 0.5 M urea 1 M urea 6 M urea 0.1 M guani-dine-HCl
0.5 M guani-dine-HCl
49 °C 43 °C 40 °C n.d. a) 48 °C 37 °C
B 0.1 % β-ME 0.01 M TCEP HCl
0.001 M DTT 0.005 M DTT 0.01 M DTT 0.02 M DTT
50 °C 52 °C 50 °C 50 °C 50 °C 50 °C
C 0.1 M Na-acetate
0.1 M Ca-acetate
0.1 M K-acetate
0.1 M NH4-acetate
0.001 M sper-midine
0.001 M sperm-ine-HCl
51 °C 48 °C 51 °C 51 °C 51 °C 51 °C
D 0.1 M Na2SO4 0.1 M MgSO4 0.1 M (NH4)2SO4
0.1 M NaH2PO4
0.1 M KH2PO4 0.005 M EDTA
50 °C 50 °C 50 °C 42 °C 43 °C 49 °C
E 0.05 M imidaz-ole
0.1 M imidaz-ole
0.3 M imidaz-ole
0.1 M NaHCO2 0.1 M KHCO2 0.1 M NH4HCO2
49 °C 48 °C 42 °C 49 °C 50 °C 51 °C
F 0.1 M Na-citrate
0.1 M Na-malonate
0.1 M NaNO3 0.1 M NaBr 0.1 M NaCl H2O (refer-ence)
50 °C 50 °C 51 °C 49 °C 51 °C 51 °C
G 0.01 M MgCl2 0.01 M CaCl2 0.001 M MnCl2 0.001 M NiCl2 0.001 FeCl3 0.001 M ZnCl2
51 °C 51 °C 51 °C n.d. a) n.d. a) 60 °C
H 0.001 M CoCl2 0.1 M LiCl 0.1 M KCl 0.1 M NH4Cl 0.1 M NaI 0.1 M KI
n.d. a) 51 °C 51 °C 51 °C 47 °C 47 °C
4.4 The structure of VFe-protein
4.4.1 Crystallization and data collection of VFe-protein
After extensive crystallization experiments without success, the first initial hit was observed solely
after usage of ZnCl2 as additive with in the commercially available initial screen MORPHEUS
[305] condition A6, containing 10 % PEG 8000, 20 % ethylene glycol, 0.03 M MgCl2, CaCl2 and
0.1 M MOPS/HEPES-Na at pH 7.5, using a protein concentration of 5.5 mg/ml (see Figure 26
A), a reservoir to protein ration of 1:1 with a drop size of 1.4 μl, 0.1 mM ZnCl2 and 5 mM DT in
protein solution and an incubation temperature of 21 °C. Crystals grew as small misshaped plates.
Results
76
Crystals could be optimized to a cubic shape and in size by fine screening and seeding (see Figure
26 B, C). Especially CaCl2, a higher protein concentration and protein impurity led to formation
of spherolites, and a high ZnCl2 concentration around 1 mM led to protein precipitation. Opti-
mal crystallization conditions are 5.5 mg/ml VFe-protein, 10-13 % PEG 8000, 0 and 15-20 %
ethylene glycol, 0.03 M MgCl2, 0.1 M MOPS/HEPES-Na at pH 7.5, 0.1 mM ZnCl2 as well as
5 mM DT in protein solution, combined with seeding. Crystals were harvested after 5-7 days with
a nylon loop, flash-cooled and stored in liquid nitrogen.
Figure 26: Crystals of VFe-protein. A) initial crystals of VFe-protein in MORPHEUS A6 with 10 % PEG 8000,
20 % ethylene glycol, 0.03 M MgCl2, CaCl2, 0.1 M MOPS/HEPES-Na at pH 7.5, ≈ 5.5 mg/ml protein, 0.1 mM
ZnCl2 and 5 mM DT in protein solution; optimized crystals of B) ‘hiDT’ VFe-protein and C) ‘loDT’ VFe-protein in
12.5 and 12 % PEG 8000, 0 and 20 % ethylene glycol, 0.03 M MgCl2, 0.1 M MOPS/HEPES-Na at pH 7.5,
≈ 5.5 mg/ml protein 0.1 mM ZnCl2 and 5 mM DT in protein solution.
Data were collected and processed as in sections 3.5.2 and 3.5.3 described. Best crystals for the
‘hiDT’ VFe-protein diffracted at a wavelength of 1.0 Å up to a resolution of 1.35 Å. and for the
‘loDT’ VFe-protein at wavelengths of 0.8, 0.9 and 1.0 Å up to a resolution of 1.20 Å. A. vinelandii
VFe-protein crystallizes in the triclinic space group P1, with unit cell parameters of a = 75.3 Å,
b = 79.8 Å and c = 107.0 Å, α = 84.0°, β = 72.6° and γ = 75.2° for the ‘hiDT’ VFe-protein and
a = 75.6 Å, b = 79.8 Å and c = 107.2 Å, α = 84.0°, β = 72.4° and γ = 75.3° for the ‘loDT’ VFe-
protein containing one complete VnfD2K2G2 heterohexamer per unit cell. Data collection and
refinement statistics are summarized in section 6.5 in Table 23, Table 24 and Figure 46.
A B C
Results
77
4.4.2 The structure of ‘hiDT’ VFe-protein (‘resting’ state)
4.4.2.1 Overall structural organization of VFe-protein
The structure of A.vinelandii VFe-protein was solved by multiple-wavelength anomalous disper-
sion (see section 3.5.3) and established the composition of a VnfD2K2G2 complex for the VFe-
protein (see Figure 27).
Figure 27: Overall structural organization of the A. vinelandii VFe-protein. In A the VnfD2K2G2 complex is visual-
ized; VnfD and VnfD’ are shown in medium and light green; VnfK and VnfK’ are shown in dark and light red;
VnfG and VnfG’ are shown in dark and light blue; the P-cluster, the FeV-cofactor and the Mg binding site between
VnfK and VnfK’ are highlighted. Surface representations are shown in B and C.
For the ‘hiDT’ VFe-protein form (see section 3.3.2) the peptide chains of all subunits are almost
fully defined in the electron density map. The model contains residues 2-474 of VnfD, 12-475 of
VnfK and 2-113 of VnfG (see Figure 28). A dimer of the VnfDK heterodimer, representing the
VnfK‘ (β‘)
VnfG (δ)
VnfD (α)
VnfG‘ (δ‘)
VnfD‘ (α‘)
VnfK (β)
Mg2+
P-clusterFeV-cofactor
A
G (δ)
K‘ (β‘)
D (α)
G‘ (δ‘)D‘ (α‘)
K (β)
B C
180 °
Results
78
protein core, resembles the NifD2K2 arrangement of the MoFe protein and aligns to it with a
root-mean-squared deviation of 1.97 Å for all atoms (see Figure 28). Both α- and β-subunits con-
sist of three domains and each domain shows a Rossman fold motif. Whereas in NifK ≈ 50 resi-
dues of the N-terminus wrap around NifD, stabilizing the NifDK dimer, these residues are miss-
ing in VnfK (see Figure 28). Instead, an additional β-strand extends the parallel β-sheet of the
third domain. The C-terminus (≈ 15 residues) of the α-subunit is flexible with no secondary
structural element and lies in a cleft between the α- and β’-subunit at the protein surface (see Fig-
ure 27 A and Figure 28 A). Although it is quite far away from the Fe-protein binding site, it might
have a specific role in VnfH recognition, or for another protein that is involved in cofactor as-
sembly.
Figure 28: Subunits VnfD (A), VnfK (B), VnfG (C) and comparison of overall architecture of VFe- and MoFe-
protein (D, E). A, B, C) The single subunits VnfD, VnfK, VnfG are coloured from the C-terminus in red to the N-
terminus in blue; the subunits positions and orientations in the VFe-protein are visualized as surface representation
in the small VnfD2K2G2 illustration beneath; the flexible C-terminus of VnfD without secondary structural elements
is visuable in A; the FeMo-co is situated between the three domains and the P-cluster is located at the interface of
VnfD and VnfK; the Mg binding site (neon green) between VnfG and VnfD can bee seen in C. D, E) Structural
similarity between NifD2K2 and VnfD2K2 is shown in the protein structure alignment in D; MoFe-protein subunits
NifD and NifD’ are shown in dark grey, NifK and NifK’ are shown in light grey; the N-terminus of NifK wrapping
around NifD which is missing in VnfK is highlighted in E.
VnfD
A B C
VnfK VnfG
180 °
D E
N-terminus of NifK
Results
79
The P-cluster and the FeV-cofactor keep their positions as in NifDK. The P-cluster is situated at
the α- and β-subunit interface of VnfDK and the FeV-cofactor is buried within the α-subunit of
VnfD (see Figure 27).
There are two additional metal sites within a VnfDKG trimer. One is located between the two β
subunits of both trimers, as in MoFe-protein, where it is assigned to either a Ca2+ or a Fe2+/3+,
depending on the given condition [113], [119]. In the VFe-protein, coordination geometry and
bond distances evidence it to be a Mg2+ (see Figure 29) All given distances are averaged and listed
in section 6.5 in Table 20 to Table 22. The coordination environment, consisting of each one
carboxyl-O of β’-Glu70 and β-Asp314 from both VnfK subunits and complemented by four
water molecules, indicates a similar role in interface stabilization of the catalytic dimer through
metal ionic interactions in VFe-protein.
Figure 29: Mg binding site between VnfK and VnfK’. VnfK and VnfK’ are shown in dark and light red, VnfD’ is
shown in light green. Mg (neon green) is coordinated by residues β-Asp314 and β’-Glu70 and the octahedral coordi-
nation environment is completed by four water molecules (red). Residues β-Asp318, α’-Lys413 and α’-Lys414 are in
the second coordination sphere. The 2Fo-Fc electron density map is contoured at 2 σ level and coloured in grey.
Another metal site is located between the α- and δ-subunits (see Figure 28 C). Similar coordina-
tion geometry and bond distances evidence this metal also to be a Mg2+. The first octahedral co-
ordination shell consists of six water molecules, whereas δ-Glu14 and δ-Glu15 and backbone
carbonyl-O of δ-Ala11 from VnfG and protein backbone carbonyl-O of α-Ala373 from VnfD
are in the second coordination sphere. This metal site might serve for stabilization of VnfD and
VnfG, although without direct protein ion ligation.
β‘-Glu70
β-Asp318 α‘-Lys413
α‘-Lys414
β-Asp314
1.99 Å
2.07 Å
Results
80
The additional δ-subunit is a globular 113 residue peptide chain and forms four α-helices (see
Figure 28). VnfG only interacts with the α-subunit VnfD, but shows no contact to the β-subunit
VnfK. The mechanistic relevance of the VnfG subunit remains unclear.
4.4.2.2 Structure of the P-cluster
The P-cluster of A. vinelandii VFe-protein is analogous to the one in MoFe-protein, retaining its
unique [8Fe-7S] cluster composition and shape (see Figure 30 A). The P-cluster is coordinated by
the conserved cluster ligand residues α-Cys49, α-Cys75 and α-Cys138 from VnfD, as well as β-
Cys31, β-Cys56 and β-Cys115 from VnfK and is in-line with the pseudosymmetry axis that re-
lates the α- and β-subunits.
Figure 30: Structure of P-cluster of V-N2ase. A) The [8Fe-7S] P-cluster of V-N2ase in the reduced PN-state. Residues
α-Cys49, α-Cys75, α-Cys138 are from VnfD (medium green) and residues β-Cys31, β-Cys56, β-Cys115 and β-Ser153
are from VnfK (dark red). B) The 2Fo-Fc electron density map is contoured at 5 σ level (grey mesh). The omit (Fo-Fc)
electron density map contoured at 6 σ level (green mesh) indicates a low proportion of the second conformation of
the P-cluster where Fe6-S1 bond is broken and Fe6 ligation by the harder β-Ser153 ligand occurs. Contrary to the
oxidized P2+state in MoFe-protein no change at Fe5 position is visible. Movement of only one Fe atom indicates this
state to be the spectroscopically observed one electron oxidized P1+ state.
The P-cluster under 2.5 mM DT-reduced conditions is present in a mixed redox state. One con-
formation resembles the PN-state in DT-reduced MoFe-protein, with two coupled [4Fe-4S] clus-
ters sharing sulfur S1. In the second conformation, the Fe6-S1 bond breaks up and formation of
a new bond between Fe6 and the harder hydroxy ligand of β-Ser153 occurs, as evidenced by a
positive omit (Fo-Fc) electron density peak at 6 σ level (see Figure 30 B). The exact occupancies of
both states are unknown, but roughly 60 – 80 % PN-state are present. Such a conformation has
β-Ser153
β-Cys115β-Cys31
β-Cys56α-Cys75
α-Cys138
α-Cys49
S1
Fe6Fe5
A B
PN/P1+
Results
81
not been observed before. Compared to the known two-electron oxidized P2+ state, only the Fe6-
S1 bond is broken, whereas the Fe5-S1 bond is retained (see Figure 5 in section 1.3.4.1). In EPR
spectroscopy of MoFe- and VFe-protein, the one electron oxidized P-cluster is denoted as P1+
[122], [123], [206] suggesting that this new state with just one Fe moved, represents the structure
of the one-electron oxidized P-cluster in the P1+ state.
4.4.2.3 Structure of the FeV-cofactor
The FeV-co, like its Mo-analogue, is situated within the α-subunit, in a cleft between the three
domains (see Figure 28 A). The cofactor is held in position by the two protein ligands α-Cys257,
ligating Fe1, and α-His423, ligating V1, at opposite ends. The octahedral V coordination envi-
ronment of three sulfides and the amine-N is complemented by two oxygen moieties from the
bidentate ligand homocitrate, identical to FeMo-co (see Figure 31).
Figure 31: Structure of the FeV-cofactor of V-N2ase. A) The [7S-8Fe-V-CO3-C]-homocitrate FeV-cofactor of the
‘hiDT’ VFe-protein. The FeV-co is ligated by α-Cys257 and α-His423 (medium green). V (grey) in FeV-co shows an
identical coordination sphere as Mo in FeMo-co. The carbonate is ligating Fe4 and Fe5 in a μ-bridging mode replac-
ing the sulfide in 3A position. Fe, sulfur and carbon atoms are labeled and coloured in dark orange, yellow and me-
dium green, respectively. B) The 2Fo-Fc electron density map is contoured at 3 σ level (grey mesh). It shows the inter-
stitial carbon and the μ-symmetry as well as the equidistant bond lengths of the carbonate. C) CO32- is slightly kinked
out of the C3 symmetry plane. D) The CO32- ligand shows almost equidistant bondlengths with one slightly shorter.
The Fe4/5-O distances match Fe-O bond lengths. The Fe4-Fe5 distance is elongated (2.61 Å to 2.77 Å) while the
Fe1-Fe4 distance is shortened (2.65 Å to 2.58 Å) compared to FeMo-co.
α-Cys257
α-His423
Fe4
Fe5
Fe1
S2A
S5A
S2B
S3B
S4A
S1A
S4B
S1BV1
Fe3
Fe2
Fe7Fe6
CO3
C1
homocitrate
A B
S2B
S5A
CO3
C
Fe4
Fe5
1.27 Å1.31 Å
1.30 Å
1.95 Å
1.94 Å
2.77 Å
Fe1
2.58 Å
D
Results
82
The exchange of Mo to V results only in slight differences in the coordination sphere. The bond
lengths of V to the adjacent sulfides S1B, S3B and S4B are almost equidistant with 2.34 Å, 2.35 Å
and 2.33 Å compared to Mo distances with 2.35 Å, 2.37 Å and 2.35 Å. The V to Fe distances for
Fe5, Fe6 and Fe7 within the [V-3Fe-4S] subpart are 2.70 Å, 2.76 Å and 2.74 Å, with an average of
2.73 Å slightly longer than in FeMo-co with 2.73 Å, 2.67 Å and 2.68 Å with an average of 2.69 Å
[116]. This confirms the similar Mo and V coordination chemistry in protein cofactors and inor-
ganic cluster models from recent EXAFS studies [322]. The electron density at this resolution
defines the interstitial carbon (see Figure 31 B), which was evidenced to also be present in VFe-
cofactor recently [323].
An unexpected but major difference concerns the cluster composition and the three bridging belt
atoms. Whereas in FeMo-co sulfides S2B, S3A, S5A ligate the prismatic Fe atoms, a C3-
symmetric tetraatomic molecule is present at the S3A position of FeV-co, ligating Fe4 and Fe5 in
a μ-bridging mode (see Figure 31).
The electron density for this ligand is well defined at 1.35 Å resolution, with almost equal bond
distances of 1.27 Å to 1.31 Å and matches a carbonate (CO32-) and the isoelectronic nitrate
(NO3-) (see Figure 31 B, D). Other possible ligands might be a bicarbonate (HCO3
-), an acetate
(H3C-COO-) and a carbamate (H2N-COO-). As the protein was isolated from a diazotrophically
grown culture with active N2ase producing bacteria, the presence of NO3- as N source in growing
condition would inhibit N2ase expression. Nitrate is thus considered unreasonable and can be
excluded. In acetate and carbamate molecules, the single bonds are longer than the mesomerically
stabilized carboxy C-O bonds, which is not observed in the electron density of the present ligand.
The C-O bond that does not coordinate Fe is even slightly shorter with 1.27 Å than the Fe4- and
Fe5-ligating C-O bonds with 1.31 Å and 1.30 Å (see Figure 31 D). This rather indicates a me-
someric form of carbonate with a little shorter C=O double bond and two C−O single bonds
with the negative charges located on the Fe-coordinating O-atoms. While bicarbonate carries just
one negative charge, a carbonate ion is two-fold negatively charged, like the exchanged S2-. For
these reasons, the molecule is tentatively assigned as carbonate. The Fe-to-ligand bond distances
are 1.95 Å (Fe4 ) and 1.94 Å (Fe5), matching typical Fe-O bond lengths [324] (see Figure 31 D).
The ligand exchange affects the Fe4-Fe5 distance by an elongation from 2.61 Å to 2.77 Å, and in
turn leads to a shortening of the Fe1-Fe4 distance from 2.65 Å to 2.58 Å. The ligand is slightly
kinked out of the cluster C3 symmetry plane (see Figure 31 C).
The ligand is situated in a pocket surrounded by a flexible loop, consisting of the polypeptide
sequence 335T–G–G–P–R–L340. The carbonate oxygen, opposing the Fe atoms, interacts with the
Results
83
protein by formation of three H-bonds (2.8 – 2.9 Å), two H-bonds with backbone amide-N from
α-Gly337 and α-Arg339 and one H-bond with the hydroxyl group of α-Thr335 (see Figure 32 A).
This constellation precludes acetate and also contradicts carbamate as a ligand, as α-Gly337 and
α-Arg339 can only act as H-bond donors. An additional fourth H-bond is created by a backbone
amide-N of α-Gly336 to the carbonate-O coordinating Fe5 (≈ 2.9 Å). Thereby the ligand is tight-
ly embedded in this position. In comparison, in FeMo-co four protein backbone amide-N from
residues α-Gly356, α-Gly357, α-Leu358 and α-Arg359 coordinate sulfide S3A by weak H-bonds
of 3.4 Å to 3.7 Å and thus also strengthen its position. The correlating polypeptide sequence in
NifDK is 355I–G–G–L–R–P360. Both loops are similar in location and conformation but residues
leucin and proline are exchanged. This results in a decrease in pocket size. A theoretical ligand
exchange in FeMo-co of sulfide to the bigger carbonate might lead to clashes between α-Pro340,
carbonate and thereby probably excluding carbonate as ligand in FeMo-co (see Figure 32 B).
Figure 32: Protein environment of the carbonate ligand. A) H-bond stabilization of the CO32- by environmental
protein residues α-Thr335, α-Gly336, α-Gly337 and α-Arg339 in VFe-protein (medium green). B) Protein environ-
mental residues in VFe- (green) and MoFe-protein (grey) and conformation of the corresponding petide sequence.
The exchange of the residues proline and leucine leads to a smaller pocket for the ligand in 3A position and might
result in a clash between carbonate and α-Pro360 in MoFe-protein.
These distinct differences in ligand stabilization between FeV-co and FeMo-co reduce the proba-
bility of an artificial presence of this ligand and reinforce the putative CO32- as a native ligand
A B
α-Thr335α-Gly336
α-Gly337
α-Arg339
VFe: 335T-G-G-P-R-L340
MoFe: 355I-G-G-L-R-P360
Results
84
with a mechanistically relevant function. The FeV-cofactor of A. vinelandii VFe-protein thus likely
consists of a [7Fe-8S-V-CO3-C]-homocitrate cluster.
4.4.3 The structure of ‘loDT’ VFe-protein (‘active’ state)
4.4.3.1 Overall structural organization and P-cluster
The overall structure of the A. vinelandii VFe-protein form purified at a low DT concentration
(see section 3.3.1), determined at a resolution of 1.20 Å is congruent with the structure of ‘hiDT’
VFe-protein, beside 12 missing residues at the flexible C-terminus of the α-subunit. Instead of a
flexible arm, there is an additional metal site interacting with VnfD and VnfK’ in ‘loDT’ VFe-
protein. Residues α-His448 and β’-His379 and two water molecules form the tetrahedral metal
coordination sphere (see Figure 33). The finding of thermal protein stabilization and successful
protein crystallization only by addition of Zn2+ as an additive (see section 4.3.4) and the coordina-
tion geometry and bond distances, matching Zn-O and Zn-N bond lengths [324] (see Table 22),
allow for the assignment of this metal side as a Zn2+. The metal site and His-residues in this al-
ternative conformation are approximately just half occupied.
Figure 33: Zn binding site in ‘loDT’ VFe-protein. VnfD and VnfK’ of ‘hiDT’ VFe-protein are coloured medium
green and light red, respectively. VnfD and VnfK’ of ‘loDT’ VFe protein are coloured dark green and beige, respec-
tively. In the hi’DT’ VFe-protein the floppy C-terminus of VnfD is located in the cleft between VnfD and VnfK’.
with residues α-His448 and β-His379 pointing away. In ‘loDT’ VFe protein the VnfD C-terminus is absent and α-
His448 and β’-His379 are ligating a Zn ion (grey). The tetrahedral coordination sphere is completed by two water
molecules (red). The 2Fo-Fc electron density map is contoured at 1 σ level (grey mesh).
VnfD C-terminus ‚hiDT‘ VFe
α-His448
‚hiDT‘ VFe
‚loDT‘ VFe
β‘-His379
‚hiDT‘ VFe
‚loDT‘ VFe
Results
85
The P-cluster shows no structural changes compared to the ‘hiDT’ VFe-protein form and is also
present in a mixed redox state of PN and P+1, with similar occupancies as in the ‘hiDT’ VFe-
protein.
4.4.3.2 The FeV-cofactor in the ‘active’ state
Three main structural and conformational changes can be observed at the substrate binding site.
One major change is the replacement of another belt sulfur atom (S2B) that links Fe2 and Fe6 by
a third ligand that coordinates both Fe in μ2-bridging mode, retaining the tetrahedral Fe coordi-
nation sphere (see Figure 34 A, B). While the displacement of S3A to carbonate resulted in a Fe4-
Fe5 distance elongation, Fe2 and Fe6 stay equidistant with 2.65 Å compared to 2.62 Å in the
‘hiDT’ FeV-co form. Anomalous difference Fourier maps were calculated with diffraction data
measured at 7100 eV, at an energy below the Fe K-edge, to enhance the anomalous scattering of
S relative to Fe. At 4.5 σ level no anomalous electron density is present at the N-ligand position,
as well as for the carbonate, but for S5A (see Figure 34 C, D). The absence of anomalous elec-
tron density at the former S2B position evidences the displacement of the sulfide. The ligand-Fe
bond length is decreased to 2.02 Å (N2B-Fe2) and 2.00 Å (N2B-Fe6) in comparison to previous
distance of 2.22 Å and 2.20 Å for S2B. A complete ligand exchange did not occur, as a positive
omit (Fo-Fc) electron density peak in the S2B position indicates the presence of a small proportion
of sulfide (see Figure 34 B). Like the ‘hiDT’ form, this ‘loDT’ form of VFe-protein was isolated
from a diazotrophically grown culture of A. vinelandii under aerobic conditions thus producing
active N2 fixing N2ase. Due to the vast abundance of native substrate N2, with 78 % in air com-
pared to a negligible concentration of the artificial substrate CO as trace gas with only 50 –
200 ppb, an intermediate of CO reduction as new ligand is very unlikely and rather an N-
intermediate from N2 reduction to NH3 can be assumed. It cannot be a N-N bond containing
ligand because the electron density defines a mono-atomic species. Therefore, possible candidates
are N3-, NH2-, NH2- and NH3. Ammonia can be excluded, as the molecule shows a tetrahedral
coordination with its three H-atoms and one free electron pair, which cannot ligate two Fe atoms
in a μ2 bridging fashion. A similar ligand exchange has been observed for the CO-bound MoFe
protein with a CO displacing sulfide S2B and coordinating Fe2 and Fe6 by carbon [140]. But
different from a likely N-species of native substrate N2 in VFe, in MoFe protein this state was
trapped by addition of the inhibitor CO under turnover conditions.
Results
86
Figure 34: Structure of the FeV-cofactor of ‘loDT‘ VFe-protein. A) The FeV-cofactor of ‘loDT’ VFe-protein (dark
green). The FeV-cofactor is ligated by α-Cys257 and α-His423. S2B sulfide is replaced by a nitrogen species (N2B)
(blue) coordinating Fe2 and Fe6. B, C) The 2Fo-Fc electron density map is contoured at 3 σ level (grey mesh). The
omit (Fo-Fc) electron density map contoured at 5 σ level (green mesh) indicates a low proportion of remaining sulfide
in S2B position. D) The anomalous electron density map contoured at 4.5 σ level (orange mesh) shows electron
density for the S5A but no density at positions of N2B and carbonate ligands evidencing the replacement to light
atoms.
4.4.3.3 The active site
Two other major changes concern the cofactor environment. At the cofactor side of the new
third ligand, residue α-Gln176, which is normally kinked downwards for H-bonding between
glutamine-N and carboxyl-O from homocitrate (see Figure 37 A), has undergone a conforma-
tional change and is coordinating the third ligand (see Figure 35 A and Figure 37 B). This rear-
rangement entails the formation of new H-bonds. While the glutamine carbamoyl-N still acts as
H-bond donor with the carboxyl-O from homocitrate and is also ligating a water molecule, the
glutamine carbamoyl-O acts as H-bond acceptor with α-His180 amine-N at 2.84 Å, and can con-
sequently also only act as acceptor with the N-intermediate ligand at 2.48 Å. Homocitrate is not
fully triply deprotonated at pH 7.5. But the carboxyl-O of homocitrate, that is H-bonding to α-
Gln176, is also H-bonding to the positively charged α-Lys361 (not shown), indicating this car-
boxyl-O to be deprotonated and thus confirming its role as H-bond acceptor. This glutamine side
chain conformation is also supported by the electron density at 1.2 Å resolution. In a 2Fo-Fc elec-
tron density map at 4 σ level there is electron density at the position coordinating the N-species
residual
‚S2B‘
A B C
D
Fe2
Fe6
S5AN2B
CO32.02 Å
2.00 Å
α-Cys257
α-His423
Results
87
and no more e-density at the position coordinating homocitrate. This evidences the electron-
richer oxygen at the position with remaining electron density and acting as acceptor in H-bond
formation with the N-species (see Figure 35 B).
Figure 35: The active site of ‘loDT’ VFe-protein. A) Coordination and H-bond network upon the conformational
rearrangement of α-Gln176 in ‘loDT’ VFe-protein (dark green). The α-Gln176 coordinates the both cofactor N-
ligand and α-His180 as H-bond acceptor via the side chain-O. It retains the donor H-bonding to homocitrate by side
chain-N. The H2S/HS- species is coordinated by backbone-N from α-Gln176, α-Gly48 and from one water molecule
(red). B) Remaining 2Fo-Fc electron density contoured at 4 σ level (grey mesh) at the side chain glutamine position
coordinating N2B indicates and strengthens the electron richer oxygen instead of the N in this position and therby
acting as H-bond acceptor.
Geometrically regarded, the carbamoyl-O is located in the plane created by Fe2, Fe6 and the lig-
and and is located close to the μ2 ligand axis with only 13° deviation, therefore creating an ap-
proximately three-fold symmetry for the N-intermediate (see Figure 36). In NH2-, the two free
electron pairs of N would ligate Fe2 and Fe6 and the two H-atoms would be in corresponding
tetrahedral coordination contradicting the observed threefold symmetry of the ligand with an H-
bond to glutamine-O. Therefore this geometrical restraint further excludes NH2- as ligand. A μ2-
nitrido coordination cannot be excluded. But it would belong to a three-fold negatively charged
N3- thus very prone to protonation to NH2- and is therefore very unlikely. Hence, the most likely
candidate is the NH2- -species. It ligates Fe2 and Fe6 with two free electron pairs, acts as H-bond
donor to carbamoyl-O, thus showing pseudo-three-fold symmetry, and is also two-fold negatively
charged, like the displaced sulfide.
α-Gly48
α-Gln176
α-His180
H2S/HS-
3.08 Å
3.23 Å
2.86 Å
2.41 Å
2.90 Å
2.48 Å
2.84 Å
A B
Results
88
Figure 36: The coordination geometry of the N-ligand. The 2Fo-Fc electron density map is contoured at 4 σ level
(grey mesh). The three atom positions of Fe4, Fe5 and N2B span the Fe-N-Fe plane (grey, left and right). The glu-
tamine-O position is not exactly in-line with the μ2 symmetry axis of N2B (dashed line) but deviates with only 13°
thereby creating an ≈ three-fold symmetry for N2B (black lines). A 90° rotation (right) shows that glutamine-O is
located exactly in the Fe-N-Fe plane indicating the planar ≈ three-fold symmetry of N2B and contradicting a tetra-
hedral coordination.
The third major change is directly related to the glutamine relocation. By this residue rearrange-
ment, some space is created at the position of the former glutamine conformation, where two
new molecules are present. While one is the aforementioned water, coordinating the glutamine
side chain N, the other one is identified as a sulfur-containing molecule. While anomalous differ-
ence Fourier maps in ‘hiDT’ FeV-co evidence the S2B sulfide, in ‘loDT’ FeV-co they evidence
the disappearance of S2B sulfide, but the occurrence of a sulfur-containing molecule in the for-
mer glutamine position, likely representing the displaced S2B sulfide (see Figure 37). The sulfur
species is stabilized by three H-bonds, two with peptide backbone N from α-Gln176 and α-Gly48
at 3.1 – 3.2 Å and one with the adjacent new water at 2.9 Å (see Figure 35 A). There are only
from one side three neutral ligands, that stabilize by H-bonding, but no positively charged lig-
ands, that could stabilize by ionic interactions. Consequently, a two-fold-negatively charged S2-
can be precluded. Rather, a hydrosulfide ion (HS-) or hydrogen sulfide (H2S) can be expected,
that both are present and stable at pH = 7.5 [325] but cannot be distinguished so far. The dis-
tance of the putative HS-/H2S to the new FeMo-co N2B site is 7.0 Å.
13°
≈ three-fold
symmetry
Fe-N-Fe
plane
Fe-N-Fe
plane
90°
Results
89
Figure 37: Active site of ‘hiDT’ ‘resting’ and ‘loDT’ ‘active’ state VFe-protein. The 2Fo-Fc electron density map is
contoured at 1 σ level (grey mesh) and the anomalous electron density map is contoured at 4.5 σ level (orange mesh).
A) N2ase active site of ‘hiDT’ ‘resting’ state VFe-protein with α-Gln176 in the normal position kinked downwards
only H-bonding with homocitrate and with sulfide in S2B position of FeV-co as evidenced by anomalous electron
density. B) N2ase active site of ‘loDT’ ‘active’ state VFe-protein with α-Gln176 conformational rearrangement coor-
dinating cofactor N2B ligand and α-His180 and with H2S/HS- in the likely sulfur storage position ≈ 7 Å distant to
N2B as evidenced by anomalous electron density.
A potential sulfur storage position for the sulfide in the CO-bound MoFe protein was proposed
≈ 22 Å away from the S2B site, where it is too far for a mechanistic relevance during catalysis
[140]. In contrast, a distance of 7.0 Å might be short enough to play a mechanistic role as tempo-
rary sulfur storage position during catalysis. Especially as this position seems to be connected
with a conformational rearrangement of the conserved α-Gln176 that is consequently coordinat-
ing an N-intermediate at the former S2B position and thus might be involved or even mediate
the ligand exchange.
A major difference between the CO-inhibited MoFe-protein and this structure is, that the corre-
sponding α-Gln191 side chain in MoFe rests in the original position, like for α-Gln176 in the
‘hiDT’ VFe, while in this structure of ‘loDT’ VFe protein the glutamine has moved and is ligating
a N-species of the modified VFe-cofactor. The reason for keeping the position in the CO-MoFe
might be two-fold. One could be the size of the bigger ligand CO compared to the putative NH2-
and thus both oxygens, from carbon monoxide and the glutamine side chain, would clash. And
secondly, the nature the oxygen atoms themselves, as both can only act as H-bond acceptor and
thus not gain a stabilization effect by H-bonding.
A B
‚hiDT‘ resting VFe-protein
α-His180
α-Gln176
S2B
‚loDT‘ active VFe-protein
7.0 Å
H2S/HS-
α-His180
N2Bα-Gln176
Results
90
With a N-intermedate bound to Fe2 and Fe6, the α-Gln176 and α-His180 in this conformation
and the presence of the nearby sulfur storage evidences this position to be the substrate binding
site at the FeV-cofactor. Furthermore, this new conformation might represent an actual interme-
diate active site state during N2 reduction. Therefore the ‘loDT’ VFe-protein form is referred to
as an ‘active’ state and the ‘hiDT’ VFe is referred to as the ‘resting’ state in the following discus-
sion.
4.5 Characterization of VFe-protein
4.5.1 Liquid chromatography mass spectrometry
Liquid Chromatography mass spectrometry (LC-MS) (see section 3.4.3) was used for unambigu-
ous identification of VFe-protein (VnfDKG) after separation by SDS-PAGE..
The protein bands in gel slices were separately digested with trypsin and extracted. The digested
peptide sequences were separated via liquid chromatography and their mass analyzed by a first
mass spectrometry run. After fragmentation of the peptide sequence, the fragmented peptides
were analyzed and identified by the second mass spectrometry. Peptide sequences and subse-
quent protein identification is performed via a protein database alignment of the identified frag-
ments and the known peptide mass with theoretical protein digestion patterns of proteins based
on their protein sequence.
The presence of VFe-protein in the extracted bands of the gel slices was verified by identification
of VnfD (474 AA; 53843 Da), VnfK (475 AA; 52742 Da) and VnfG (113 AA; 13364 Da) with a
protein sequence coverage of 41 %, 47 % and 81 %., respectively.
4.5.2 Nitrogenase acetylene reduction assay
An acetylene reduction activity of 1003 ± 93 nmol C2H2 min-1 mg-1 for NifDK with NifH and
454 ± 21 nmol C2H2 min-1 mg-1 for VnfDKG with VnfH was obtained. This decreased C2H2 re-
duction activity compared to Mo-N2ase of 45 % is higher than the reported 28 % for V-N2ase.
An activity of 454 nmol C2H2 min-1 mg-1 corresponds to 1080 nmol C2H2 min-1 nmol-1 and is 8x
higher than the reported 136 nmol C2H2 min-1 nmol-1 [193]. Furthermore, a specific activity for
ethane formation was obtained with 4.93 ± 0.64 nmol C2H2 min-1 mg-1. This 1 % C2H6 formation
Results
91
is lower than the earlier observed 3 % [193] or 5 % [198], [199]. These activity data were kindly
provided by Michael Rohde and obtained during his Master thesis.
4.5.3 CW-EPR spectroscopy
The EPR spectrum of VFe-protein in the as-isolated state is more complex and weaker in signal
strength than the spectrum of the corresponding MoFe-protein. While the MoFe-protein in the
as-isolated state (0.2-2 mM DT) has just a single, apparent S = 3/2 spin system with a rhombic
EPR signal at g = 4.35, 3.65 and 2.01 (see Figure 38 C), in the VFe-protein apparently several
paramagnetic species are present with a signal magnitude of only 1/10 of that of MoFe-protein.
The two purification methods result in two different forms of the VFe-protein (see section 3.3.2
for ‘hiDT’/’resting’ state and 3.3.1 for ‘loDT’/‘active’ state VFe-protein). At first, the ‘resting’
state VFe protein (see Figure 38 B, red spectra) and secondly the ‘active’ state VFe-protein (see
Figure 38 A, green spectra) will be explained.
4.5.3.1 ‘hiDT’/’resting’ state VFe-Protein
‘hiDT’ + as-isol: The shape of the as-isolated ‘resting‘ state VFe-protein (see Figure 38 B, dark
red) is similar to the known VFe-proteins from A. chroococcum [194] and from A. vinelandii [326],
[193]. The spectrum is dominated by an axial S = ½ ground state spin system [327], [186] with
signals at g| = 2.04 and g⊥ = 1.93. In low field, signals at g = 5.74, 5.40, 4.35 and 3.82 are associ-
ated with an S = 3/2 spin system. The combined feature at g = 4.35 and 3.82 shows a different
temperature and redox behavior than the signals at g = 5.74 and 5.40 indicating that these signals
arise from a mixture of S = 3/2 spin systems [206], [193]. The signals at g = 5.74 and 5.40 itself
show shape changes depending on different temperatures, indicating to arise from ground and
excited state doublets [327]. And furthermore, in contrast to chemical reduction, enzymatic re-
duction results in a change in rhombicity observed by a shift of the g-factors [206]. The spectrum
is complemented by a very weak signal at g = 6.64, that is assigned to an inverted S = 5/2 spin
system from an excited state, as determined from temperature variation studies [206], [193].
S = 3/2: The signals of the S = 3/2 spin systems at g = 5.74, 5.40, 4.35 and 3.82 have been as-
signed to the FeV-cofactor in analogy to the S = 3/2 spin system of FeMo-cofactor in MoFe-
protein [194], [327]. These different S = 3/2 spin systems are presumably associated with differ-
ent environments of the FeV-cofactor [186]. Interestingly, while in VFe-protein from Hodgson,
Hedman and Ribbe the signal at g = 5.74 and 5.40 is just a single feature at g = 5.5 [193], the
Results
92
VFe-protein of Hales and coworkers miss the inflection at g = 3.82, but shows a broad peak at
g ≈ 4.3, which might be just misinterpreted [326], [206].
Figure 38: X-band perpendicular mode CW-EPR spectra of ‘lo-DT’, ‘hiDT’ VFe-protein and MoFe-protein. A)
Spectra of ‘loDT’ ‘active’ state VFe-protein as isolated (dark green) and reduced (light green). B) Spectra of ‘hiDT’
‘resting’ state VFe-protein as isolated (dark red) and reduced (light red). A, B) Signals at g = 1.93, 2.04 are assigned to
a S = ½ spin system, signals at g ≈ 3.82 – 5.74 to at least two S = 3/2 spin systems, the signal at g = 6.64 to a
S = 5/2 spin system and the signals at g =1.83, 1.97, 2.02 are unassigned yet. The g values are shown. C) EPR spec-
trum of reduced MoFe-protein (black) with the typical rhombic S = 3/2 signal. The g-values are shown.
S = ½, 5/2: The origin of the S = ½ and S = 5/2 spin systems are still under debate. In oxida-
tive titrations with the MoFe protein, the S = ½ signal at g = 2.06, 1.95 and S = 5/2 signal at
g = 6.67 were associated singly oxidized P-cluster (P1+) [123]. The presence of analogous signals,
although under DT-reduced conditions, gives rise to that in VFe-protein both signals origin from
P1+ as well [206] and suggests that the P-cluster in the VFe-protein may exist in a more oxidized
state than in the MoFe-protein. Assigning the S = ½ signal to the P-cluster is supported by EPR
experiments with a FeV-co deficient but P-cluster containing VFe-protein form that also shows
such a S = ½ signal at g = 2.08 and 1.89 [208]. Mössbauer spectroscopy indicated that under the
same reducing as-isolated conditions in VFe-protein, a mixture of oxidized and reduced P-cluster
and FeV-cofactor is present [204], whereas in the MoFe-protein both clusters are reduced, with
the P-cluster in the fully reduced all-ferrous state being diamagnetic with an S = 0 spin state
500 1000 1500 2000 2500 3000 3500 4000 4500
x 1/10
2.01
3.65
4.35
Field [G]
MoFe
6.64
loDT/active:
- as-isol.
- reduced
1.972.02
1.834.09
4.24 1.932.04
3.82
4.35
5.405.74
x5
hiDT/resting:
- as-isol.
- reduced
C
B
A
x5
x5
Results
93
[328]. In case the S = ½ and S = 5/2 spin systems can be assigned to the P1+ state, the Mössbau-
er results support the finding that the signals from these two spin states are present in the as-
isolated VFe-protein. They even cannot be removed with 10 mM DT and/or addition of methyl
viologen and benzyl viologen. Only enzymatic reduction via MgATP and Fe-protein led to full
removal of both S = 1/2 and S = 5/2 signals without change of the FeMo-co S = 3/2 signals,
also in line with the hypothesis that enzymatic reduction by Fe-protein reduces P-clusters [206].
‘hiDT’ + red: Upon further reduction to 20 mM DT (see Figure 38 B, light red), the axial S = ½
signal at g = 2.04 and 1.93 increases, whereas the S = 3/2 signals at g = 4.35, 3.82 and g = 5.74,
5.40 equally decrease in magnitude. The very weak S = 5/2 signal at g = 6.64 increases slightly.
Furthermore, two weak signals at g = 4.09 and 4.24 become more distinct.
4.5.3.2 ‘loDT’/’active’ state VFe-protein
‘loDT’ + as isol: The ‘active’ state as-isolated VFe-protein (see Figure 38 A, dark breen) has a
similar shape to the ‘resting’ state VFe-protein and has also the S = ½ EPR spin system, with
EPR signals at g = 2.04 and 1.93, the mixed S = 3/2 spin systems with signals at g = 5.74, 5.40,
4.35 and 3.82 as well as the S = 5/2 spin state at g = 6.64, although very weak. However, it shows
distinct differences. The S = ½ signal is drastically decreased in magnitude, not dominating the
spectrum anymore, but at a similar magnitude as the low-field signals. Additionally two further
signals within the g ≈ 2 region occur at g = 2.02 and 1.97, and another small signal at g = 1.83.
Furthermore, the shapes of the S = 3/2 signals are different. While in the ‘resting’ state VFe-
protein both S = 3/2 spin system EPR signals have a similar magnitude, as for the known VFe-
proteins [194], [206], [193], in the ‘active’ state as-isolated VFe-protein the g = 4.35 and 3.82 sig-
nals increase and have approximately three times the magnitude of the g = 5.74 and 5.40 signals.
‘loDT’ + red: Upon further reduction with 8 mM DT (see Figure 38 A, light green), the axial
S = ½ signal increases drastically, dominating again the spectrum and becoming as strong as in
the spectrum of the ‘resting’ state VFe-protein. The new features at g = 2.02 and 1.97, as well as
at g = 1.83 are not clearly distinguishable anymore. Therefore it is not known, whether they are a
feature of the as-isolated ‘active’ state VFe-protein or are more often present and are just not
visible as they are very small in magnitude and hidden within the dominant g = 2.04 and 1.93
ground state EPR signals. In contrast to the ‘resting’ state VFe-protein, the EPR signals for the
S = 3/2 spin systems increase equally upon reduction. The weak S = 5/2 EPR signal at g = 6.64
becomes more visible, like for the ‘resting’ state VFe-protein.
Results
94
4.6 Comparison of EPR spectra and structures of VFe-protein
The structure determination of V-N2ase in the ‘resting’ and the ‘active’ state enables - in combi-
nation with the corresponding EPR-spectra, which provide information about the cluster’s elec-
tronic structure - an approach to the question of the different substrate reactivity and the related
altered redox properties of MoFe- and VFe-protein and their clusters. To do so, a comparison of
EPR-spectra with both structures considering earlier results is necessary to gain an improved
interpretation of the EPR-spectra and to finally draw more proper conclusions from spectra and
structures to identify the actually present redox states. Afterwards, the interpreted observations
can be compared to standing hypotheses in order to analyse where indeed differences between
the clusters of Mo- and V-N2ase are present.
The EPR-spectra are taken under two conditions: ‘as isolated’ means working under O2 exclusion
and the presence of 0.2-2 mM DT, and thus in principle reducing conditions; ‘reduced’ means
that the as isolated protein was complemented with 8 or 20 mM DT (‘active’ or ‘resting’ state)
and thus the condition is more reduced than as isolated. For crystallization, directly prior to set-
ting up the experiment, the as isolated protein solution was complemented with additional 5 mM
DT and thus the protein was once more reduced. Hence, the crystallization condition of the pro-
tein structures rather resembles the ‘reduced’ conditions of the EPR-spectra.
S = ½ und 5/2: It is a fact that in both ‘resting’ and ‘active’ state structures the P-cluster exists in
a mixed redox state of PN and P1+ and thus the one-electron oxidized P1+ state is definitely pre-
sent (see Figure 30). Therefore corresponding signals also have to appear in the EPR-spectra. If
the S = ½ at g = 2.04 and 1.93 and the S = 5/2 at g = 6.64 can be assigned to the P1+, it matches
the ‘reduced’ EPR spectra as both signals are present. The S = 5/2 signal at g = 6.64 arises from
an excited state doublet and is thus temperature- and power-dependent [206], [193]. Maximum
signal amplitude was observed at 15 K and at 50 mW [193], whereas our spectra were measured
at 10 K and at 2 mW (see Figure 38 and section 3.6.1). This could explain a lower population of
the excited state, and hence a weaker signal in our spectra. As mentioned, the P1+ state, as evi-
denced by the structures, is present under the (more) reduced condition. Interestingly, the S = ½
signal (and the S = 5/2 signal slightly as well) increases upon further reduction in both forms (see
Figure 38). This means, that the P-cluster of VFe-protein in the as-isolated condition is partially
present in the P2+ state and will be reduced to P1+. Hence, theoretically, the characteristic P2+ in-
teger spin S = 3 signal at g ≈ 11.5 could be observed [206]. But this is not the case in both as-
isolated spectra. An explanation for this observation is that this signal, and in general integer spin
signals, are only observed in ‘parallel’ mode EPR [186], [316], while our spectra were measured in
Results
95
‘perpendicular’ mode EPR. In conclusion, 1) the presence of analogous S = ½ and S = 5/2 sig-
nals in MoFe-protein, which are assigned to the P1+ state, 2) the presence of the P1+ state in the
both structures and the observation of the S = ½ and 5/2 signals in EPR-spectra and 3) the FeV-
co deficient but P-cluster-containing VFe-protein, also showing the S = ½ signal [208], gives
strong evidence that the assignment of at least the S = ½ signal to the P1+ state of P-cluster is
correct.
S = 3/2 at g = 4-6): In the ‘resting’ state VFe-protein, the signals at g = 5.74, 5.40 and g = 4.35,
3.82 are similar in magnitude, whereas in the ‘active’ state the signals at g = 4.35, 3.82 are approx-
imately three times higher in magnitude (see Figure 38). This feature has not been observed be-
fore [206], [194], [193]. While there is no alteration at the P-cluster in the ‘active’ state, the sulfide
at the FeV-co in the S2B position is exchanged to an N-intermediate (see Figure 34). Therefore it
can be concluded that this signal change is related to the structural change in the FeV-co and the
S = 3/2 signal at g = 4.35, 3.82 can be assigned to FeV-co. This evidences that at least this
S = 3/2 signal belongs to the FeV-co but does not confirm that the signal at g = 5.74, 5.40 be-
long to the FeV-co as well.
double feature at g = 2 in ‘active’ state: Interestingly, for the as isolated ‘active’ state VFe-
protein a clear double feature at g ≈ 2 can be observed, next to the g = 2.04, 1.93 is a signal at
g = 2.02, 1.97 (see Figure 38). The FeV-co in the ‘active’ state structure shows additional Fo-Fc
(omit map) electron density in the former S2B position, indicating a mixed state with the pres-
ence of a small proportion of ‘resting’ state with present sulfide (see Figure 34). The source of
this signal is not known yet. One reason could be that the new signal arises from FeV-co and is
created due to the bound intermediate or maybe other intermediates that are not reflected in the
structure as they occur just in a minor concentration. Another source would be an inter-cluster
interaction. The FeV-co-deficient VFe-protein showed a S = ½ signal at g = 2.08 and 1.89 [208]
instead of the common values of g = 2.04 and 1.93. Hence, the presence or absence of FeV-co
seems to affect the P-cluster, indicating that an interaction between both clusters exists. Besides,
while in MoFe both clusters oxidize sequentially [329], in VFe both clusters are simultaneously
oxidized and a paramagnetic interaction between the oxidized P-cluster and FeV-co has been
proposed [206]. As evidenced by the different S = 3/2 signal proportions, the electronic struc-
tures of ‘active’ and ‘resting’ state differ as well. Therefore, two different electronic structures of
FeV-co could impact the P-cluster and result in an altered S = ½ signal through the proposed
cluster interaction. Or it has other still unknown sources and thus cannot be assigned.
Discussion
96
5. Discussion
5.1 Purification of VFe-protein in two states
The question arises why the VFe-protein can be purified in two different forms from normal
diazotrophically grown cultures that produce active N2ase. The common purification is the
‘loDT’ version (see section 3.3.1) and delivers the ‘active’ state form of VFe-protein. The ‘hiDT’
purification (see section 3.3.2) was performed only once and delivered the ‘resting’ state form.
The chemical differences are just the higher DT concentrations in lysis buffer (10 mM to 2 mM)
(see Table 4) and loading/elution buffer (5 mM to 2 mM) during cell lysis, performance of the
HiTrapQ HP, the first ResourceQ and storage overnight at 4°C in 5 mM DT containing loading
buffer. The following steps were performed with buffers containing only 2.5 mM DT. This indi-
cates that the DT concentration influences the conversion of ‘active’ to ‘resting’ state. The reduc-
tion potential of a solution with DT as reducing agent shows pH-, temperature- and concentra-
tion-dependence [330]. Temperature- and pH- dependence are not relevant, as these parameters
remain constant, but alteration of the DT concentration accordingly leads to a change in reduc-
tion potential of the solution. For example, the calculated midpoint potential for DT at pH 7 and
25°C is -660 mV in a solution with 10 nM DT, whereas the theoretical potential of a solution of
1 M DT was calculated to be only -386 mV [330]. While 10 nM to 1 M is a huge change of a
10.000-fold, a change of 2 mM to 10 mM is within the same order of magnitude and is not to be
expected to have such a huge influence on the reduction potential of the solution.
While DT in buffers is used to ensure anoxic conditions, it is also used as a reducing agent. EPR
probes of reduced ‘active’ state VFe-protein were prepared by adding 8 mM DT to the as isolated
VFe-protein sample. After an incubation of 10 - 30 min, the reduced sample was refrozen in liq-
uid dinitrogen. The EPR spectra show the reduction of the sample, while keeping the 3:1 signal
magnitude ratio of the g = 4.35, 3.82 to the g = 5.74, 5.40 signals, which was identified to be
characteristic for the ‘active’ state (see section 4.5.3.2). This means, that an incubation of
10 - 30 min at room temperature in 8 mM DT and subsequent freezing and storage in liquid dini-
trogen did result in a reduction but did not initiate a change from ‘active’ to ‘resting’ state.
On the other side, in thermofluor assays (see section 4.3.3) the impact of DT on VFe-protein
stability showed slight differences. While melting temperatures with 1 mM, 2 mM and 4 mM DT
were identical with 51°C, DT concentrations of 10 mM and 20 mM slightly differed, with melting
temperatures of 50°C and 48 °C, respectively (see Table 13).
Discussion
97
In experiments for FeMo-co maturation, an effect of varied DT concentrations has been recog-
nized as well. An increase of 2 mM to 20 mM DT leads to an optimized maturation of the
NifEN-associated precursor to a ‘FeMo-co’ which thereupon shows the same activity abilities as
isolated FeMo-co [180].
Although FeMo-co maturation and thermofluor assays are not directly comparable to the trans-
formation of FeV-co with bound intermediate to its ‘resting’ state, there appears to be a relation
or indication that the DT concentration even between 2 mM, 10 mM and 20 mM somehow has
an influence other than just serving as a electron donor. In conclusion, due to the concentration-
dependence of DT the effect is supposed to be related to the reduction potential of the protein
solution. Although in EPR probes no impact besides electron delivery was observed, a longer
incubation time and higher DT concentrations have to be investigated.
Maybe the reduction potential of the crude extract, where also Fe-protein and MgATP are still
present, has an influence on whether one or two further electron transfer steps between Fe-
protein and VFe-protein can take place, and therewith the ‘resting’ state with bound sulfide can
be recovered.
Interestingly, an electron transfer within VFe-protein between P-cluster and FeV-co has been
proposed to take place during oxidative redox titration of the pure protein [206]. Hence, depend-
ing on the oxidation state of both clusters and the amount of added reducing agent or the redox
potential of the solution, an electron transfer between P-cluster and FeV-co seems to be possible
without participation of any other protein.
Finally, there is no answer so far why at the current state the common ‘loDT’ purification results
in the ‘active’ state VFe-protein and why once at the ‘hiDT’ purification the ‘resting’ state VFe-
protein was received. Anyway, the ‘hiDT’ VFe-protein purification has to be repeated and
checked for reproducibility. But it seems, that for V-N2ase a) the reduction pathway and b) the
interaction between P-cluster and FeV-co is not as ‘static or restricted to the resting state’ as for
the MoFe-protein, because a) maybe by alteration of the reduction potential of the crude extract,
a conversion of at least the ‘active’ into the ‘resting’ state appears possible, and b) electron trans-
fer between P-cluster and FeV-co can be triggered, only by addition of reducing equivalents
[206]. These additional features favor V-N2ase as compared to Mo-N2ase as a more versatile
model for investigating catalytic intermediates and consequently the mechanism of N2-fixation by
N2ase.
Discussion
98
5.2 FeV-cofactor biosynthesis
The VFe-protein structure shows that the active site cofactors do not just differ in the metal
composition, that FeV-cofactor also has a CO32- in a belt ligand position, in contrast to a sulfide
present in the FeMo-cofactor of MoFe-protein. As there exists a whole assembly machinery for
the cofactor biosynthesis, the question arises how the altered FeV-cofactor is made. A compari-
son of the nif and vnf gene-cluster shows the absence of several genes necessary for cofactor bio-
synthesis. Mutational studies proposed that genes such as nifU, nifS, nifV, nifM, and nifB are also
required for diazotrophic growth via alternative N2ases [331], [97]. Transcriptional profiling under
N2-fixing conditions for either Mo-, V- or Fe-dependent growth, compared to fixed-N replete
growth, supports the usage of nif-gene products of the biosynthetic machinery by verifying a high
transcription level of genes nifU, nifS, nifV, nifM, nifB and iscA, nifW, nifZ, nifQ also for V- and Fe-
dependent N2 fixation [94].
The question arises at which stage of maturation and from which proteins the modification is
performed. The absence of NifB homologues for vnf indicates its shared usage and suggests a
common NifB-co precursor. Differentiation in cofactor assembly will thus occur afterwards. The
scaffold protein VnfEN itself seems not to perform the cofactor manipulation, as vnfEN deletion
strains maintained diazotrophic growth [332].
5.2.1 The putative metallocluster carrier protein VnfY
A vnfY modified strain has shown, that VnfY is required for optimal V-dependent diazotrophy
[333]. VnfY belongs to the family of nitrogenase cofactor binding proteins like NifX, NafY and
additionally NifY, the C-terminal domain of NifB and VnfX [334], [333]. While NifX binds
FeMo-co precursors (NifB-co or L-cluster; see Figure 8) and NafY is proposed to bind and
transfer FeMo-co to apo-NifDK, the role of NifY is probably similar to NafY [335], [336], but is
still under debate [87]. VnfX is proposed to carry an analogous V-containing precursor that ac-
cumulates on VnfX during FeV-co biosynthesis [337]. Upon addition of homocitrate, the newly
formed cluster can be transferred to apo-NifDK (apo-Mo-N2ase), which is then able to reduce
C2H2 [338]. A mutation in vnfY resulted in only 12 % of 49V radiolabel accumulating on V-N2ase,
and an according tenfold decrease in C2H2 reduction activity compared to wild-type [333].
Whereas mutations in nafY, nifY, nifX or vnfX have little or no effect on the formation of active
N2ase under metal-sufficient growth conditions [336], [339], [334], [332] the loss of VnfY func-
tion has a clear impact on the maturation of V-N2ase, which is reflected in a lower level of FeV-
co incorporation and consequently in lower N2ase activity. This suggests that VnfY is involved in
Discussion
99
biosynthesis or insertion of FeV-co into VFe-protein. Furthermore, in vnfDKG deletion strains it
has been shown that the vnfY mutation led to a decreased accumulation of 49V radiolabel on
VnfX. This indicates that VnfY participates in an earlier step of FeV-co maturation [333].
In addition, there are significant differences in gene transcription levels under N2-fixing Mo- or
V-dependent growth compared to fixed-N saturated growth. While under Mo-dependent growth,
genes nifX (2-fold) and nifY (8-fold) are lowly transcribed, under V-dependent growth, transcrip-
tion levels are high for vnfX (17-fold) and very high for vnfY (147-fold; highest transcription level
of vnf-genes). This implies that VnfX and certainly VnfY have a similar, but also different, more
significant role in FeV-co-biosynthesis, than NifX and NifY in FeMo-co assembly [94]. This sug-
gests that VnfY in FeV-co biosynthesis has a specific and distinctly different task than in FeMo-
co biosynthesis, such as possibly the insertion of V or the carbonate production and exchange of
the belt sulfide.
5.2.2 The unidentified hypothetical protein Avin_02580
Another protein of A. vinelandii which has an increased transcription level (34-fold) under V-
dependent N2-fixing conditions is the hypothetical protein Avin_02580. It is located between
vnfY (Avin_02570) and vnfK (Avin_02590) [94]. A protein sequence BLAST [340] of non-
redundant protein sequences yielded only 16 hits for bacteria that show a sequence identity be-
tween 100 % and 22 % (see in section 6.3 in Table 18).
High sequence identity of this protein is found within the three Azotobacter species (A. vinelandii.
100 %, A. chroococcum 90 %, A. beijerinckii 85 %). The vnf-gene clusters of A. vinelandii [60] and A.
chroococcum [341] exhibit an identical arrangement for the genes vnfYKGDFH, including the hypo-
thetical proteins (A. vinelandii: Avin_02580; A. chroococcum: Achr_RS01250) which are located at
the very same position embedded within the vnf genes (see Figure 39).
Eight hits for this protein are present in the N2-fixing and for alternative V-N2ase encoding (de-
fined by presence vnfD in genome) bacterial strains (Azospirillum brasilense, Methylocystis parvus,
Methylocystis bryophila, Phaeospirillum fulvum, Rhodomicrobium vannielii, Rhodopseudomonas palustris,
Tolumonas lignilytica, Desulfobacter curvatus), with a sequence identity between 47 % and 33 %, indi-
cating to be homologues. Five bacterial strains have a sequence identity of only 24 % to 21 %.
Interestingly, these bacteria were either not N2-fixers or, in case of Klebsiella pneumoniae, do not
have the genes for the alternative V-N2ase.
Discussion
100
Figure 39: The vnf-gene cluster of A. vinelandii and A. chroococcum. Both gene clusters contain the hypothetical protein
(Avin_02580 or Achr_RS01250) located at the same position between vnfY and vnfK. Genes vnfXNEAU have only
an old locus tag but not yet a new locus tag and therefore their position could not be dedicated on the genome indi-
cated by grey brackets.
Hence, homologuos proteins of Avin_02580 are only present in N2-fixing diazotrophs that en-
code for the V-N2ase. Interestingly, in all strains this protein was denoted as ‘hypothetical pro-
tein’ or ‘uncharacterized protein’ without further description. Therefore this seems to be an en-
tirely new kind of protein, that is not identified or characterized so far. A CO32-, being not just
simply in a single metal bidentate coordination, but as iron ligand coordinating in complex multi-
nuclear clusters in a μ2-bridging mode, has not been seen in biological complex metallocluster
chemistry yet and is therefore entirely new as well.
The fact that this hypothetical and unidentified protein is highly transcribed under V-dependent
N2-fixing conditions, that it is located in the vnf gene cluster directly between vnfK and vnfY, that it
shows very high sequence identity within the Azotobacter species and that homologues do only
exist in other for V-N2ase-encoding diazotrophs is a strong evidence that this protein is of crucial
relevance for V-N2ase. Additionally, a whole series of proteins are involved in FeMo-cofactor
assembly and only a few of them are commonly used, but there are obviously also specified pro-
teins neccessary for FeV-co maturation. As FeV-co differs in metal composition, it seems likely
that homologous but specific vnf-proteins will be responsible for the analogous process of inser-
tion of V instead of Mo. However, as both cofactors exhibit a difference that has in general in
metallocluster biochemistry not been seen before, such as the exchange of a sulfide for a μ2-
bridging carbonate coordinating two Fe atoms, it might even be reasonable and symptomatic that
not a known protein of known functions is performing an apparently unknown process, but ra-
vnfY
(Avin_02570)
hypothetical protein
(Avin_02580)
vnfK
(Avin_02590)
vnfG
(Avin_02600)
vnfD
(Avin_02610)
vnfF
(Avin_02650)
vnfH
(Avin_02660)
vnfX
(Avin_02740)
vnfN
(Avin_02750)
vnfE
(Avin_02770)
vnfA
(Avin_02780)
vnfY
(Achr_RS01245)
(Achr_2490)
A. vinelandii
A. chroococcum(old locus tag)
hypothetical protein
(Achr_RS01250)
(Achr_2500)
vnfK
(Achr_RS01255)
(Achr_2510)
vnfG
(Achr_RS01260)
(Achr_2520)
vnfD
(Achr_RS01265)
(Achr_2530)
vnfF
(Achr_RS01275)
(Achr_2550)
vnfH
(Achr_RS01280)
(Achr_2560)
vnfX
(Achr_38780)
vnfN
(Achr_38770)
vnfE
(Achr_38760)
vnfA
(Achr_38750)
vnfU
(Achr_38740)
vnfU
(Avin_02790)
Discussion
101
ther an unknown protein which is able to perform a process, that has not been seen so far.
Therefore it seems likely that this hypothetical and completely unidentified protein Avin_02580 is
responsible for the yet unknown process of carbonate production and insertion into the most
complex metal cluster in biochemistry.
5.3 Different redox properties of the V- and Mo-N2ase P-cluster and
cofactor
Although the V-N2ase P-cluster is actually identical in composition, architecture and ligand coor-
dination by the equivalent conserved His-ligands to its MoFe-protein analogue, the structure and
according EPR spectra confirm that the P-cluster, even under more reducing conditions than the
yet reduced as isolated conditions, is generally present in a more oxidized state than the MoFe-
protein analogue [204], [206] as it is present in the mixed redox state of PN and the P1+ state.
Thus a proposed more negative reduction potential of V-N2ase P-cluster than Mo-N2ase P-
cluster is also strengthened.
An indication for the aforementioned simultaneous oxidation of both clusters in VFe-protein
[206] in contrast to the sequential oxidation in MoFe-protein [329] is given by EPR-spectra as
well. Although no oxidative redox titration was performed, but just a single reduction step, in
both protein forms signals dedicated to FeV-co (S = 3/2) and to P-cluster (S = 1/2) change sim-
ultaneously without disappearance of one of the signals (see Figure 38). An interaction between
both clusters cannot be confirmed, as this was just speculation.
But the main question is the distinctly different reactivity of VFe- and MoFe-protein towards the
non-physiological substrate CO (see section 1.3.8). Why is it a substrate for V-N2ase, while it is
an inhibitor for Mo-N2ase? This question is most likely related to the substrate binding site and
its redox properties. And actually, major changes are observed in cofactor composition, architec-
ture and electronic structure between the FeV-co and the FeMo-co. Mo is exchanged to a V, a
single-atomic sulfide is displaced by a C3-symmetric tetraatomic carbonate ligand, and conse-
quently the electronic situation, displayed via EPR-spectroscopy, alters from a single, rhombic
S = 3/2 signal at g = 4.3, 3.7 and 2.0 to apparently two S = 3/2 spin systems at g = 5.74, 5.40
and g = 4.35, 3.82.
Although V instead of Mo has only little effects on the cluster geometry, it obviously affects the
electron distribution among the metal atoms. Studies on inorganic structural model complexes
Discussion
102
assigned both metals to be Mo3+ and V3+, and thus one remaining electron has to be located on a
Fe atom, resulting in an oxidation of Fe3+ to Fe2+ [322].
The metal ligands have no direct influence on the electronic structure of the cluster, but they do
affect the ligand field splitting of the coordinating metals. This in turn influences the electronic
structure of each metal atom and thereby also impacts the electronic structure of the entire clus-
ter, as the metal ions couple with each other, which finally determines the cluster electronic struc-
ture and its redox properties. There is little known about carbonate as metal ligand in complex
multinuclear clusters. Hence it is hard to predict or conclude how carbonate, in comparison to
sulfide, affects the properties of the FeV-co. Carbonate shows a C3 symmetry and accordingly
the atoms can be considered as sp2-hybrids, exhibiting p-orbitals that could lead to a π-
backbonding between iron and oxygen. This could explain the known bigger ligand field splitting
compared to sulfide. Furthermore, the S2- to CO32- exchange alters the geometric environment of
the Fe atoms leading to a distortion of the formerly tetrahedral Fe-coordination (see Figure 40).
In consequence the Fe-ligand orbital interactions alter, which impacts the formation of molecular
orbitals and their corresponding energy levels, like a Jahn-Teller-distortion in octahedral coordi-
nation. Although carbonate as a Fe-S-cluster ligand has not been seen before, there are examples
of [4Fe-4S]-clusters which show a similar ligand coordination, like ligation by aspartate [342]. In
the dark-operative Protochlorophyllide oxidoreductase (DPOR), the inter-subunit [4Fe-4S] clus-
ter has a 1Asp/3Cys coordination [343]. A mutation of the aspartate ligand to a normal cysteine
ligand led to an almost complete loss of catalytic activity, and the broad S = 3/2 EPR signal
changed to a rhombic, normal S = ½ spin system. The aspartate is suggested to enhance the re-
ducing power by lowering the cluster reduction potential [344]. Although the cluster confor-
mation appeared to be almost the same, a slight structural distortion seemed to be sufficient to
support a change from the high-spin to the low-spin state. Anyway, the spin state of this [4Fe-
4S]-cluster has changed, which might have two reasons. Either the electronic structure of the
coordinating Fe-atom has changed due to the ligand exchange, that led to an altered coupling
between all Fe-atoms. Or it caused by the small structural change.
As very similar ligand alterations take place in FeV-co (see Figure 40), a likewise impact on the
Fe-atoms, on their electronic structure and eventually on the electronic structure of the cluster is
possible. This would explain the differences in EPR-spectroscopy between the FeV-co and
FeMo-co, especially concerning the S = 3/2 signals, and accordingly it would support that the
signal at g = 5.74 and 5.40 also belongs to FeV-co. In addition, a cysteine to aspartate ligand re-
placement was reported to significantly decrease the redox potential of a [4Fe-4S] cluster in PsaC
of photosystem I from -440 to -630 mV [345]. Taken together, the enhanced reducing power
Discussion
103
and/or the lower reduction potential in these two [4Fe-4S] clusters, due to a modification of cys-
teine to aspartate ligand, matches the situation of Mo- and V-N2ase very well. Therefore it ration-
alizes the different, more negative redox potentials of FeV-co compared to FeMo-co and thus
gives a reasonable explanation for the altered catalytic reactivity of VFe or MoFe-protein towards
the non-physiological substrate or inhibitor CO.
Figure 40: Tetrahedral Fe coordination. Although a tetrahedral coordination for Fe4 is retained upon S3A ligand
exchange to carbonate-O it is distorted accompanied with a bond length decrease. Furthermore the ligand nature
changed from sulfide to carboxyl-like-O having a bigger ligand field splitting and being able for π-backbonding.
5.4 Mechanistic insights in to the active site of N2ase
5.4.1 Mechanistic relevance of α-Gln176
Although α-Gln191 mutants have not been extensively studied, the investigation of N2 reduction
activity, acetylene reduction activity, H2 inhibition of N2-reduction, HD-formation and electron
flux with wild-type MoFe and three single site MoFe protein mutants with mutations at α-
His195Asn or α-His195Gln or α-Gln191Lys have been performed [234], [224]. α-His195 and α-
Gln191 are the corresponding residues to α-His180 and α-Gln176 in VFe-protein.
Wild-type MoFe protein binds and reduces N2 at high levels, N2 reduction is inhibited by addition
of H2, HD formation takes place and there is no electron flux inhibition with N2 or azide as sub-
strate. The MoFe mutants behaved differently concerning these properties. A main difference
concerns N2 binding and reduction. While the α-His195Gln mutant does bind and reduce N2,
although at lower rates than wild-type, α-His195Asn binds N2 but does not reduce it and α-
Gln191Lys does not even bind N2 and consequently does not reduce it. Although both α-
His195Asn and α-Gln191Lys show no N2 reduction activity, they show different H2 evolution
rates under N2 atmosphere compared to Ar atmosphere. Whereas for α-His195Asn the H2-
production rate is decreased, no change is observed for α-Gln191Lys. A similar behavior was
S3A
S4A
S1A
Fe4
C
1.95 Å
2.24 Å
Discussion
104
observed for N2 addition to C2H2 reduction assays. α-His195Asn MoFe shows an inhibitory ef-
fect by N2, while there is no rate reduction for the α-Gln191Lys mutant. Although α-His195Asn
MoFe does not reduce N2, unlike α-His195Gln MoFe, it does interact with N2 [234]. N2 seems to
act as a reversible ‘competitive’ inhibitor, because electron flux inhibition with substrates N2 and
azide occurs for α-His195Gln and α-His195Asn mutants, but not for wild-type [233]. Further-
more, N2 apparently binds to the normal binding site, because adding H2 relieved the N2 inhibi-
tion of C2H2, and H2 is a known specific inhibitor for N2 reduction. For a better comprehension
results are summarized in Table 15.
Table 15: Activity assays with MoFe-protein mutants [224], [234].
properties
with dinitrogen with azide
MoFe pro-tein
binding reduction rate
H2 inhibi-tion
HD for-mation
rate flux inhibi-tion
wild-type yes high yes yes high no
α-His195Gln yes very low yes yes low yes; ≈ 65%
α-His195Asn yes zero yes no low yes; ≈ 20%
α-Gln195Lys no zero N/A N/A low no
In reconsideration of these results based on the ‘active’ state VFe-protein structure, new conclu-
sions can be drawn. Both α-His195Gln and α-His195Asn mutants with intact α-Gln191 bind N2,
although only α-His195Gln shows small N2 reduction rates. Thus, both proteins reach states E3
and/or E4. But α-His195Asn shows decreased H2 production or C2H2 reduction rates when N2 is
present, compared to α-Gln191Lys MoFe that shows no decrease, and both α-His195Asn and α-
His195Gln mutants show electron flux inhibition in contrast to wild-type. These results indicate
that α-His195 might be involved in H2 production, which α-Gln191 seems not. α-Gln191Lys with
intact α-His195 does not bind N2, but H2 production and C2H2 reduction rates are not affected.
These results indicate that by exchange of α-Gln191 to α-Gln191Lys either the active site is just
blocked for N2 binding, although it binds and reduces C2H2, or presumably it just does not reach
E3 and/or E4 states, but it is not related to H2 production.
Based on these assumptions the results could be explained as follows. N2 binds at its normal
binding site (E3 or E4) for α-His195Asn and α-His195Gln mutants, because α-Gln191 is intact.
Simultaneously, α-His195 is altered in α-His195Gln and α-His195Asn and thus H2 production is
hindered. Therefore there is either no (α-His195Asn) or very low (α-His195Gln) N2 reduction. It
Discussion
105
does lead in both cases to an inhibition of electron flux. The addition of H2 as specific N2 inhibi-
tor competes against the bound, but not or barely reduced N2, and eventually H2 binds by releas-
ing N2 and thus C2H2 can normally bind and be reduced.
After evidence for H2 inhibition of N2 binding with α-His195Asn was obtained, HD formation
was tested under a D2/N2 atmosphere. While HD formation was observed with α-His195Gln,
unexpectedly no HD could be detected with α-His195Asn MoFe, although the E3/E4 states are
reached. Only a relief of N2-induced electron flux inhibition could be observed upon D2 addition
(see Table 15). These results could be interpreted as follows. H2 inhibition of N2 binding can only
take place if N2 can bind or is bound. This requires states E3 or E4, which for their part can ap-
parently just be achieved with intact α-Gln191. This means, that the reaction of H2 inhibition and
achieving states E3 and E4 might be controlled by α-Gln191. On the other site, for HD formation
achieving E3/E4 states and D2 binding is prerequisite, but not sufficient. HD formation only
takes place in wild-type protein with intact α-His195 and in α-His195Gln MoFe, but not in α-
His195Asn MoFe. This indicates a necessary interaction with α-His195 or α-His195Gln, which
does not occur with α-His195Asn. Histidine and glutamine have the same length concerning the
amine-N and amide-N able to reach the cluster for H-bonding, while asparagine is shorter and
cannot [233]. Thus, the NH→S2B hydrogen bond in wild-type [113], [114] is likely to be main-
tained by α-Gln195, but broken in the α-His195Asn MoFe [224]. Such a difference can affect the
electronic structure of the cluster. EPR spectroscopy is related to the electronic structure and
similar spectra for wild-type and α-His195Gln MoFe and an altered spectrum for the α-
His195Asn MoFe do support a difference [346]. Glutamine might be sufficiently long and aspar-
agine might be too short for an interaction and thus no HD can be created in α-His195Asn Mo-
Fe. That means HD formation might be controlled through interaction with α-His195. This miss-
ing but required interaction would also be the explanation for the α-His195Asn MoFe not being
able to reduce N2. The plausible idea is that α-His195 is involved in proton delivery during sub-
strate reduction [235], that is necessary for N2-reduction. And the different N2 reduction rates
could arise from the very different pKa values of histidine amine-NH and glutamine amide-NH
[224].
The main point of these activity assays with the MoFe mutants is that apparently α-Gln191 is an
essential residue for N2 fixation, as it seems to be responsible for achieving the crucial states E3
and E4 for N2 binding. And the new structure of the ‘active’ state VFe-protein, with a rearrange-
ment of the corresponding α-Gln176 residue in VFe, displacing the S2B sulfide and H-bonding
an N-intermediate, gives strong support to this hypothesis. Additionally, next to having con-
firmed a property of α-Gln191, further conclusions for the relevance and function of α-His195
Discussion
106
can be drawn. HD formation requires H2 inhibition, which only takes place under N2 reduction
turnover conditions and itself prerequisites high electron flux. During HD formation, no ‘D’ ex-
change with the solvent takes place [223]. Normal ‘H’ exchange with solvent is assumed to occur
via protons bound to sulfides. This could be interpreted as, upon D2 binding, by reversible N2
exchange, ‘D’ is just not present as protons, but as commonly seen as deuteride ‘D-‘, which is also
supported by reductive elimination and electron storage as hydrides [214], [80], [104]. Consider-
ing that under N2 turnover conditions (high electron flux) no HD formation occurs with the
shorter α-His195Asn, but only with α-His195Gln and α-His195, this finding strengthens the hy-
pothesis, α-His195 is responsible for HD formation by protonation of deuterides D-, and
strengthens the role of α-His195 in proton delivery, maybe by the first or several protonation
step in N2 fixation.
The essential relevance is also displayed by the sequence S Q S X G H H containing the residues
glutamine and histidine being conserved in all known MoFe-protein sequences [114] and in at
least the six V-N2ases and one Fe-only-N2ase used for an protein sequence alignment (see Table
19), indicating the same mechanism of N2-fixation by all three N2ases.
With the possibility of a conformational change of α-Gln176, which is related to the S2B dis-
placement, how does this residue affect the FeV-co to achieve the necessary E3 and E4 states?
Does it have an influence on the electronic structure or does it impact the structural geometry of
the cofactor, that allows attaining the essential states E3 and E4. The comparison of EPR spectra
of α-His195Gln and α-His195Asn mutants shows that the presence or absence of an H-bond
affects the electronic structure [346] and could manipulate FeV-co in case of α-Gln191. Or does
the rearrangement support or even mediate S2B displacement, and thus modifies the cofactor
structure by revealing a Fe-Fe edge, that is thought to be more reactive to N2 binding and reduc-
tion (see section 1.4.4.2) [275].
The structure of ‘active’ state VFe protein represents a state where N2 is already cleaved, but it
does not show how N2 was bound to the FeMo-co, and therefore gives no or only little infor-
mation about the electronic or geometrical structure of FeMo-co in states E3 or E4. Considering
the computational mechanistic model for N2 reduction, including an H2S removal of S2B in sec-
tion 1.4.4.2 [275], the ‘active’ state VFe structure matches the theoretical states until approximate-
ly E5 and especially E4 very well. It is even predicted and emphasized that a supporting ligand, or
an appropriate docking site for stabilization of H2S, would further decrease the energy barrier of
the crucial hydride formation. Both can be found in the ‘active’ state. However, this model does
Discussion
107
not deal with different substrate binding properties concerning E1/E2 and E3/E4 states and it
does not give an explanation for the different reduction rates of CO and N2.
To current knowledge, it seems that it always needs to be considered that binding of N2 is re-
versible and inhibition by re-exchange to H2 takes place. For a structural rearrangement this
means that it must be easily reversible as well. Besides N2 and H2, non-physiological substrates
such as C2H2 and CO bind to E2, which is intrinsically confirmed by the fact that H2 only inhibits
the reduction of N2 and no other substrate. But what does it mean that only N2 and H2 bind to
E3 or E4 and non-physiological substrates like C2H2 and CO to E2? Do E3 and E4 states not occur
during reduction of other substrates? Why do not or cannot other substrates bind to E3/E4, or
do they? How can these states be discriminated and what is the difference? Does S2B displace-
ment take place for other substrates as well, like indicated from CO-MoFe, or does the cluster
stay intact? If other substrate reduction is performed via S2B displacement as well, where is the
mechanistical difference in substrate binding and reduction of N2 and other substrates? If CO
doesn’t bind to E3/E4 and is a non-competitve inhibitor for N2, what do we see in the S2B dis-
placed CO-MoFe structure? Or, how relevant and actually representing reality is the dogma
E1/E2 vs E3/E4 and does it need to be scrutinized?
Or, which other factors might play a role besides the electronic or geometrical structure of the
FeMo-cofactor? In earlier pre-steady-state stopped-flow spectrophotometry studies, an absorb-
ance increase at 430 nm has been observed under N2-fixing conditions and was assigned to P-
cluster oxidation [347]. Interestingly, under low-electron flux (not N2-fixing condition) with nei-
ther MoFe-proteins this 430 nm absorbance increase was observed [348], and under high-
electron flux (N2-fixing conditions) the 430 nm absorbance increase was only observed with wild-
type protein, the α-His195Gln and α-His195Asn variants, but not with the α-Gln191Lys variant.
That means that besides the electronic or geometric structure of the cofactor, the electron trans-
fer pathway itself plays a crucial role. Either the electron transfer is mediated by P-cluster oxida-
tion, leading to N2-reduction, or without P-cluster oxidation, resulting in no N2-reduction possi-
bility. As the glutamine residue is situated between the P-cluster and the cofactor, the glutamine
might be involved in this step. This would rationalize that P-cluster oxidation monitored by the
430 nm increase is only observed in MoFe-proteins with intact α-Glu191. The fact that the α-
His195Asn does not reduce N2 can be explained by the too short α-HisAsp195 residue, which is
not able to support the first crucial protonation step. Furthermore, the 430 nm absorbance in-
crease was observed with the substrates N2 and H+, but not with C2H2. As N2 and H2 are so far
the only molecules proposed to bind via reductive elimination to the cofactor, this observation
could hint to a relevant role in N2-binding via reductive elimination. On the other side, it is
Discussion
108
claimed that this P-cluster oxidation is necessary for the first and irreversible reduction and pro-
tonation step of N2 [347].
This finding also indicates a different reduction mechanism for C2H2 and N2. While C2H2 binding
is supposed to be side-on at one Fe-atom [225], and consequently reduction could take place at
an intact cofactor on a five-fold coordinated Fe-atom. N2 binding is supposed to be end-on, and
according to the ‘active’ state structure a Fe-Fe-edge is involved and presumably necessary. Inter-
estingly, CO binds also end-on and at the Fe-Fe-side, indicating a similar binding and reduction
mechanism like N2. Is CO really a non-competitive inhibitor with different binding behavior than
N2? Maybe the binding and reduction mechanism for both is similar, but distinct from C2H2. And
V-N2ase, simply due to the lower reduction potentials, reduces CO, while Mo-N2ase does not.
Comparable kinetics of V- and Mo-N2ase do not exist yet. Drawing conclusions about a differen-
tiation of the reduction mechanism between these three substrates might be possible with the
VFe-protein, because it actually reduces all three substrates, and apparently resides in a stable
intermediate electronic state with a product intermediate bound to the cofactor. Substrate turno-
ver under Ar and subsequent EPR spectroscopy and structure determination might show differ-
ent or no product intermediates bound to the cofactor, thereby hinting to altered reduction
mechanisms for these three substrates.
Structures of E3 and E4 states are unknown. And although it is also unknown how α-Gln176 in
VFe- and corresponding α-Gln191 in MoFe-protein perform their mechanistic function, appar-
ently this residue must be considered for the active site and the FeMo-co can’t anymore isolated
be regarded as active site.
5.4.2 Mechanistic outlook based on the structure of ‘active’ state VFe-protein
Based on the argumentation in section 4.4.3.2 “The FeV-cofactor in the ‘active’ state” and 4.4.3.3
“The active site”, the bound N-intermediate is tentatively assigned to a NH2- ligand. The question
arises, whether it is an artificial structure or it represents a naturally occurring intermediate state
(E1-E7) during N2-fixation.
Based on the model of ligand field theory and the assumption that N possesses an electron octet,
the existence of NH2- implies an N-III oxidation state of nitrogen. That means for the eight elec-
tron reduction of N2 that two electrons are in the required molecule of H2, three are in first NH3
molecule, and the remaining three electrons are in this situation already placed on the second N,
which thus is fully reduced. As it is still bound to FeV-co and an intermediate state is assumed,
Discussion
109
FeV-co has to be in an electron-deficient state, and all remaining electron transfer steps deliver
electrons to FeV-co to restore its resting state E0. Possible intermediate states are E6 or E7.
Figure 41: Possible intermediate state E7. This illustration is based on ligand field theory with an electron octet for N.
It shows the possible intermediate state E7 with the FeV-cofactor core in a one electron deficit (green) and nitrogen
in the oxidation state N-III (red). In A, the ‘active’ state structure with the N-ligand, as putative NH2- species, is dis-
played with a strong donor H-bond to α-Gln176-O (blue dashed line), and two coordinative bonds with μ2-
symmetry to both Fe atoms (solid blue lines). Upon protonation (B) the three-fold planar coordination of the N-
ligand would change to a tetrahedral environment, breaking the strong H-bond to the glutamine ligand. Thereby a
switch from the μ2 Fe-N-Fe coordination to a single Fe-N ligation and concomitantly the rebinding of H2S/HS- is
enabled (C, blue dashed arrow). State A represents the most stable conformation due to the stabilizing strong H-
bond, and thus rationalizes its observation in the protein structure. Anyway, upon the last e-/H+ transfer step via
state C, NH3 could be produced and released by recoordination of H2S/HS- and thus the resting state of FeV-co
would be regenerated.
Depending on the protonation state of the N-species, the state E7, with the core of FeV-co in a
one electron oxidized state, could be displayed as in Figure 41. Conformation A represents the
solved ‘active’ state structure. Upon protonation of [NH]2- to [NH2]1- the H-bond to α-Gln176-O
can break and the μ2 Fe-N-Fe coordination can switch to a single Fe-N coordination which ena-
bles the recoordination of H2S/HS- to the second Fe. Conformation A is observed in the ‘active’
state structure, because the H-bond to α-Gln176-O stabilizes this state as the most stable one of
these three forms. Formation of the second NH3, recoordination of S2B sulfur ligand and recov-
ery of the resting state E0 would be initialized by the last and eighth e-/H+ transfer via state C. In
the EPR spectra, two S = 3/2 signals, dedicated to the FeV-co, appear in both the ‘active’ and
‘resting’ state. Although both signals differ in magnitude, they stay S = 3/2 signals. Based on the
assumption, that the ‘resting’ state structure corresponds to E0, the ‘resting’ state exhibits two
non-integer S = 3/2 signals. Regarding neighboring intermediate states, like one electron reduced
(E1) or a one electron deficit (E7), at least one integer spin signal has to appear. This is not the
+ H+
- H+
N-III
H
Fe Fe
N-III
H
Fe Fe
H
N-III
H
Fe Fe
HH2S/HS-E7
1+ 1+ 1+
A
[N-IIIH]2-
‚active‘ state structure
B
[N-IIIH2]1-
C
[N-IIIH2]1-
Gln176
O
C
H2N
2.5 Å
2.0 Å2.0 Å
Discussion
110
case in the EPR spectrum of the ‘active’ state and argues against the presence of E7 as the ‘active’
state.
It rather indicates an electronic state of the cluster with an even amount of electrons, compared
to E0, and thus points to E6. This would imply that the FeV-co is in a two electron oxidized state,
compared to the resting state E0. First, at current state of research, the cofactor is thought to
work with one redox couple, switching only between two oxidation states [269], [270] during one
cycle of catalytic N2 fixation. Secondly, it is very unlikely that the FeV-co, with a two electron
deficit, exhibits the same EPR signals, than the intact FeV-co in the ‘resting’ state VFe-protein,
concerning the type of spin states (S = 3/2). Both argues against a two electron oxidized FeV-co.
On the other side, three different oxidation states are evidenced for the cofactor of MoFe-
protein, namely the resting state MN, the one electron oxidized state Mox and the one electron
reduced state Mr, that is only observed under N2 turnover (see section 1.3.4.2) [132], [122], [133],
[134]. Considering these three states in E6, the FeV-co would be in the Mox state and could adopt
the Mr state upon the remaining two e-/H+ transfers. This implies that the resting state E0 of
FeV-co would be the Mr state, which is for FeMo-co an integer spin system. The EPR-spectrum
of the ‘resting’ state VFe-protein shows S = 3/2 signals, proposed to arise from resting state MN
from the FeV-co. Assuming analogous spin states of FeV-co and FeMo-co in corresponding
electronic states (Mr → integer S = 2; MN → half-integer S = 3/2; Mox → diagmagnetic S = 0),
the resting state of FeV-co cannot be the Mr state. Therefore this also argues against E6.
A potentiometric redox titration and EPR experiments under turnover for VFe-protein could
help to identify FeV-co oxidation states. In conclusion, the observed EPR spin states seem to
mismatch the electronic state of the proposed intermediate state (E6 or E7) of the structure. That
means, based on ligand field theory and a nitrogen electron octet the ‘active’ state structure, EPR
and a certain intermediate state do not match so far.
Another possibility is that oxidation numbers are not as clear as assumed and the Fe-N bond is
not a pure dative bond, but shows also covalent character. In such a model, the electrons in the
Fe-N bonds of the NH2- ligand would not only belong to N (N-III). They would also partially
count to the Fe atoms and show a kind of a reducing character, and in reverse bring nitrogen to a
oxidation number like N-I or N-II. Such a behavior is not considered with ligand field theory, but
it is under discussion [349], [350]. How such a model would influence the electronic structure,
indicated by EPR spectra, is also not clear.
Additionally it might be possible, that this ‘active’ state structure actually reflects an intermediate
state like E6 or E7, but electronically the FeV-co recovered to the resting state E0, and by accident
Discussion
111
the putative N-intermediate, NH2-, kept bound or rebound to its position. This would mean, we
see a FeV-co in the electronic resting state with an exchanged S2B ligand to a N-species, that is
well stabilized by the Gln176 and shows the same double negative charge than the original sul-
fide. This also rationalizes the presence of the same S = 3/2 spin systems, just differing in magni-
tude due to the altered ligand.
A further alternative, instead of a nitride (NH2-) with N-III species with an electron octet, is the
presence of an imidogen (NH) that belongs to the nitrenes with a N-I species, that shows an elec-
tron sextet. Metal clusters with a (N-R)-mojeties are known [351], [352]. This would also rational-
ize an E6 state with two missing electrons. In that case, the two-electron deficit would not be
located on the FeV-co core but on the N-ligand (Figure 42). Accordingly, the cluster could be
present in the resting state with the two S = 3/2 signals, just differing in magnitude due to the
altered ligand NH for S2-. This would also agree with the three-fold symmetry of the N-species.
Nitrenes are highly reactive and mainly short-lived species, but it would be stabilized by the
strong H-bonding to glutamine.
Figure 42: Possible intermediate state E6 with a bound nitrene, NH. In this model N-species has an electron sextet
with the formal oxidation state (red) N-I, and needs two further reduction steps. Accordingly, the ‘active’ state struc-
ture would represent the intermediate state E6 with the FeV-co (green) in the resting state. After an e-/H+ transfer
(E7), the FeV-co would be in the one-electron-reduced state. Upon the last reduction step, a reaction cascade via
transition states, e.g. ‘E0’, is initiated and results in the release of the second NH3 and the FeV-co resting state, E0, is
restored. This second e-/H+ transfer could lead again to generation of a bridging hydride, like as described for states
E2 and E4, but in that case, next to a very reactive NH with a two-electron deficit. Subsequent reduction of the
nitrene by the H- would lead to N-IIIH2-, Such a species could break the Fe-N-Fe coordination and lead to a single
Fe-N ligation and consequently enable the rebinding of the H2S/HS-. After proton shuttling to NH2-, NH3 could be
released and the FeV-co with bound sulfide would be restored.
E6
[N-IH]
‚active‘ state structure
Gln176
O
C
H2N
N-I
H
Fe Fe0
e-/H+
E0
S2-
resting state
E7
[N-IH]
N-I
H
Fe Fe1-
‘E0’
[N-IIIH2]-
e-/H+NH3
S
Fe Fe0
N-III
H
Fe Fe0
H2S/HS-H
Gln176
O
C
H2N
Discussion
112
As long as the electronic state of the Fe-atoms, and especially the nature of the N-ligand, is not
clarified, such as presence of nitride (N-I to N-III) or a nitrene (N-I), it is hard to denote this state
either to E6, E7 or even E8, with just an exchanged ligand. The EPR spectra also always have to
be considered. Therefore at current state a discussion about the mechanism is hard, as none of
these possibilities can be excluded leaving open too many alternatives for this state.
Appendix
113
6. Appendix
6.1 Index of abbreviations
6.1.1 General abbreviations
(v/v) volume fraction per volume
(w/v) weight fraction per volume fraction
ARA acetylene reduction assay
A. vinelandii Azotobacter vinelandii
APS ammonium persulfate
ATP adenosine triphosphate
bc bond cleavage
BCA 2,2’-Bichinoline-4,4’-dicarboxylic acid
C-BNF cultivated induced biological nitrogen fixation
CV column volumes
DMSO dimethyl sulfoxide
DTT dithiothreitol
EDTA ethylenediaminetetraacetic acid
EPR electron paramagnetic resonance
HC homocitrate
HCl hydrochloride
IEC ion exchange chromatography
LC-MS liquid chromatography-mass spectrometry
β-ME β-mercaptoethanol
mi migratory insertion
Mo molybdenum
pmf proton motive force
PCET proton coupled electron transfer
re reductive elimination
RT room temperature
SAM S-adenosyl-L-methionine
DT sodium dithionite (Na2S2O4)
SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis
SEC size exclusion chromatography
TCEP tris(2-carboxyethyl)phosphine
Appendix
114
TEMED N,N,N’,N’-tetramethylethylenediamine
Tris Tris(hydroxymethyl)aminomethane
V vanadium
6.1.2 Units
% percent
°C degree Celsius
A ampere
Å angstrom
Au absorption unit
Da dalton
g gram
h hours
Hz hertz
J joule
K kelvin
l liter
M molarity
min minutes
rpm rounds per minute
T tesla
V volt
×g multiple of gravity
6.1.3 Prefixes
G giga 109
M mega 106
k kilo 103
c centi 10-2
m milli 10-3
μ micro 10-6
n nano 10-9
p pico 10-12
Appendix
115
6.2 Thermofluor assay melting curves and temperatures
Figure 43: Melting curves of ‚Optimal pH and NaCl concentration’.
Figure 44: Melting curves of ‚Influence of Na2S2O4 concentration’.
20 30 40 50 60 70 80
-1200
-1000
-800
-600
-400
-200
0
200
400
600
-(d
RF
U)/
dT
Temperature [°C]
pH6,0 0mM
pH6,0 100mM
pH6,0 200mM
pH6,0 300mM
pH6,0 400mM
pH6,0 500mM
20 30 40 50 60 70 80
-1000
-800
-600
-400
-200
0
200
400
600
800
-(d
RF
U)/
dT
Temperature [°C]
pH6,5 0mM
pH6,5 100mM
pH6,5 200mM
pH6,5 300mM
pH6,5 400mM
pH6,5 500mM
20 30 40 50 60 70 80
-1000
-800
-600
-400
-200
0
200
400
600
-(d
RF
U)/
dT
Temperature [°C]
pH7,0 0mM
pH7,0 100mM
pH7,0 200mM
pH7,0 300mM
pH7,0 400mM
pH7,0 500mM
20 30 40 50 60 70 80
-1000
-800
-600
-400
-200
0
200
400
-(d
RF
U)/
dT
Temperature [°C]
pH7,5 0mM
pH7,5 100mM
pH7,5 200mM
pH7,5 300mM
pH7,5 400mM
pH7,5 500mM
20 30 40 50 60 70 80
-1000
-800
-600
-400
-200
0
200
400
600
800-(
dR
FU
)/d
T
Temperature [°C]
pH8,0 0mM
pH8,0 100mM
pH8,0 200mM
pH8,0 300mM
pH8,0 400mM
pH8,0 500mM
20 30 40 50 60 70 80
-600
-400
-200
0
200
400
-(d
RF
U)/
dT
Temperature [°C]
pH8,5 0mM
pH8,5 100mM
pH8,5 200mM
pH8,5 300mM
pH8,5 400mM
pH8,5 500mM
20 30 40 50 60 70 80
-500
-400
-300
-200
-100
0
100
200
300
-(d
RF
U)/
dT
Temperature [°C]
pH9,0 0mM
pH9,0 100mM
pH9,0 200mM
pH9,0 300mM
pH9,0 400mM
pH9,0 500mM
20 30 40 50 60 70 80
-1000
-500
0
500
1000
1500
-(d
RF
U)/
dT
Temperature [°C]
0 mM DT
1 mM DT
2 mM DT
4 mM DT
10 mM DT
20 mM DT
Appendix
116
Table 16: Melting temperatures (Tm) of ‚Influence of Na2S2O4 concentration’.
0 mM DT 1 mM DT 2 mM DT 4 mM DT 10 mM DT 20 mM DT
52 °C 51 °C 51 °C 51 °C 49 °C 48 °C
52 °C 51 °C 51 °C 51 °C 50 °C 48 °C
52 °C 51 °C 51 °C 51 °C 50 °C 48 °C
52 °C 51 °C 51 °C 51 °C 50 °C 49 °C
Figure 45: Melting curves of ‚Influence of additives’. The melting curves of additive ZnCl2 is coloured in red
Table 17: Melting temperatures (Tm) of ‚Influence of additives’. Tm are given in °C. a) For 6 M urea, 1 mM NiCl2,
1 mM FeCl3 and 1 mM CoCl2 no Tm could be determined from the melting curves.
1 2 3 4 5 6
A 0.1 M urea 0.5 M urea 1 M urea 6 M urea 0.1 M guani-dine-HCl
0.5 M guani-dine-HCl
49, 49, 48, 49 43, 43, 43, 43 41, 40, 39, 39 n.d. a) 49, 48, 49, 47 37, 38, 37, 37
B 0.1 % β-ME 0.01 M TCEP HCl
0.001 M DTT 0.005 M DTT 0.01 M DTT 0.02 M DTT
50, 51, 50, 50 52, 52, 51, 51 50, 51, 50, 50 51, 50, 50, 50 50, 50, 50, 50 50, 50, 50, 49
C 0.1 M Na-acetate
0.1 M Ca-acetate
0.1 M K-acetate 0.1 M NH4-acetate
0.001 M sper-midine
0.001 M sperm-ine-HCl
51, 51, 50, 50 48, 48, 48, 46 51, 51, 50, 51 52, 51, 50, 51 51, 51, 51, 51 52, 51, 51, 51
D 0.1 M Na2SO4 0.1 M MgSO4 0.1 M (NH4)2SO4
0.1 M NaH2PO4
0.1 M KH2PO4 0.005 M EDTA
50, 50, 49, 49 50, 50, 49, 50 50, 50, 49, 50 43, 42, 42, 42 43, 43, 42, 42 49, 49, 48, 49
E 0.05 M imidaz-ole
0.1 M imidazole 0.3 M imidazole 0.1 M NaHCO2 0.1 M KHCO2 0.1 M NH4HCO2
49, 49, 48, 49 48, 48, 47, 47 42, 43, 42, 42 50, 49, 49, 49 50, 50, 49, 49 51, 51, 50, 50
F 0.1 M Na- 0.1 M Na- 0.1 M NaNO3 0.1 M NaBr 0.1 M NaCl H2O (refer-
20 30 40 50 60 70 80
-3000
-2500
-2000
-1500
-1000
-500
0
500
1000
-(d
RF
U)/
dT
Temperature [°C]
20 30 40 50 60 70 80
-3000
-2000
-1000
0
1000
2000
-(d
RF
U)/
dT
Temperature [°C]
Appendix
117
citrate malonate ence)
50, 50, 50, 50 50, 50, 49, 49 51, 51, 50, 50, 49, 49, 49, 49 51, 51, 50, 50 51, 51, 51, 50
G 0.01 M MgCl2 0.01 M CaCl2 0.001 M MnCl2 0.001 M NiCl2 0.001 FeCl3 0.001 M ZnCl2
52, 51, 51, 51 51, 51, 51, 51 51, 51, 51, 51 n.d. a) n.d. a) 60, 60, 59, 60
H 0.001 M CoCl2 0.1 M LiCl 0.1 M KCl 0.1 M NH4Cl 0.1 M NaI 0.1 M KI
n.d. a) 51, 51, 50, 50 51, 51, 50, 50 51, 51, 50, 50 47, 47, 48, 47 47, 47, 48, 47
6.3 BLAST
Table 18: Protein sequence BLAST of non-redundant protein sequences with protein sequence of Avin_02580.
protein description [bacterial strain] sequence identity nif-genes vnf-genes
hypothetical protein [Azotobacter vinelandii] 100 % yes yes
hypothetical protein [Azotobacter chroococcum] 90 % yes yes
hypothetical protein [Azotobacter beijerinckii] 85 % yes yes
hypothetical protein [Azospirillum brasilense] 47 % yes yes
hypothetical protein [Methylocystis parvus] 45 % yes yes
hypothetical protein [Methylocystis bryophil]a 39 % yes yes
hypothetical protein [Phaeospirillum fulvum] 43 % yes yes
hypothetical protein [Rhodomicrobium vannie]lii 40 % yes yes
hypothetical protein [Rhodopseudomonas palustris] 43 % yes yes
hypothetical protein [Tolumonas lignilytica] 39 % yes yes
hypothetical protein [Desulfobacter curvatus] 33 % yes yes
hypothetical protein [Bacillus thuringiensis] 24 % no no
hypothetical protein [Bacillus cereus] 24 % no no
hypothetical protein [Paeniclostridium sordellii] 21 % no no
uncharacterized protein [Klebsiella pneumoniae] 23 % yes no
hypothetical protein [Stenotrophomonas maltophilia] 22 % no no
Appendix
118
6.4 Nitrogenase protein sequence alignment
Table 19: Nitrogenase protein sequence alignment. Nitrogenase α- and β-subunits were used. 13 x NifDK (Azotobac-
ter vinelandii, Azotobacter chroococcum, Azotobacter beijerinckii, Pseudomonas stutzeri, Klebsiella pneumoniae, Clostridium pasteuri-
anum, Gluconacetobacter diazotrophicus, Azospirillum brasilense, Methylocystis parvus, Methylocystis bryophila, Rhodomicrobium
vannielii, Rhodopseudomonas palustris, Tolumonas lignilytica), 6 x VnfDK (A. vinelandii, A. chroococcum, A. beijerinckii, A. bra-
silense, R. vannielii, R. palustris) and 1 x AnfDK (A. vinelandii) was used. The residues SQSXGHH including the corre-
sponding α-Gln176 and α-His180 in VnfDKG from A. vinalndii (bold) are conserved in all used N2ases and are high-
lighted (yellow).
A_vinelandii_AnfDK --------------------MPHHEFECSKVIPERKKHAVIKGKGET-----------LA
A_vinelandii_VnfDK --------------------MPMVLLECDKDIPERQKHIYLKAPNED-----------TR
A_chroococcum_VnfDK --------------------MPMVLLECDKDIPERQKHIYLKAPNED-----------TR
A_beijerinckii_VnfDK --------------------MPMVLLECDKDIPERQKHIYLKAPNED-----------TR
R_vannielii_VnfDK --------------------MPMVLLKCDTDIPERQKHIYLKVDGED-----------TR
A_brasilense_VnfDK --------------------MPMVLLKCDKDIPEREKHIYLKAPDED-----------TR
R_palustris_VnfDK --------------------MPMVLLKCDKDIPEREKHIYLKVDGED-----------TR
C_pasteurianum_NifDK --------------MSENLKDEILEKYIPKTKKTRSGHIVIKTEE-------------TP
P_stutzeri_NifDK -------MTGMSREEVESLIQEVLEVYPEKARKDRAKHLSPNDPALE-----------QS
A_vinelandii_NifDK -------MTGMSREEVESLIQEVLEVYPEKARKDRNKHLAVNDPAVT-----------QS
A_chroococcum_NifDK -------MSGMSREEVESLIQEVLEVYPEKARKDRAKHLAVNDQSVT-----------QS
A_beijerinckii_NifDK -------MSGMSREEVESLIQEVLEVYPEKARKDRAKHLAVNDPAVT-----------QS
K_pneumoniae_NifDK ------MMTNATGERNLALIQEVLEVFPETARKERRKHMMVSDPKMK-----------SV
T_lignilytica_NifDK -------MTNRTREENLALIQEVLEAYPEKARKDRKKHLTVNDPSLE-----------KA
A_brasilense_NifDK MSLSVNE-----GVDVKGLVDKVLEAYPEKSRRRRAKHLNVLEAE-------------AK
G_diazotrophicus_NifDK MS--LDEDKTNDSAFHARLIAEVLEAYPDKARKRRQKHLNVAGHAEADAQDTGEEGAMLS
R_palustris_NifDK MSTAVAESPADIKERNKKLIGEVLEAYPDKSAKRRAKHLNTYDAE-------------KA
R_vannielii_NifDK MSLGLA-ESEDLKAKNKALVEEVLKVYPDKTAKRRAKHLGVYQNG-------------KP
M_parvus_NifDK MSVAQAESIEDIKARNKELVQEVLKAYPEKTAKRRAKHLGTFEDG-------------KP
M_bryophila_NifDK MSLAPSQSVAEIKARNKALIEEVLKVYPEKTAKRRAKHLNVHEAG-------------KQ
. * *
A_vinelandii_AnfDK DALPQGYLNTIPGSISERGCAYCGAKHVIGTPMKDVIHISHGPVGCTYDTWQTKRYIS--
A_vinelandii_VnfDK EFLPIANAATIPGTLSERGCAFCGAKLVIGGVLKDTIQMIHGPLGCAYDTWHTKRYPT--
A_chroococcum_VnfDK EFLPIANAATIPGTLSERGCAFCGAKLVIGGVLKDTIQMIHGPLGCAYDTWHTKRYPT--
A_beijerinckii_VnfDK EFLPIANAATIPGTLSERGCAFCGAKLVIGGVLKDTIQMIHGPLGCAYDTWHTKRYPT--
R_vannielii_VnfDK DFLPVSNAPTIPGTLSERGCSFCGAKLVIGGVLKDTIQMIHGPVGCAYDTWHTKRYPS--
A_brasilense_VnfDK DYLPLSNAATIPGTLSERGCAFCGAKLVIGGVLKDTIQMIHGPLGCAYDTWHTKRYPT--
R_palustris_VnfDK DYLPVSNAPTIPGTLSERGCAFCGAKLVIGGVLKDTIQMIHGPIGCAYDTWHTKRYPS--
C_pasteurianum_NifDK NPEIVANTRTVPGIITARGCAYAGCKGVVMGPIKDMVHITHGPIGCSFYTWGGRRFKSKP
P_stutzeri_NifDK KKCITSNKKSQPGLMTIRGCAYAGSKGVVWGPIKDMIHISHGPVGCGQYSRAGRRNYYIG
A_vinelandii_NifDK KKCIISNKKSQPGLMTIRGCAYAGSKGVVWGPIKDMIHISHGPVGCGQYSRAGRRNYYIG
A_chroococcum_NifDK KKCITSNKKSQPGLMTIRGCAYAGSKGVVWGPIKDMIHISHGPVGCGQYSRAGRRNYYIG
A_beijerinckii_NifDK KKCITSNKKSQPGLMTIRGCAYAGSKGVVWGPIKDMIHISHGPVGCGQYSRAGRRNYYIG
K_pneumoniae_NifDK GKCIISNRKSQPGVMTVRGCAYAGSKGVVFGPIKDMAHISHGPAGCGQYSRAERRNYYTG
T_lignilytica_NifDK SKCITSNRKSLPGVMTIRGCAYAGSKGVVFGPIKDMIHLSHGPVGCGQYSRAGRRNYYTG
A_brasilense_NifDK DCGVKSNIKSIPGVMTIRGCAYAGSKGVVWGPIKDMIHISHGPVGCGYYSWSGRRNYYVG
G_diazotrophicus_NifDK ECDVKSNVKSVPGVMTIRGCAYAGSKGVVWGPVKDMVHISHGPVGCGQYSWSQRRNYYIG
R_palustris_NifDK ECSVKSNIKSIPGVMTIRGCAYAGSKGVVWGPIKDMVHISHGPVGCGQYSWGSRRNYYKG
R_vannielii_NifDK DCGVKSNIKSIPGVMTIRGCAYAGSKGVVWGPIKDMIHISHGPVGCGQYSWGSRRNYYVG
M_parvus_NifDK DCGVKSNIKSIPGVMTIRGCAYAGSKGVVWGPIKDMIHISHGPVGCGQYSWASRRNYYIG
M_bryophila_NifDK DCGVKSNIKSIPGVMTIRGCAYAGSKGVVWGPIKDMIHISHGPVGCGQYSWAARRNYYIG
. : ** :: ***::.*.* *: :** :: *** ** : :*
A_vinelandii_AnfDK DND--NFQLKYTYATDVKEKHIVFGAEKLLKQNIIEAFKAFPQIKRMTIYQTCATALIGD
A_vinelandii_VnfDK DNG--HFNMKYVWSTDMKESHVVFGGEKRLEKSMHEAFDEMPDIKRMIVYTTCPTALIGD
A_chroococcum_VnfDK DNG--HFNMKYVWSTDMKESHVVFGGEKRLEQSMHEAFDEMPDIKRMIVYTTCPTALIGD
A_beijerinckii_VnfDK DNG--HFNMKYVWSTDMKESHVVFGGEKRLEKSMHEAFDEMPDIKRMIVYTTCPTALIGD
R_vannielii_VnfDK DNG--HFQMKYVWSTDMKESHIVFGGDKRLEKAINEAFDEMPDVKRMIVYTTCPTALIGD
A_brasilense_VnfDK DNG--HFNMKYVWSTDMKESHIVFGGEKRLEKSMHEAFDAMPDVKRMIVYTTCPTALIGD
R_palustris_VnfDK DNG--HFQMKYVWSTDMKESHIVFGGDKRLEKSIHEAFDEMPHIKRMIVYTTCPTALIGD
C_pasteurianum_NifDK ENGTGLNFNEYVFSTDMQESDIVFGGVNKLKDAIHEAYEMFHP-AAIGVYATCPVGLIGD
P_stutzeri_NifDK TTGV-NAFVTMNFTSDFQEKDIVFGGDKKLAKLIDEIETLFPLNKGISVQSECPIGLIGD
A_vinelandii_NifDK TTGV-NAFVTMNFTSDFQEKDIVFGGDKKLAKLIDEVETLFPLNKGISVQSECPIGLIGD
A_chroococcum_NifDK TTGV-NAFVTMNFTSDFQEKDIVFGGDKKLAKLIDEVETLFPLNKGISIQSECPIGLIGD
A_beijerinckii_NifDK TTGV-NAFVTMNFTSDFQEKDIVFGGDKKLAKLIDEVETLFPLNKGISIQSECPIGLIGD
K_pneumoniae_NifDK VSGV-DSFGTLNFTSDFQERDIVFGGDKKLSKLIEEMELLFPLTKGITIQSECPVGLIGD
T_lignilytica_NifDK LTGV-NSFGTMNFTSDFQEKDIVFGGDKKLTKIIEEMEMLFPLSKGITVQSECPVGLIGD
A_brasilense_NifDK DTGV-DKLGTMHFTSDFQEKDIVFGGDKKLHKVIEEINELFPLVNGISIQSECPIGLNGD
G_diazotrophicus_NifDK NTGV-DSFVTMQFTSDFQEKDIVFGGDKKLEKIIDEIDELFPLAKGISVQSECPIGLIGD
Appendix
119
R_palustris_NifDK TTGV-DTFGTMQFTSDFQEKDIVFGGDKKLGKIIDEIQELFPLSKGISVQSECPIGLIGD
R_vannielii_NifDK TTGI-DSFGTMQFTSDFQENDIVFGGDKKLSKLMDEVQELFPLNKGLSVQSECPIGLIGD
M_parvus_NifDK TTGI-DTFGTMQFTSDFQEKDIVFGGDKKLAKIIDEIQDLFPLNKGISVQSECPIGLIGD
M_bryophila_NifDK TTGI-DTFVTMQFTSDFQEKDIVFGGDKKLAKIMDEIQELFPLNNGITVQSECPIGLIGD
.. :::*.:* .:***. : * . : * : : : * .* **
A_vinelandii_AnfDK DINAIAEEVMEEMPEVDIFVCNSPGFAGPSQSGGHHKINI---AWINQKVGT--VEPEIT
A_vinelandii_VnfDK DIKAVAKKVMKDRPDVDVFTVECPGFSGVSQSKGHHVLNI---GWINEKVET--MEKEIT
A_chroococcum_VnfDK DIKAVAKKVMKERPDVDVFTVECPGFSGVSQSKGHHVLNI---GWINEKVET--MEKEIT
A_beijerinckii_VnfDK DIKAVAKKVMKDRPDVDVFTVECPGFSGVSQSKGHHVLNI---GWINEKVET--MEKEIT
R_vannielii_VnfDK DIRHTVKKVMGERPDVDIFTVECPGFAGVSQSKGHHVLNI---GWINDKVGQ--VEPEIK
A_brasilense_VnfDK DIKAVAKKVMDARPDVDVFTVECPGFSGVSQSKGHHVLNI---GWINEKVGT--LEPEIT
R_palustris_VnfDK DIKATVKRVMEVRPDVDIFTCECPGFAGVSQSKGHHVLNI---GWVNEKVGT--VEPEIT
C_pasteurianum_NifDK DILAVAATASKE-IGIPVHAFSCEGYKGVSQSAGHHIANNTVMTDIIGKGNK------EQ
P_stutzeri_NifDK DIEAVAKKKAAE-HETTVVPVRCEGFRGVSQSLGHHIANDAIRDWVLDKRDD--DTSFET
A_vinelandii_NifDK DIESVSKVKGAE-LSKTIVPVRCEGFRGVSQSLGHHIANDAVRDWVLGKRDE--DTTFAS
A_chroococcum_NifDK DIESVSKVKGAE-HNTTVVPVRCEGFRGVSQSLGHHIANDAVRDWVLGNRDD--DNSFQT
A_beijerinckii_NifDK DIESVSKVKGAE-HNTTVVPVRCEGFRGVSQSLGHHIANDAVRDWVLGNRDD--DNSFQT
K_pneumoniae_NifDK DISAVANASSKA-LDKPVIPVRCEGFRGVSQSLGHHIANDVVRDWILNNREG---QPFET
T_lignilytica_NifDK DIESVAKQAKAK-IGKSVVPVRCEGFRGVSQSLGHHIANDVVRDWILGNRDE---QEFEG
A_brasilense_NifDK DIEGVSKAKSEE-LGKPVVPVRCEGFRGVSQSLGHHIANDVIRDWILPKKTE-PKEGFVS
G_diazotrophicus_NifDK DIEAVSRKKKKE-IGKTIVPVRCEGFRGVSQSLGHHIANDAIRDWVFDGED--KHAAFET
R_palustris_NifDK DIEAVSKAKSKQYDGKPIIPVRCEGFRGVSQSLGHHIANDVIRDWVFDKAGE-KNAGFQS
R_vannielii_NifDK DIEAVSKKKSKEYGGKPIVPVRCEGFRGVSQSLGHHIANDAVRDWVFDKAEG-KPARFEA
M_parvus_NifDK DIEAVSKAKTKEYEGKTIVPVRCEGFRGVSQSLGHHIANDAIRDWVFDKSEG-KPIRFEP
M_bryophila_NifDK DIEAVSKAKSKEYDGKTIVPVRCEGFRGVSQSLGHHIANDAIRDWVFDKMDPNAAPRFEP
** : . *: * *** *** * :
A_vinelandii_AnfDK GDHVINYVGEYNIQGDQEVMVDYFKRMGIQVLSTFTGNGSYDGLRAMHRAHLNVLECARS
A_vinelandii_VnfDK SEYTMNFIGDFNIQGDTQLLQTYWDRLGIQVVAHFTGNGTYDDLRCMHQAQLNVVNCARS
A_chroococcum_VnfDK SEYTMNFIGDFNIQGDTQLLQTYWDRLGIQVVAHFTGNGTYDDLRCMHQAQLNVVNCARS
A_beijerinckii_VnfDK SEYTMNFIGDFNIQGDTQLLQTYWDRLGIQVVAHFTGNGTYDDLRCMHQAQLNVVNCARS
R_vannielii_VnfDK SEYTINFIGDYNIQGDTQLLQQYWDRLGIQVIAHFTGNANYDELRSMHRAQLNVVNCARS
A_brasilense_VnfDK SPYTMNFIGDFNIQGDTQLLQTYWDRLGIQVIAHFTGNGTYDDLRKMHRAQLNVVNCARS
R_palustris_VnfDK SPYTINFIGDYNIQGDTQLLQQYWDKLGIQVIAHFTGNANYDDLRAMHRAQLNVVNCARS
C_pasteurianum_NifDK KKYSINVLGEYNIGGDAWEMDRVLEKIGYHVNATLTGDATYEKVQNADKADLNLVQCHRS
P_stutzeri_NifDK TPYDVSIIGDYNIGGDAWSSRILLEEMGLRVVAQWSGDGTISEMELTPKVKLNLVHCYRS
A_vinelandii_NifDK TPYDVAIIGDYNIGGDAWSSRILLEEMGLRCVAQWSGDGSISEIELTPKVKLNLVHCYRS
A_chroococcum_NifDK TPYDVAIIGDYNIGGDAWSSRILLEEMGLRCVAQWSGDGSIAEIELTPKVKLNLVHCYRS
A_beijerinckii_NifDK TPYDVAIIGDYNIGGDAWSSRILLEEMGLRCVAQWSGDGSIAEIELTPKVKLNLVHCYRS
K_pneumoniae_NifDK TPYDVAIIGDYNIGGDAWASRILLEEMGLRVVAQWSGDGTLVEMENTPFVKLNLVHCYRS
T_lignilytica_NifDK TPYDVAIIGDYNIGGDAWSSRILLEEIGLRVVAQWSGDGTLAEMENTPKVKMNLVHCYRS
A_brasilense_NifDK TPYDVTIIGDYNIGGDAWASRILLEEIGLRVIAQWSGDGTLAELENQPKAKVNLIHSYRS
G_diazotrophicus_NifDK TPYDVNVIGDYNIGGDAWSSRILLEEMGLRVVGNWSGDATLAEIERAPKAKLNLIHCYRS
R_palustris_NifDK TPYDVAIIGDYNIGGDAWASRILLEEMGLRVIAQWSGDGTIAELENTPKAKLNILHCYRS
R_vannielii_NifDK TEYDIAILADYNIGGDAWASRILLEEMGLRVVAQWSGDGTIAELEATPRAKLNVLHCYRS
M_parvus_NifDK SPYDVAIIGDYNIGGDAWSSRILLEEMGLRVVAQWSGDGTIAELEATPKAKLNVLHCYRS
M_bryophila_NifDK TPYDVAIIGDYNIGGDAWSSRILLEEMGLRVIAQWSGDGSLAELEATPKAKLNVLHCYRS
: : :.::** ** ..:* : . :*:.. :. ..:*::.. **
A_vinelandii_AnfDK AEYICNELRVRYGIPRLDIDGFGFKPLADSLRKIGMFFG---IEDRAKAIIDEEVARWKP
A_vinelandii_VnfDK SGYIANELKKRYGIPRLDIDSWGFNYMAEGIRKICAFFG---IEEKGEELIAEEYAKWKP
A_chroococcum_VnfDK SGYIANELKKRYGIPRLDIDSWGFSYMAEGIRKICAFFG---IEEKGEALIAEEYAKWKP
A_beijerinckii_VnfDK SGYIANELKKRYGIPRLDIDSWGFSYMAEGIRKICAFFG---IEEKGEALIAEEYAKWKP
R_vannielii_VnfDK AGYIANELKKRYGIPRLDIDSWGFNYMAEGIRKVCAFFG---IEERGEALIAEEYAKWKP
A_brasilense_VnfDK SGYIANELKKQYGIPRLDIDSWGFNYMAEGIRKICAFFG---IEEKGEALIAEEYALWKP
R_palustris_VnfDK AGYIANELKKRYGIPRLDIDSWGFNYMAEGIRKVCAFFG---IEEKGEALIAEEYAKWKP
C_pasteurianum_NifDK INYIAEMMETKYGIPWIKCNFIGVDGIVETLRDMAKCFDDPELTKRTEEVIAEEIAAIQD
P_stutzeri_NifDK MNYISRHMEEKYGIPWMEYNFFGPTKTAESLRAIAEHFDDS-IKAKCEQVIAKYQSEWEA
A_vinelandii_NifDK MNYISRHMEEKYGIPWMEYNFFGPTKTIESLRAIAAKFDES-IQKKCEEVIAKYKPEWEA
A_chroococcum_NifDK MNYISRHMEEKYGIPWMEYNFFGPTKTIESLRAIAAKFDES-IQKKCEEVIAKYKPEWEA
A_beijerinckii_NifDK MNYISRHMEEKYGIPWMEYNFFGPTKTIESLRAIAAKFDES-IQKKCEEVIAKYKPEWEA
K_pneumoniae_NifDK MNYIARHMEEKHQIPWMEYNFFGPTKIAESLRKIADQFDDT-IRANAEAVIARYEGQMAA
T_lignilytica_NifDK MNYISRHMEEKYDIPWMEYNFFGPTKIAESLRKIAAQFDET-IQKKAEEVIAKYEAQTAA
A_brasilense_NifDK MNYIARHMEEKFGIPWMEYNFFGPSQIAESLRKIAALFDDT-IKENAEKVIAKYQPMVDA
G_diazotrophicus_NifDK MNYICRHMEEKYNIPWTEYNFFGPSQIAASLRKIAALFDEK-IQEGAERVIAKYQPLVDA
R_palustris_NifDK MNYITRHMEEKFGIPWVEYNFFGPTKIEASLREIASKFDDK-IKEGAERVIAKYKPQMEA
R_vannielii_NifDK MNYVARHMEEKYGVPWVEYNFFGPTKILESLRKIAAQFDDK-IKENAEKVIAKYQPLMDA
M_parvus_NifDK MNYISRHMEEKYGIPWCEYNFFGPSKIEESLRKIASHFDDK-IKEGAERVIAKYRPLMDA
M_bryophila_NifDK MNYISRHMEEKYGIPWCEYNFFGPSKIAESLRKIASFFDDK-IKEGAERVIAKYQPLMDA
*: . :. :. :* . : * :* : *. : : :* .
A_vinelandii_AnfDK ELDWYKERLMGKKVCLWP--GGSKLWHWAHVIEEEMGLKVVSVYIKFGHQGDMEKG----
A_vinelandii_VnfDK KLDWYKERLQGKKMAIWT--GGPRLWHWTKSVEDDLGVQVVAMSSKFGHEEDFEKV----
A_chroococcum_VnfDK KLDWYKERLQGKKMAIWT--GGPRLWHWTKSVEDDLGIQVVAMSSKFGHEEDFEKV----
A_beijerinckii_VnfDK KLDWYKERLRGKKMAIWT--GGPRLWHWTKSVEDDLGIQVVAMSSKFGHEEDFEKV----
R_vannielii_VnfDK QLDWYKERLKGKKMAIWT--GGPRLWHWTKSVEDDMGMTVVAMSSKFGHQEDFEKV----
A_brasilense_VnfDK KLDWYKERLKGTRMAIWT--GGPRLWHWTKSVEDDLGVEVVAMSSKFGHEEDFEKV----
Appendix
120
R_palustris_VnfDK QLDWYKERLKGTKMAIWT--GGPRLWHWTKSVEDDLGVQVVAMSSKFGHQEDFEKV----
C_pasteurianum_NifDK DLDYFKEKLQGKTACLYV--GGSRSHTYMNML-KSFGVDSLVAGFEFAHRDDYEGREVIP
P_stutzeri_NifDK VIAKYRPRLEGKRVMLYV--GGLRPRHVIGAY-EDLGMEVVGTGYEFGHNDDYDRT----
A_vinelandii_NifDK VVAKYRPRLEGKRVMLYI--GGLRPRHVIGAY-EDLGMEVVGTGYEFAHNDDYDRT----
A_chroococcum_NifDK VVAKYRPRLEGKRVMLYI--GGLRPRHVIGAY-EDLGMEVVGTGYEFAHNDDYDRT----
A_beijerinckii_NifDK VVAKFRPRLEGKRVMLYI--GGLRPRHVIGAY-EDLGMEVVGTGYEFAHNDDYDRT----
K_pneumoniae_NifDK IIAKYRPRLEGRKVLLYM--GGLRPRHVIGAY-EDLGMEIIAAGYEFAHNDDYDRT----
T_lignilytica_NifDK VIAKYKPRLEGKKVMLYV--GGLRPRHVIGAY-EDLGMEIVGAGYEFAHNDDYDRT----
A_brasilense_NifDK VVGDAALRAHGTKGRVPLKPLGPRHRPIVDAY-HDLGMEIVGTGYEFAHNDDYQRT----
G_diazotrophicus_NifDK VIEKFRPRLAGKKVMLYV--GGLRPRHVVNAY-NDLDMEIVGTGYEFGHNDDYQRT----
R_palustris_NifDK VIAKYRPRLEGKKVMLYV--GGLRPRHVIGAY-EDLGMEVVGTGYEFGHNDDYQRT----
R_vannielii_NifDK VVAKYRPRLEGKKVMLYV--GGLRPRHVIGAY-EDLGMEVVGTGYEFAHNDDYQRT----
M_parvus_NifDK VIAKYRPRLEGKKVMLYV--GGLRPRHVIGAY-EDLGMEVVGTGYEFGHNDDYQRT----
M_bryophila_NifDK VIAKYRPRLEGKTVMLYV--GGLRPRHVIGAY-EDLGMEVVGTGYEFGHNDDYQRTA---
: : * : * : ..:.: : :*.*. * :
A_vinelandii_AnfDK ------------------------------------------------IARCGEGTLAID
A_vinelandii_VnfDK ------------------------------------------------IARGKEGTYYID
A_chroococcum_VnfDK ------------------------------------------------IARGKEGTYYID
A_beijerinckii_VnfDK ------------------------------------------------IARGKEGTYYID
R_vannielii_VnfDK ------------------------------------------------IARGAAGTYYVD
A_brasilense_VnfDK ------------------------------------------------IARGREGTYYID
R_palustris_VnfDK ------------------------------------------------IARGQSGTYYVD
C_pasteurianum_NifDK TIKIDADSKNIPEITVTPDEQKYRVVIPEDKVEELKKAGVPLSSYGGMMKEMHDGTILID
P_stutzeri_NifDK ------------------------------------------------LKEMGNATLLYD
A_vinelandii_NifDK ------------------------------------------------MKEMGDSTLLYD
A_chroococcum_NifDK ------------------------------------------------IKEMGDSTLLYD
A_beijerinckii_NifDK ------------------------------------------------MKEMGDSTLLYD
K_pneumoniae_NifDK ------------------------------------------------LPDLKEGTLLFD
T_lignilytica_NifDK ------------------------------------------------LKELPEATLLFD
A_brasilense_NifDK ------------------------------------------------QHYVKEGTLIYD
G_diazotrophicus_NifDK ------------------------------------------------GHYVKEGTLIYD
R_palustris_NifDK ------------------------------------------------THYVKDGTLIYD
R_vannielii_NifDK ------------------------------------------------THYIKDGTLVYD
M_parvus_NifDK ------------------------------------------------THYVKDGTLIYD
M_bryophila_NifDK ------------------------------------------------QHYVKDGTIIYD
.* *
A_vinelandii_AnfDK DPNELEGLEALEMLKPDIILTGKRPGEVAKKVRVPYLNAHAYH-NGPYKGFEGWVRFARD
A_vinelandii_VnfDK DGNELEFFEIIDLVKPDVIFTGPRVGELVKKLHIPYVNGHGYH-NGPYMGFEGFVNLARD
A_chroococcum_VnfDK DGNELEFFEIIDLVKPDVIFTGPRVGELVKKLHIPYVNGHGYH-NGPYMGFEGFVNLARD
A_beijerinckii_VnfDK DGNELEFFEIIDLVKPDVIFTGPRVGELVKKLHIPYVNGHGYH-NGPYMGFEGFVNLARD
R_vannielii_VnfDK DGNELEFFEILQHVHPDVIFTGPRVGELVKKLHIPYINGHAYH-NGPYMGFEGFVNMARD
A_brasilense_VnfDK DGNELEFFEIIDLVKPDVIFTGPRVGELVKKQHIPYVNGHAYH-NGPYMGFEGFVNLARD
R_palustris_VnfDK DGNELEFFEIIDLVKPDVIFTGPRVGELVKKLHIPYVNGHAYH-NGPYMGFEGFVNLARD
C_pasteurianum_NifDK DMNHHDMEVVLEKLKPDMFFAGIKEKFVIQKGGVLSKQLHSYDYNGPYAGFRGVVNFGHE
P_stutzeri_NifDK DVTGYEFEEFVKRIKPDLIGSGIKEKYIFQKMGIPFRQMHSWDYSGPYHGFDGFAIFARD
A_vinelandii_NifDK DVTGYEFEEFVKRIKPDLIGSGIKEKFIFQKMGIPFREMHSWDYSGPYHGFDGFAIFARD
A_chroococcum_NifDK DVTGYEFEEFVKKVKPDLIGSGIKEKFIFQKMGIPFRQMHSWDYSGPYHGFDGFAIFARD
A_beijerinckii_NifDK DVTGYEFEEFVKKVKPDLIGSGIKEKFIFQKMGIPFRQMHSWDYSGPYHGFDGFAIFARD
K_pneumoniae_NifDK DASSYELEAFVKALKPDLIGSGIKEKYIFQKMGVPFRQMHSWDYSGPYHGYDGFAIFARD
T_lignilytica_NifDK DVTGYEFEEFVKAVKPDLIGSGIKEKFIFQKMGIPFRQMHSWDYSGPYHGYDGFAIFARD
A_brasilense_NifDK DVTAFELEKFVELMRPDLVASGIKEKYVFQKMGLPFRQMHSWDYSGPYHGYDGFAIFARD
G_diazotrophicus_NifDK DVTGYELEKFIEGIRPDLVGSGIKEKYPVQKMGIPFRQMHSWDYSGPYHGYDGFAIFARD
R_palustris_NifDK DVTGYEFEKFVEKVRPDLVGSGVKEKYIFQKMGVPFRQMHSWDYSGPYHGYDGFGIFARD
R_vannielii_NifDK DVTAYELEKFVEAIKPDLVGSGIKEKYVFQKMGIPFRQMHSWDYSGPYHGYDGFAIFARD
M_parvus_NifDK DVTGYEFEKFVEKIQPDLVGSGIKEKYVFQKMGVPFRQMHSWDYSGPYHGYDGFAIFARD
M_bryophila_NifDK DVTGYEFEKFVEKIQPDLVGSGIKEKYVFQKMGVPFRQMHSWDYSGPYHGYDGFAIFARD
* . : :. ::**:. :* : :* : : *.:. .*** *: * :.::
A_vinelandii_AnfDK IYNAIYSPIHQLSGIDITKDNAPEWGNGFRTRQMLSDGNLSDAVRNSETLRQYTGGYDSV
A_vinelandii_VnfDK MYNAVHNPLRHLAAVDIRDKSQTTPVI--------VRGAA-----------------MSN
A_chroococcum_VnfDK TYNAVHNPLRHLAAVDIRDSSQTTPVI--------VRGAA-----------------MSN
A_beijerinckii_VnfDK TYNAVHNPLRHLAAVDIRDSSQTTPVI--------VRGAA-----------------MSN
R_vannielii_VnfDK IYNATNNPLLSLAAKDIRGAKKTAPVL--------E---A-----------------AMG
A_brasilense_VnfDK MYNAVNNPLLKLAAKDIRGANRIAYKE--------A---A-----------------EMS
R_palustris_VnfDK MYNAVHNPLLKLAATDIRGETSTRLLE--------A---A-----------------EMG
C_pasteurianum_NifDK LVNGIYTPAWKMITPPWKKASSESKVV--------VGGE---------------------
P_stutzeri_NifDK MDMTLNNPCWKKLQAPWQKAEESAEKV--------AASAMSQQVDNIK---------PSY
A_vinelandii_NifDK MDMTLNNPCWKKLQAPWEAS-EGAEKV--------AASAMSQQVDKIK---------ASY
A_chroococcum_NifDK MDMTLNNPCWKKLQAPWEAS-EAAEKV--------AASAMSQQVDNIK---------ASY
A_beijerinckii_NifDK MDMTLNNPCWKKLQAPWQQADEAAEKV--------AASAMSQQVDNIK---------ASY
K_pneumoniae_NifDK MDMTLNNPAWNELTAPWLK------------------SAMSQTIDKIN---------SCY
T_lignilytica_NifDK MDMTLNNPCWSKQTAPWKK------------------SAMSQNIENIK---------SCY
A_brasilense_NifDK MDLAINNPVWGIMKAPFMSMS--HP--------------VSQSADKVI---------DHF
G_diazotrophicus_NifDK MDLAINNPVWSLFKAPWKNA------------------AMPQNVDKIL---------DHA
R_palustris_NifDK MDIAINAPVWKLTKAPWS---------------------MTETAEKIR---------DHF
R_vannielii_NifDK MDMAINSPVWNLTKAPF----------------------MVQNAENVV---------DHF
Appendix
121
M_parvus_NifDK MDMAINAPVWKMARAPWA--------------------AMPQNADNVL---------DHF
M_bryophila_NifDK MDMAINSPVWKMTKAPWKSEP--APLL--------QAAEMPQNAENVL---------DHF
*
A_vinelandii_AnfDK SKLREREYPAFERKVGMT-----------------------CEVKEKGRVGTINPIFTCQ
A_vinelandii_VnfDK C--------ELTVLKPAE-----------------------VKLSPRDREGIINPMYDCQ
A_chroococcum_VnfDK C--------ELTVLKPAE-----------------------VKLVKREREGIINPMYDCQ
A_beijerinckii_VnfDK C--------ELTVLKPAE-----------------------VKLSPRDREGIINPMYDCQ
R_vannielii_VnfDK C-----------------------------------------EVVSKDRVGVINPMYDCQ
A_brasilense_VnfDK C-----------------------------------------EITSKDRAGIINPMFDCQ
R_palustris_VnfDK C-----------------------------------------EVISKERVGVINPMYDCQ
C_pasteurianum_NifDK ----------------AMLDAT-------------------PKEIVERKALRINPAKTCQ
P_stutzeri_NifDK PLFRDEDYKDMLAKKRDAFEEK-HPQDKIDEVFQWTTTQEYQELNFQREALTVNPAKACQ
A_vinelandii_NifDK PLFLDQDYKDMLAKKRDGFEEK-YPQDKIDEVFQWTTTKEYQELNFQREALTVNPAKACQ
A_chroococcum_NifDK PLFLDEDYKDMLAKKRDGFEEK-HPQEKIDEVFQWTTTEEYKELNFQREALTINPAKACQ
A_beijerinckii_NifDK PLFLDQDYKDMLAKKRDGFEEK-HPQEKIDEVFQWTTTQEYQELNFQREALTINPAKACQ
K_pneumoniae_NifDK PLFEQDEYQELFRNKR-QLEEA-HDAQRVQEVFAWTTTAEYEALNFRREALTVDPAKACQ
T_lignilytica_NifDK PLFEQDEYQNLFASKRANYEEA-HDAAKVREVFEWTTTEEYKELNFKREALTIDPAKACQ
A_brasilense_NifDK TLFRQPEYKELFERKKTEFEYGPTPTKEVARVSAWTKTEEYKEKNLPVEAVVINPTKACQ
G_diazotrophicus_NifDK PLFREPEYQEMLAEKAK-LENM-PPADKVVEIADWTKSWEYREKNFARESLSVNPAKACQ
R_palustris_NifDK ELFREPQYEELMENKRKNFENY-VGDAEVTRVADWTKTKEYQDKNFAREALVINPAKACQ
R_vannielii_NifDK QLFRGPEYREMFKNKRDNFENG-TPDEEVNRVAEWTKTVEYQEKNFAREALTINPAKACQ
M_parvus_NifDK NLFRQPEYREMFENKKKNFENP-VADNELERVREWTKTEEYKEKNFAREALTVNPAKACQ
M_bryophila_NifDK DLFRGPEYQQMLANKKKMFENP-RDPAEVERIREWAKTPEYKEKNFAREALVVNPAKACQ
. ::* **
A_vinelandii_AnfDK PAGAQFVSIGIKDCIGIVHGGQGCVMFVRLIFSQHYKESFELASSSLHEDGAVFGACGRV
A_vinelandii_VnfDK PAGAQYAGIGIKDCIPLVHGGQGCTMFVRLLFAQHFKENFDVASTSLHEESAVFGGAKRV
A_chroococcum_VnfDK PAGAQYAGIGVKDCIPLVHGGQGCTMFVRLLFAQHFKENFDVASTSLHEESAVFGGAKRV
A_beijerinckii_VnfDK PAGAQYAGIGVKDCIPLVHGGQGCTMFVRLLFAQHFKENFDVASTSLHEESAVFGGAKRV
R_vannielii_VnfDK PAGAQYAGIGIKDCIPLVHGGQGCTMFVRLLFAQHFKENFDIASSSLHEDSAVFGGHDRI
A_brasilense_VnfDK PAGAQYAGIGVKDSIPLVHGGQGCSMFVRLLFAQHFKENFDIASTSLHEESAVFGGNKRM
R_palustris_VnfDK PAGAQYAGIGVKDCIPLVHGGQGCTMFVRLLFAQHFKENFDIASTSLHEDSAVFGGHQRI
C_pasteurianum_NifDK PVGAMYAALGIHNCLPHSHGSQGCCSYHRTVLSRHFKEPAMASTSSFTEGASVFGGGSNI
P_stutzeri_NifDK PLGSVLCALGFEKTMPYVHGSQGCVAYFRTYFNRHFKEPISCVSDSMTEDAAVFGGQQNM
A_vinelandii_NifDK PLGAVLCALGFEKTMPYVHGSQGCVAYFRSYFNRHFREPVSCVSDSMTEDAAVFGGQQNM
A_chroococcum_NifDK PLGAVLCALGFEKTMPYVHGSQGCVAYFRSYFNRHFREPVSCVSDSMTEDAAVFGGQQNM
A_beijerinckii_NifDK PLGAVLCALGFEKTMPYVHGSQGCVAYFRSYFNRHFREPVSCVSDSMTEDAAVFGGQQNM
K_pneumoniae_NifDK PLGAVLCSLGFANTLPYVHGSQGCVAYFRTYFNRHFKEPIACVSDSMTEDAAVFGGNNNM
T_lignilytica_NifDK PLGAVLCSLGFEKTLPYVHGSQGCVAYFRTYFNRHFREPVACVSDSMTEDAAVFGGHANM
A_brasilense_NifDK PIGAMLAAQGFEGTLPFVHGSQGCVSYYRTHLTRHFKEPNSAVSSSMTEDAAVFGGLNNM
G_diazotrophicus_NifDK PLGAVFVASGFERTMSFVHGSQGCVAYYRSHLSRHFKEPSSAVSSSMTEDAAVFGGLNNM
R_palustris_NifDK PLGAVFAAVGFEKTLPFVHGSQGCVAYYRSHFTRHFKEPTSCVSSSMTEDAAVFGGLNNM
R_vannielii_NifDK PLGAVFAAVGFEGTLPFVHGSQGCVAYYRSHFSRHFKEPTSAVSSSMTEDAAVFGGLTNM
M_parvus_NifDK PLGAVFAAVGFESTIPFVHGSQGCVAYYRSHFSRHFKEPTSCVSSSMTEDAAVFGGLNNM
M_bryophila_NifDK PLGAVFAAIGFEETIPFVHGSQGCVAYYRSHLSRHFKEPTSCVSSSMTEDAAVFGGLNNM
* *: . *. : **.*** : * : :*::* : *: * .:***. .:
A_vinelandii_AnfDK EEAVDVLLSRYPDVKVVPIITTCSTEIIGDDVDGVIKKLNEGLLKEKFPDREVHLIAMHT
A_vinelandii_VnfDK EEGVLVLARRYPNLRVIPIITTCSTEVIGDDIEGSIRVCNR-ALEAEFPDRKIYLAPVHT
A_chroococcum_VnfDK EEGVLVLARRYPELRLIPIITTCSTEVIGDDIEGTINVCNR-ALAAEFPERKIYLAPVHT
A_beijerinckii_VnfDK EEGVLVLARRYPELRVIPIITTCSTEVIGDDIEGCIRVCNK-ALESEFPERKIYLAPVHT
R_vannielii_VnfDK EDGIVVLAQRYPELRVIPLITTCSTEVIGDDCEGAVVRVQK-KLKEMFPNRKFHIPVVHT
A_brasilense_VnfDK EEGVQVLVNRYPELRVIPIITTCSTEVIGDDVEGTINVLNR-WLKENHPYRKVHLVPVHT
R_palustris_VnfDK IDGVLTLARRYPELRVIPVITTCSTEIIGDDIEGNIGKANA-ALKKEFPGRKIHVVPVHT
C_pasteurianum_NifDK KTAVKNIFSLYN-PDIIAVHTTCLSETLGDDLPTYISQMED----AGSIPEGKLVIHTNT
P_stutzeri_NifDK KDGLANCKATYK-PDMIAVSTTCMAEVIGDDLNAFINNSKK----EGFIPEDYPVPYAHT
A_vinelandii_NifDK KDGLQNCKATYK-PDMIAVSTTCMAEVIGDDLNAFINNSKK----EGFIPDEFPVPFAHT
A_chroococcum_NifDK KDGLQNCKATYK-PDMIAVSTTCMAEVIGDDLNAFINNSKK----EGFIPDEYPVPFAHT
A_beijerinckii_NifDK KDGLQNCKATYK-PDMIAVSTTCMAEVIGDDLNAFINNSKK----EGFIPDEYPVPFAHT
K_pneumoniae_NifDK NLGLQNASALYK-PEIIAVSTTCMAEVIGDDLQAFIANAKK----DGFVDSSIAVPHAHT
T_lignilytica_NifDK NDGLQNALALYK-PEMIAVSTTCMAEVIGDDLQAFTNNAKK----DGFVPQDFPVPYAHT
A_brasilense_NifDK IDGLQ-RYALYK-PKMIAVLTTCMAEVIGDDLSGFINNAKN----KESVPADFPVPFAHT
G_diazotrophicus_NifDK VDGLANTYKLYD-PKMIAVSTTCMAEVIGDDLHAFIQTAKG----KGSVPEEFDVPFAHT
R_palustris_NifDK IDGLANAYALYK-PKMIAVSTTCMAEVIGDDLNAFIKNAKE----KGSVPQEFDVTYAHT
R_vannielii_NifDK VDGLSNAYNMYK-PKMIAVSTTCMAEVIGDDLNAFIKTSRE----KGSIPMDYDVTFAHT
M_parvus_NifDK IDGLANTYNMYK-PKMISVSTTCMAEVIGDDLNAFIKTSKE----KGSVPQEFDVPFAHT
M_bryophila_NifDK IDGLANTYNMYK-PKMIAVSTTCMAEVIGDDLNAFIKTSKE----KGSVPGEYDVPFAHT
.: * :: : *** :* :*** . : :*
A_vinelandii_AnfDK PSFVGSMISGYDVAVRDVVRHFAK---------REAPNDKINLLTGWVNP--GDVKELKH
A_vinelandii_VnfDK PSFKGSHVTGYAECVKSVFKTITDAHG------KGQPSGKLNVFPGWVNP--GDVVLLKR
A_chroococcum_VnfDK PSFKGSHVTGYAECVKSMFKTITEVHG------KGQPSGKLNVFPGWVNP--GDVVLLKR
A_beijerinckii_VnfDK PSFKGSHVTGYAECVKSMFKTVTEVHG------KGQPSGKLNVFPGWVNP--GDVVLLKR
R_vannielii_VnfDK PSFKSSQVGGYAECVLSVVKDIAK--T------KTTPSGKLNFFPGWINP--GDLVQLKH
A_brasilense_VnfDK PSFKGSHVSGYDECVKAMVKTMSK--T------KAKPNGKLNVFPGWVNP--GDVVLLKS
R_palustris_VnfDK PSFKASQVGGYAECMQAIIKTICD--H------KVAPTGKLNVFPGWVNP--GDVVLLKH
C_pasteurianum_NifDK PSYVGSHVTGFANMVQGIVNYLSENTG--------AKNGKINVIPGFVGP--ADMREIKR
Appendix
122
P_stutzeri_NifDK PSFVGSHVTGWDNMFEGIARYFTLNHM---DDKVVGSNHKINVVPGFETY-LGNFRVIKR
A_vinelandii_NifDK PSFVGSHVTGWDNMFEGIARYFTLKSM---DDKVVGSNKKINIVPGFETY-LGNFRVIKR
A_chroococcum_NifDK PSFVGSHVTGWDNMFEGIARYFTVKSM---DDKVVGSNGKLNIVPGFETY-LGNFRVIKR
A_beijerinckii_NifDK PSFVGSHVTGWDNMFEGIARYFTVKSM---DDKVVGSNGKLNIVPGFETY-LGNFRVIKR
K_pneumoniae_NifDK PSFIGSHVTGWDNMFEGFAKTFTAD-Y---QG-QPGKLPKLNLVTGFETY-LGNFRVLKR
T_lignilytica_NifDK PSFVGSHITGWDNMFEGFANTFTAKDV---EY-KPGSNGKLNIVTGFETY-LGNYRVIKR
A_brasilense_NifDK PAFVGSHIVGYDNMIKGVLTHFWGTS----ENFDTPKNETINLIPGFDGFAVGNNRELKR
G_diazotrophicus_NifDK PAFVGSHVTGYDNMLKGILEHFWKGQTA-------VPNRSVNIIPGFDGFAVGNNRELKR
R_palustris_NifDK PAFVGSHITGYDNTMKGIVEHFWDGKSGTVEKLERKPNESINFLGGFDGYTVGNIREIKR
R_vannielii_NifDK PAFVGSHITGYDNTMKGVVEYFWGGKAGLAPKLERTPNESINFFGGFDGYTVGNLREVKR
M_parvus_NifDK PAFVGSHITGYDNVMKGVLEHFWDGKAGTEPKLERTPNNSINFLGGFDGYTVGNLREVKR
M_bryophila_NifDK PAFVGSHVTGYDNALKGVLEHFWDGKAGTVDKLERKPNEKINFIGGFDGYTVGNTREIKR
*:: .* : *: . . . .:*.. *: .: :*
A_vinelandii_AnfDK LLGEMDIEANVLFEIE-SFDSPILPDGSAVSHGNTTIEDLIDTGNARATFALNRYEGTKA
A_vinelandii_VnfDK YFKEMDVEANIYMDTE-DFDSPMLPNKSIETHGRTTVEDIADSANALATLSLARYEGNTT
A_chroococcum_VnfDK YFKEMGVDATVFMDTE-DFDSPMLPNKSIETHGRTTVEDIADSANALATLALARYEGATT
A_beijerinckii_VnfDK YFTEMGVDATVFMDTE-DFDSPMLPNKSIETHGRTTVEDIADSANALATLSLARYEGATT
R_vannielii_VnfDK YVKEMGVDAHFFFDIE-DFDSPMMPDKSISTHGRTTVEDIADAPNALASISLARYESDPA
A_brasilense_VnfDK YLKDMGVDATVFMDTE-DFDSPMLPDKTNHTHGRTTVEDFQGAPNAVGSIALARYEGASA
R_palustris_VnfDK YFNEMGVDATIFMDTE-DFDSPMLPDKSIHTHGRTTVEDIKHASGAIASLALAKYEGGST
C_pasteurianum_NifDK LFEAMDIPYIMFPDTSGVLDGPTTGEYKMYPEGGTKIEDLKDTGNSDLTLSLGSYASDLG
P_stutzeri_NifDK MLKEMDVGYSLLSDPEEVLDTPADGQFRMYS-GGTTQDEIKDAPNALNTLLLQPWQLEKT
A_vinelandii_NifDK MLSEMGVGYSLLSDPEEVLDTPADGQFRMYA-GGTTQEEMKDAPNALNTVLLQPWHLEKT
A_chroococcum_NifDK MLSEMGVGYSLLSDPEEVLDTPADGQFRMYA-GGTTQEEMKDAPNALSTVLLQPWQLEKT
A_beijerinckii_NifDK MLSEMGVGYSLLSDPEEVLDTPADGQFRMYA-GGTTQEEMKDAPNALSTVLLQPWQLEKT
K_pneumoniae_NifDK MMEQMAVPCSLLSDPSEVLDTPADGHYRMYS-GGTTQQEMKEAPDAIDTLLLQPWQLLKS
T_lignilytica_NifDK MLTEMGVPFSMLSDPSEVLDTPSDGQYRMYA-GGTTQAEMKDAPNAIDTLMLQPWHLVKT
A_brasilense_NifDK IAGLFGIQMTILSDVSDNFDTPADGEYRMYD-GGTPLEATKEAVHAKATISMQEYCTPQS
G_diazotrophicus_NifDK ILGMMGVQYTILSDVSDQFDTPSDGEYRMYD-GGTKIEAARDAVNADFTISLQEYCTPKT
R_palustris_NifDK IFELMGVDYTIFGDNSDVWDTPADGEFRMYD-GGTTLEQAANAVHAKATISMQEFCTEKT
R_vannielii_NifDK IFDLIGADYTIIGDNSDVWDTPADGEYRMYD-GGTKLEDVANAVNAKATISMQQYCTEKT
M_parvus_NifDK IFESMGVEYTLIGDNSDVWDTPTDGEFRMYD-GGTKLEDVANALHAKATISMQEYCTEKT
M_bryophila_NifDK IFSLMGVEATILADNSEVLDTPTDGEFRMYS-GGTTLEETAAALNAKGTISMQEYCTEKT
: . : . * * . * * : : :. : :
A_vinelandii_AnfDK AEYLQKKFEIPAIIGPTPIGIRNTDIFLQNLKKATGKPIPQSLAHERGVAIDALADLTHM
A_vinelandii_VnfDK GELLQKTFAVPNALVNTPYGIKNTDDMLRKIAEVTGKEIPESLVRERGIALDALADLAHM
A_chroococcum_VnfDK GEYLEKTFAVPNSLVNTPYGIKNTDDMLRKIAEITGKEIPESLVRERGIALDALADLAHM
A_beijerinckii_VnfDK GDLLQKTFAVPNALVNTPYGIKNTDDMLRKIAELTGKEIPESLVRERGIALDALADLAHM
R_vannielii_VnfDK AQHLQNAFGVPAAKLATPYGIANTDAMLEEISRITGKAIPDSLAHERGVAIDALQDLAHM
A_brasilense_VnfDK AEYLRDEFEVPAIVSPTPYGIKNTDDMLEAISRLTGREIPDSLVRERGIALDALQDLAHM
R_palustris_VnfDK ADYLKTEFEVPSHLVSTPYGIANTDAMLKRISEITGKPIPDSLVKERGIALDALQDLAHM
C_pasteurianum_NifDK AKTLEKKCKVPFKTLRTPIGVSATDEFIMALSEATGKEVPASIEEERGQLIDLMID-AQQ
P_stutzeri_NifDK KKFVEGTWKHETPKLSIPMGLDWTDEFLMKVSEITGQPIPESLAKERGRLVDMMTD-SHT
A_vinelandii_NifDK KKFVEGTWKHEVPKLNIPMGLDWTDEFLMKVSEISGQPIPASLTKERGRLVDMMTD-SHT
A_chroococcum_NifDK KKLVEGTWKHEVPKLNIPMGLDWTDEFLMKVSEISGQPIPASLTKERGRLVDMMTD-SHT
A_beijerinckii_NifDK KKLVEGTWKHEVPKLNIPMGLDWTDEFLMKVSEISGQPIPASLTKERGRLVDMMTD-SHT
K_pneumoniae_NifDK KKVVQEMWNQPATEVAIPLGLAATDELLMTVSQLSGKPIADALTLERGRLVDMMLD-SHT
T_lignilytica_NifDK KKVVKETWGQPGTELNIPMGLEWTDEFLMKVSALTGKAIPASLELERGRLVDMITD-SHT
A_brasilense_NifDK LQFIKREGPAGRQAYNYPMGVTGTDELLMKLAELSGKPSR-GVKLERGRLVDAIAD-SHT
G_diazotrophicus_NifDK LEYCQSFGQK-TASFHYPLGIGATDDLLQKLSEISGKPVPQELEMERGRLVDAPAD-SQA
R_palustris_NifDK LATIADHGQE-VVAFNHPVGIAGTDRFLQAVSRITGKAIPEALTKERGRLVDAIGD-SSA
R_vannielii_NifDK LDYIKTWGQE-TVALNHPIGVAGTDKFLMEVSRLTGKPIPDALKKERGRLVDAIAD-SHN
M_parvus_NifDK LPYIATKGQE-VVALNHPVGVAATDKFLMEISRLSGKPISEELTKERGRLVDAIAD-SSA
M_bryophila_NifDK LPLIQAHGQE-VAAFNHPIGVAATDKFLLEVSRMTGKPIPAELEKERGRLVDAMAD-SSA
* *: ** :: : :*: : *** :* * :
A_vinelandii_AnfDK FLAEKRVAIYGAPDLVIGLAEFCLDLEMKPVLLLLGDDN-SKYVDDPRIKALQENVDYGM
A_vinelandii_VnfDK FFANKKVAIFGHPDLVLGLAQFCMEVELEPVLLLIGDDQGNKYKKDPRIEELKNTAHFDI
A_chroococcum_VnfDK FFANKKVAIFGHPDLVLGLAQFCLEVELEPVLLLIGDDQGSKYKKDPRLQELKDAAHFDM
A_beijerinckii_VnfDK FFANKKVAIFGHPDLVFGLAQFCLEVELEPVLLLIGDDQGNKYKKDPRLQELKDAAHFDM
R_vannielii_VnfDK FFADKKVAIYGHPDLVLGLADFCTEVELKPTILMLGDDN-GRYKRDSRIKDLKDKVNWDM
A_brasilense_VnfDK FFADKKVALFGHPDLVIGLAQFCREVQLKPVLLLLGDDN-NKYKKDPRILELKNSVDHDM
R_palustris_VnfDK FFADKKVALFGHPDLVIGLAEFCLEVEMKPVLMLLGDDN-SRYKKDPRILALKNKCNWDM
C_pasteurianum_NifDK YLQGKKVALLGDPDEIIALSKFIIELGAIPKYVVTGTPG-MKFQKEIDAMLAEAGI-EGS
P_stutzeri_NifDK WLHGKRFALWGDPDFVMGMAKFLLELGAEPVHILAHNGN-KRWKKAMDAILESSPYGKNC
A_vinelandii_NifDK WLHGKRFALWGDPDFVMGLVKFLLELGCEPVHILCHNGN-KRWKKAVDAILAASPYGKNA
A_chroococcum_NifDK WLHGKRFALWGDPDFVMGMVKFLLELGCEPVHILCNNGS-KRWKKAVDAILEASPYGKNA
A_beijerinckii_NifDK WLHGKRFALWGDPDFVMGMVKFLLELGCEPVHILCNNGN-KRWKKAVDAILEASPYGKNA
K_pneumoniae_NifDK WLHGKKFGLYGDPDFVMGLTRFLLELGCEPTVILSHNAN-KRWQKAMNKMLDASPYGRDS
T_lignilytica_NifDK WLHGKKFGVYGDPDFVEGLVKFLLELGCEPTVILCNNGS-KKWKKSIEKMLADSPYGQGS
A_brasilense_NifDK HLHGKRFAVYGDPDFCLGMSKFLMELGAEPVHILSTSGS-KKWEKQVQKVLDACRSASSG
G_diazotrophicus_NifDK YLHGKTYAIYGDPDFVYGMARFVLETGGEPKHCLATNGN-KAWEAKMQELFDSSPFGAGC
R_palustris_NifDK HIHGKKFAIYGDPDLCYGLAEFILELGGEPVHILATNGN-KAWEAKVQALLDSSPFGAGC
R_vannielii_NifDK HIHGKKFAIYGDPDLCLGVAAFLLELGAEPTTILSTNGG-KSWEKKVRELLASSPYGKDC
M_parvus_NifDK HIHGKKFAIYGDPDLCYGLAAFLLELGAEPIHVLATNGG-KGWAEKMQRLFDSSPFGKGC
M_bryophila_NifDK HIHGKKFAIYGDPDLCLGLAGFLLELGAEPTHVLATNGN-KAWAQKVQALFDSSPFGKDC
Appendix
123
: * .: * ** .: * : * : : .
A_vinelandii_AnfDK EIVTNADFWELENRIKNEGLELDLILGHSKGRFISIDY-------NIPMLRVGFPTYDRA
A_vinelandii_VnfDK EIVHNADLWELEKRIN-AGLQLDLIMGHSKGRYVAIEA-------NIPMVRVGFPTFDRA
A_chroococcum_VnfDK EIVHNADLWELEKRIN-DGLQLDLIMGHSKGRYVAIEA-------NIPMVRVGFPTFDRA
A_beijerinckii_VnfDK EIVHNADLWELEKRIN-NGLQLDLIMGHSKGRYVAIEA-------NIPMVRVGFPTFDRA
R_vannielii_VnfDK EVVCNADLWELEKRCKDPATKPDIILGHSKGRYIAIDA-------NIPMVRVGFPSFDRA
A_brasilense_VnfDK QVVCNADLWDLERRIRDKEVELDLIMGHSKGRYIAIDN-------NIPMVRVGFPTFDRA
R_palustris_VnfDK EVVCNADLWELEKRALDKDYGIDLIMGHSKGRYVAIDA-------NIPMVRVGFPTFDRA
C_pasteurianum_NifDK KVKVEGDFFDVHQWIKNE--GVDLLISNTYGKFIAREE-------NIPFVRFGFPIMDRY
P_stutzeri_NifDK TVYIGKDLWHMRSLVFTD--KPDFMIGNSYGKFIQRDTLHKGKEFEVPLIRLGFPIFDRH
A_vinelandii_NifDK TVYIGKDLWHLRSLVFTD--KPDFMIGNSYGKFIQRDTLHKGKEFEVPLIRIGFPIFDRH
A_chroococcum_NifDK TVYVGKDLWHLRSLVFTD--KPDFMIGNSYGKFIQRDTLHKGKEFEVPLIRIGFPIFDRH
A_beijerinckii_NifDK TVYIGKDLWHLRSLVFTD--KPDFMIGNSYGKFIQRDTLHKGKEFEVPLIRIGFPIFDRH
K_pneumoniae_NifDK EVFINCDLWHFRSLMFTR--QPDFMIGNSYGKFIQRDTLAKGKAFEVPLIRLGFPLFDRH
T_lignilytica_NifDK EVYVGNDLWHFRSLMFTN--KPDFMIGNSYGKFIQRDTLAKGKAFEVPLIRLGFPIFDRH
A_brasilense_NifDK KAYGAKDLWHLRSLVFTD--KVDYIIGNSYGKYLERDT-------KIPLIRLTYPIFDRH
G_diazotrophicus_NifDK KAWGGKDLWHMRSLLATE--KVDLLIGNSYGKYLERDT-------DTPLIRLMFPIFDRH
R_palustris_NifDK KVYAGKDLWHLRSLLFTE--PVDFMIGNTYGKYLERDT-------GTPLIRLGFPVFDRH
R_vannielii_NifDK EVYAGKDLWHMRSLLFTR--PVDFLIGNTYGKYLERDT-------GTPLIRLGFPIFDRH
M_parvus_NifDK KAYAGKDLWHMRSLLFTE--PVDYLIGNTYGKYLDRDT-------GTPLIRIGFPIFDRH
M_bryophila_NifDK QVYAGKDLWHMRSLLFTE--PVDFLIGNTYGKYLERDT-------GTPLIRIGFPIFDRH
*::... * ::.:: *::: : *::*. :* **
A_vinelandii_AnfDK GLFRYPTVGYGGAIWLAEQMANTLFADMEHKKNKEWVLNV----W-
A_vinelandii_VnfDK GLYRKPSIGYQGAMELGEMIANAMFAHMEYTRNKEWILNT----W-
A_chroococcum_VnfDK GLYRKPSIGYQGAMELGEMIANAMFAHMEYTRNKEWILNT----W-
A_beijerinckii_VnfDK GLYRKPTIGYQGAMELGEMIANAMFAHMEYTRNKEWILNT----W-
R_vannielii_VnfDK GLYRHPLMGYRGAMLLGESIANALFTHMEYTKDREWLLNT----W-
A_brasilense_VnfDK GLYRKPTIGYKGAMELGEAIANALFAHMEYNKNKEWILNT----W-
R_palustris_VnfDK GLYRHPTMGYRGAMLLGETIANTLFAHMEYNKDREWLLNT----W-
C_pasteurianum_NifDK GHYYNPKVGYKGAIRLVEEITNVILDKIERECT-----EEDFEVVR
P_stutzeri_NifDK HLHRQTTLGYEGAMQMLTTLVNAVLERLDDETRGMQSTDYNYDLVR
A_vinelandii_NifDK HLHRSTTLGYEGAMQILTTLVNSILERLDEETRGMQATDYNHDLVR
A_chroococcum_NifDK HLHRQTTMGYEGAMQILTTLVNSILERLDEETRGMQATDYNHDLVR
A_beijerinckii_NifDK HLHRQTTMGYEGAMQILTTLVNSILERLDEETRGMQATDYNHDLVR
K_pneumoniae_NifDK HLHRQTTWGYEGAMNIVTTLVNAVLEKLDSDTSQLGKTDYSFDLVR
T_lignilytica_NifDK HLHRMTTLGYEGAMYMLTTLVNAVMEKLDSETMELGKTDYNFDLVR
A_brasilense_NifDK HHHRYPTWGYQGALNVLVRILDRIFEDMDANTNIVGETDYSFDLVR
G_diazotrophicus_NifDK HHHRFPVWGYQGALRVLVTLLDKIFDRLDDDTIQAGVTDYSFDLTR
R_palustris_NifDK HHHRSPVWGYQGSMNVLVKILDKIFDEMDKATNTAGKTDVSFDIIR
R_vannielii_NifDK HLHRYPVWGYQGALNVLVWILDKVFSEMDAHSNVAGKTDISFDIIR
M_parvus_NifDK HKHRYPVWGYQGSMNVLVWILDAIFEDIDRNTNVVAKSDYSFDIIR
M_bryophila_NifDK HHHRYPVWGYQGGMNTLVWVLDRIFESIDANTNVPAKSDYSFDIIR
. ** *.: : : :: :: :
6.5 Atom distances in N2ase structures
Table 20: Atom-atom distances within FeMo-cofactor of Mo-N2ase. Chain A from PDB-ID 3U7Q [116] was used
atom-atom distance measurments.
atom → atom distances [Å]
Mo → S1B/S3B/S4B 2.35 / 2.37 / 2.35
Mo → Fe5/Fe6/Fe7//Fe1 2.73 / 2.67 / 2.68 (av ≈ 2.69) // 7.00
Fe → Fe (prism: upper layer /
downwards via S2B, S3A, S5A) /
lower layer)
2.64, 2.65, 2.67 /
2.58, 2.61, 2.58 /
2.60. 2.63, 2.63
Fe → Fe (Fe1 - Fe2/Fe3/Fe4) 2.67 / 2.67 / 2.65
Fe → S (within ‘[4Fe-4S]’) 2.22 – 2.30
Fe → S (in belt S2B/S3A/S5A) 2.21 (Fe2) and 2.17 (Fe6) /
2.24 (Fe4) and 2.26 (Fe5) /
Appendix
124
2.22 (Fe3) and 2.21 (Fe7)
Fe → C 1.98 – 2.02
Table 21: Atom-atom distances in FeV-cofactor of ‘resting’ state VFe-protein structure and at the Mg binding sites.
atom → atom chain A [Å] chain D [Å] average [Å]
V → S1B/S3B/S4B 2.35 / 2.34 / 2.33 2.32 / 2.36 / 2.32 2.34 / 2.35 / 2.33
V → Fe5/Fe6/Fe7//Fe1 2.70 / 2.77 / 2.73 // 7.11 2.69 / 2.75 / 2.74 // 7.09 2.70 / 2.76 / 2.74 (av all 2.73) // 7.10
Fe → Fe (prism: upper layer / downwards via S2B, CO3, S5A/ lower layer)
2.62 , 2.63, 2.63 /
2.61, 2.76, 2.60 /
2.57, 2.60, 2.63
2.63, 2.64, 2.65 /
2.63, 2.78, 2.60 /
2.57, 2.60, 2.64
2.63, 2.64, 2.64 /
2.62, 2.77, 2.60 /
2.57, 2.60, 2.64
Fe → Fe (Fe1 – Fe2/Fe3/Fe4) 2.71 / 2.66 / 2.58 2.68 / 2.65 / 2.57 2.70, 2.66 / 2.58
Fe → S (within ‘[4Fe-4S]’) 2.21 – 2.34 2.21 – 2.35 2.21 – 2.35
Fe → S (in belt S2B [Fe2 & Fe6] / S5A [Fe3 & Fe7])
2.21 & 2.17 / 2.23 & 2.27 2.23 & 2.21 / 2.23 & 2.25 2.22 & 2.20 / 2.23 & 2.26
Fe → O (to CO3) (Fe4 & Fe5) 1.97 & 1.93 1.93 & 1.94 1.95 & 1.94
Fe → C 1.95 – 2.05 1.98 – 2.04 1.95 – 2.05
C → O (in CO3) (OFe4/OFe5/O) 1.29 / 1.30 / 1.23 1.32 / 1.30 / 1.30 1.31 / 1.30 / 1.27
Mg → β-Glu70 / β’-Asp314 1.95 / 2.05 2.03 / 2.08 1.99 / 2.07
Mg → 6xH2O (between α-and δ-subunit)
2.06 – 2.12 2.04 – 2.16 2.04 – 2.16
Table 22: Atom-atom distances in FeV-cofactor and the active site of ‘active’ state VFe-protein structure and at the
Zn binding site. n.d. not defined as electron density is not good enough defined.
atom → atom chain A [Å] chain D [Å] average [Å]
V → S1B/S3B/S4B 2.35 / 2.37 / 2.31 2.36 / 2.37 / 2.32 2.36 / 2.37 / 2.32
V → Fe5/Fe6/Fe7//Fe1 2.71 / 2.79 / 2.76 // 7.15 2.70 / 2.77 / 2.76 // 7.13 2.71 /2.78 / 2.76 // 7.14
Fe → (prism: upper layer / down-wards via N2B, CO3, S5A /lower layer)
2.63, 2.64, 2.64 /
2.65, 2.79, 2.59 /
2.58, 2.58, 2.64
2.63, 2.64, 2.64 /
2.65, 2.78, 2.60 /
2.57, 2.57, 2.63
2.63, 2.64, 2.64 /
2.65, 2.79, 2.60 /
2.58, 2.58, 2.64
Fe → Fe (Fe1 - Fe2/Fe3/Fe4) 2.69 / 2.67 / 2.59 2.68 / 2.67 / 2.59 2.69 / 2.67 / 2.59
Fe → S (within ‘[4Fe-4S]’) 2.23 – 2.32 2.24 – 2.34 2.23 -2.34
Fe → S (in belt S5A [Fe3 & Fe7]) 2.23 & 2.25 2.23 & 2.25 2.23 & 2.25
Fe → O (to CO3) (Fe4 & Fe5) 1.96 & 1.97 1.96 & 1.98 1.96 & 1.98
Fe→ C 1.97 – 2.06 1.99 – 2.07 1.97 – 2.07
Fe → N2B (Fe2 & Fe6) 1.96 & 2.00 2.07 & 2.00 2.02 & 2.00
C → O (in CO3) (OFe4/OFe5/O) 1.28 / 1.28 / 1.26 1.28 / 1.30 / 1.27 1.28 / 1.29 / 1.27
Appendix
125
HS-/H2S to N2B 7.04 6.88 6.96
H2S to H2O
Carbamide-N Gln176
Carbamide-N Gly48
2.90
3.24
3.11
2.81
3.21
3.04
2.86
3.23
3.08
Gln176-O → N2B
His-180-N
2.54
2.84
2.42
2.83
2.48
2.84
Gln176-N → H2O
homocitrate
2.40
2.88
2.41
2.92
2.41
2.90
Zn → N α-His448, β’-His379 /
H2O
1.96, 2.01 /
2.09, 2.16
2.05, 2.07 /
n.d. / 2.18
2.01, 2.04 /
‘2.09’, 2.17
6.6 Data collection statistics
Table 23: Data collection, phasing and refinement statistics for ‘resting’ state VFe-protein. Values in parentheses are
for highest resolution shell. B-value analysis has been performed with program Moleman2.
Native MAD data
Data collection
Peak inflection
Space group P1 P1 P1
Cell dimensions
a, b, c (Å) 75.25, 79.79, 106.97 74.75, 79.29, 106.98 74.87, 79.36, 107,17
α, β, γ (°) 84.06, 72.62, 75.15 84.00, 72.83, 75.04 84.04, 72.85, 75.04
Wavelength (Å) 0.9000 1.7357 1.7413
Resolution (Å) 46.52 – 1.35 (1.37 – 1.35) 48.03 – 2.55 (2.61 – 2.55) 48.11 – 2.55 (2.61 – 2.55)
Rsym or Rmerge 0.088 (1.542) 0.089 (0.310) 0.104 (0.450)
Rp.i.m. [353] 0.055 (0.931) 0.030 (0.106) 0.035 (0.153)
CC(1/2) [354] 0.999 (0.559) 0.999 (0.988) 0.999 (0.976)
I / σI 10.8 (1.0) 26.0 (8.1) 22.1 (5.7)
Completeness (%) 96.1 (92.0) 91.0 (83.6) 90.6 (80.5)
Redundancy 7.0 (7.2) 19.0 (17.7) 18.9 (17.6)
unique reflections 484 439 (22 953) 67 220 (4 164) 67 245 (4 018)
Refinement
Rwork / Rfree 0.1054 / 0.1437
r.m.s. bond lengths (Å) 0.0299
Appendix
126
r.m.s. bond angles (Å) 4.0666
No. atoms 19 130
Protein 16 777
Ligand/ion 104
Water 2 249
Average B values (Å2) 18.457
Protein 17.44
Ligand/ion 10.36 – 11.16
Water 31.28
Wilson plot 11.808
Figure 46: The Ramachandran analysis was done with the program molprobity. No outliers are found in A) ‘resting’
state or B) ‘active’ state VFe-protein.
Table 24: Data collection and refinement statistics for ‘active’ state VFe-protein. Values in parentheses are for high-
est resolution shell. B-value analysis has been performed with program Moleman2.
Data collection
Space group P1
Cell dimensions
a, b, c (Å) 75.61, 79.75, 107.16
α, β, γ (°) 84.05, 72.44, 75.25
Wavelength (Å) 1.0000 and 0.8000
BA
Appendix
127
Resolution (Å) 48.38 – 1.20 (1.22 – 1.20)
Rsym or Rmerge 0.115 (0.901)
Rp.i.m. [353] 0.072 (0.686)
CC(1/2) [354] 0.998 (0.338)
I / σI 10.1 (1.2)
Completeness (%) 99.6 (99.1)
Redundancy 6.5 (4.3)
No. reflections 636 485 (31 312)
Refinement
Rcryst / Rfree 0.1174 / 0.1437
r.m.s. bond lengths (Å) 0.0311
r.m.s. bond angles (°) 4.0650
No. atoms 19 299
Protein 16 605
Ligand/ion 108
Water 2 586
average B values (Å2) 15.132
Protein 14.23
Water 28.56
Ligand/ion 8.39 – 15.53
Wilson plot 9.548
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Acknowledgement
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Acknowledgement
Vielen, vielen Dank an Prof. Dr. Oliver Einsle, der es mir ermöglicht hat meine Doktorarbeit auf
dem Gebiet der Nitrogenase durchzuführen. Ich danke Dir für die Unterstützung, die
aufgebrachte Geduld all die aufschlussreichen und bereichernden Diskussionen.
Many thanks to Prof. Dr. Susana Andrade that she took over the task of the second reviewer.
Vielen Dank an Prof. Dr. Kurz für die Übernahme des Drittprüfers.
PD Günter Fritz: Ich möchte PD Günter Fritz danken, der einerseits in seiner Beamtime einen
Datensatz von meinem Kristall aufgenommen hat und damit konkret zu meiner Dissertation
beigetragen hat. Anderseits für seine Tips and Tricks für Datenaufnahme und –auswertung.
Ich möchte den beiden Laborleitern Dr. Gerhardt und Dr. Wohlwend (Stephan und Wohli)
danken, die sich entweder durch den richtigen Rat, den notwendigen Tritt in den Hintern oder
einfach deren Fachkenntnis ausgezeichnet haben.
Dank gebührt natürlich unseren liebevollen Sekretärinnen Chrissi, Vero und Linda, die sich um
all die organisatorischen Probleme kümmern.
Ich möchte den mehr oder weniger konstanten Laborkollegen Frau Weiser, Elke und Toni
danken, die zur Ordnung im Labor beitragen, sich um „Labormaterial“ kümmern und vor allem
auch immer hilfsbereit sind. Dazu gehört natürlich auch der Phil, der sich hervorragend um das
Praktikum sowie meinen Zucker-und Koffeinspiegel kümmert. Dank gilt auch Herrn Hamacher
und vor allem an unseren Systemadministrator Manuel der wirklich bei allen Computerprobleme
immer geholfen hat.
Ich möchte mich bei meinen Diplom-, Bachelor- und Praktikums-Studenten/innen, Johanna
Mattay, Florian Schneider, Matthias Gschell, Erik Springer und Michael Rohde für deren
Unterstützung bedanken.
Ein Dankeschön an die Teams der Swiss Light Source und ESRF für deren großartige
Unterstützung bei der Datenaufnahme vor Ort.
Einen riesengroßen Dank an unsere EPR-Experten, Eva, Julia und Beni, die Tage im Keller
verbringen, um die Proben der anderen zu messen, und in letzter Zeit vor allem meine!
Acknowledgement
149
Ich danke einfach allen Laborkollegen, die für diese so angenehme Atmosphäre im Arbeitskreis
sorgen. Das sind vor allem die langjährigen Wegbegleiter Simon, Flo, Anton, Nikola, Martin, Lin,
Lorena, Nienke, Fabian aber natürlich auch die der neueren Generation um Beni, Christoph,
Prachi und Agostina. Natürlich gehört hier auch der AK Friedrich dazu.
Ich bedanke mich bei denen, die mich schon viel länger aushalten und einfach die Zeit in
Freiburg schon seit fast 12 Jahren so schön machen? Vor allem Danke an Lisa, Lucas, Mariam,
Markus, WoPe und Roman.
Danke an die Southside-Crew!
Einen großen Dank an das Nitrogenase-Team, Julia, Eva, Laure, Ivana, Chistian, Michael und
Jakob. Es war eine riesen Freude mit euch die lange Zeit zu arbeiten, zu diskutieren und Spaß
daran zu haben.
Ich möchte ausdrücklich meinen „Ziehvätern“ Julian, Thomas, Tobi & Tobi danken, die stets
Vorbild waren, mir als junger Padawan immer geholfen und gute Ratschläge gegeben haben. Hier
muss ich vor allem an die beiden Tobis denken, die einen in den stressigen Situationen wieder
runter geholt haben, die nicht unbeteiligt an einprägendsten Abenden in der langen Zeit waren
und einfach großartige Menschen sind! Und wenn es um prägende und großartige Menschen
geht, dann will ich genau hier der Bianca danken, auch wenn wir ab und zu vielleicht eine
geringfügig andere Sicht der Dinge haben ;-).
: Ich will so sehr meiner ganzen Familie danken, meinen Eltern, meinem Bruder „und meinem
Schwager“, meiner Oma und meinem Opa, meiner Tante Ria sowie Christoph und Tina, die
mich alle einfach immer unterstützt haben, egal was war, die mich wieder aufgebaut haben, wenn
ich in einem richtigen Tief war und die einem unglaublichen Halt geben!
Und Danke Anja‼! :-P
