8
559 Nitrogenase catalyzes the ATP-dependent reduction of dinitrogen to ammonia, which is central to the process of biological nitrogen fixation. Recent progress towards establishing the mechanism of action of this complex metalloenzyme reflects the contributions of a combination of structural, biochemical, spectroscopic, synthetic and theoretical approaches to a challenging problem with implications for a range of biochemical and chemical systems. Addresses *Howard Hughes Medical Institute, Division of Chemistry and Chemical Engineering, 147-75CH, California Institute of Technology, Pasadena, CA 91125, USA; e-mail: [email protected] Department of Biochemistry, University of Minnesota, Minneapolis, MN 55455, USA; e-mail: [email protected] Current Opinion in Chemical Biology 2000, 4:559–566 1367-5931/00/$ — see front matter © 2000 Elsevier Science Ltd. All rights reserved. Abbreviation EPR electron paramagnetic resonance Introduction Rationality notwithstanding, anyone studying nitrogenase should be excused for occasionally wondering whether a Faustian bargain might be required to establish the mecha- nism of dinitrogen reduction by this enzyme. The obstacles to studying nitrogenase are multiple and varied: they include the extreme oxygen lability of the proteins; the size and complexity of the iron-sulfur containing metallocen- ters; the similar and nondescript visible spectroscopic properties of multiple clusters, precluding facile monitoring of changes in individual groups; the absence of model com- pounds to evaluate potential mechanistically relevant states and intermediates; and the kinetic complexities of a multi- substrate, multi-component enzyme system with different simultaneously populated states. The contrast between studies on nitrogenase and metalloenzymes that react with dioxygen is informative; for enzymes reacting with dioxy- gen, the interplay between enzymology and chemical studies has led to the identification of key intermediates and a bountiful stream of model compounds that provide a secure chemical foundation for a detailed mechanistic understanding. Nevertheless, there are grounds for opti- mism that nitrogenase may enjoy similar levels of insight based upon the increasingly higher resolution structures of individual proteins and their varied complexes, augmented by genetic manipulation of the proteins and by spectro- scopic studies. Together with developments in the chemistry of relevant metalloclusters and in the theoretical modeling of these clusters, we may yet be able to peer inside the nitrogenase black box and achieve a detailed mechanistic description. Following a brief overview, this review highlights outstanding issues in our understanding of nitrogenase. Due to space limitations, only recent or selected references can be cited. Introduction to nitrogenase Nitrogenase is a two protein component system that cat- alyzes the reduction of dinitrogen to ammonia coupled to the hydrolysis of ATP (reviewed in [1–5]). Rather remark- ably, even after 35 years of study, the overall reaction stoichiometry is still not unambiguously determined. The uncertainties are expressed in the following equation for the overall enzyme reaction: N 2 + (6+2n)H + + (6+2n)e + p(6+2n)ATP 2NH 3 + nH 2 + p(6+2n)ADP + p(6+2n)P i In the ‘standard’ model, the evolution of one molecule of dihydrogen is coupled to the reduction of one molecule of dinitrogen, and two molecules of ATP are hydrolyzed per electron transferred, so that n = 1 and p = 2. However, there has not been a compelling demonstration of an obligatory mechanistic coupling of dihydrogen evolution and dinitrogen reduction, although dihydrogen is always observed as a product with ammonia, and a limiting ratio of 2 ammonia to 1 dihydrogen is obtained at high dinitro- gen gas pressure [6]. Because nitrogenase can also reduce protons to dihydrogen in the absence of dinitrogen, the stoichiometry of the observed product distribution could represent the outcome of two parallel paths from a com- mon branch in the electron transfer process. Additionally, while p = 2 represents the apparent limiting stoichiometry of ATP hydrolyzed per electron transferred, under many conditions the coupling is much less efficient and ratios >2 are observed. There are also reports of ratios approaching 1 when an all-ferrous form of Fe-protein is used as the electron source (see below). The most extensively studied form of nitrogenase is the molybdenum-containing system that consists of two com- ponent metalloproteins, the molybdenum-iron (MoFe-) protein and the iron (Fe-) protein, where the Fe-protein is the nucleotide-binding and electron-donating component, and the MoFe-protein contains the substrate-reducing site. Alternate nitrogenases exist that are homologous to this system, but with the molybdenum apparently substi- tuted by vanadium or iron [7]. We now have available X-ray crystallographic structures for the Fe-protein [8–10] and MoFe-protein [11–13,14 ] as well as for two complexes between the two proteins [15,16]. The Fe-protein is a dimer of two identical subunits that symmetrically coordi- nates a single [4Fe:4S] cluster. The isolated Fe-protein can bind MgADP or MgATP at a stoichiometry of two nucleotides per dimer, and participates intimately in the coupling between ATP hydrolysis and electron transfer to the MoFe-protein. Structurally, Fe-protein adopts a Nitrogenase: standing at the crossroads Douglas C Rees* and James B Howard

Nitrogenase: standing at the crossroads

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559

Nitrogenase catalyzes the ATP-dependent reduction ofdinitrogen to ammonia, which is central to the process ofbiological nitrogen fixation. Recent progress towardsestablishing the mechanism of action of this complexmetalloenzyme reflects the contributions of a combination ofstructural, biochemical, spectroscopic, synthetic andtheoretical approaches to a challenging problem withimplications for a range of biochemical and chemical systems.

Addresses*Howard Hughes Medical Institute, Division of Chemistry andChemical Engineering, 147-75CH, California Institute of Technology,Pasadena, CA 91125, USA; e-mail: [email protected]†Department of Biochemistry, University of Minnesota, Minneapolis,MN 55455, USA; e-mail: [email protected]

Current Opinion in Chemical Biology 2000, 4:559–566

1367-5931/00/$ — see front matter© 2000 Elsevier Science Ltd. All rights reserved.

AbbreviationEPR electron paramagnetic resonance

IntroductionRationality notwithstanding, anyone studying nitrogenaseshould be excused for occasionally wondering whether aFaustian bargain might be required to establish the mecha-nism of dinitrogen reduction by this enzyme. The obstaclesto studying nitrogenase are multiple and varied: theyinclude the extreme oxygen lability of the proteins; the sizeand complexity of the iron−sulfur containing metallocen-ters; the similar and nondescript visible spectroscopicproperties of multiple clusters, precluding facile monitoringof changes in individual groups; the absence of model com-pounds to evaluate potential mechanistically relevant statesand intermediates; and the kinetic complexities of a multi-substrate, multi-component enzyme system with differentsimultaneously populated states. The contrast betweenstudies on nitrogenase and metalloenzymes that react withdioxygen is informative; for enzymes reacting with dioxy-gen, the interplay between enzymology and chemicalstudies has led to the identification of key intermediatesand a bountiful stream of model compounds that provide asecure chemical foundation for a detailed mechanisticunderstanding. Nevertheless, there are grounds for opti-mism that nitrogenase may enjoy similar levels of insightbased upon the increasingly higher resolution structures ofindividual proteins and their varied complexes, augmentedby genetic manipulation of the proteins and by spectro-scopic studies. Together with developments in thechemistry of relevant metalloclusters and in the theoreticalmodeling of these clusters, we may yet be able to peerinside the nitrogenase black box and achieve a detailedmechanistic description. Following a brief overview, thisreview highlights outstanding issues in our understanding

of nitrogenase. Due to space limitations, only recent orselected references can be cited.

Introduction to nitrogenaseNitrogenase is a two protein component system that cat-alyzes the reduction of dinitrogen to ammonia coupled tothe hydrolysis of ATP (reviewed in [1–5]). Rather remark-ably, even after 35 years of study, the overall reactionstoichiometry is still not unambiguously determined. Theuncertainties are expressed in the following equation forthe overall enzyme reaction:

N2 + (6+2n)H+ + (6+2n)e– + p(6+2n)ATP →2NH3 + nH2 + p(6+2n)ADP + p(6+2n)Pi

In the ‘standard’ model, the evolution of one molecule ofdihydrogen is coupled to the reduction of one molecule ofdinitrogen, and two molecules of ATP are hydrolyzed perelectron transferred, so that n = 1 and p = 2. However,there has not been a compelling demonstration of anobligatory mechanistic coupling of dihydrogen evolutionand dinitrogen reduction, although dihydrogen is alwaysobserved as a product with ammonia, and a limiting ratioof 2 ammonia to 1 dihydrogen is obtained at high dinitro-gen gas pressure [6]. Because nitrogenase can also reduceprotons to dihydrogen in the absence of dinitrogen, thestoichiometry of the observed product distribution couldrepresent the outcome of two parallel paths from a com-mon branch in the electron transfer process. Additionally,while p = 2 represents the apparent limiting stoichiometryof ATP hydrolyzed per electron transferred, under manyconditions the coupling is much less efficient and ratios >2are observed. There are also reports of ratios approaching1 when an all-ferrous form of Fe-protein is used as theelectron source (see below).

The most extensively studied form of nitrogenase is themolybdenum-containing system that consists of two com-ponent metalloproteins, the molybdenum−iron (MoFe-)protein and the iron (Fe-) protein, where the Fe-protein isthe nucleotide-binding and electron-donating component,and the MoFe-protein contains the substrate-reducingsite. Alternate nitrogenases exist that are homologous tothis system, but with the molybdenum apparently substi-tuted by vanadium or iron [7]. We now have availableX-ray crystallographic structures for the Fe-protein [8–10]and MoFe-protein [11–13,14•] as well as for two complexesbetween the two proteins [15,16]. The Fe-protein is adimer of two identical subunits that symmetrically coordi-nates a single [4Fe:4S] cluster. The isolated Fe-protein canbind MgADP or MgATP at a stoichiometry of twonucleotides per dimer, and participates intimately in thecoupling between ATP hydrolysis and electron transfer tothe MoFe-protein. Structurally, Fe-protein adopts a

Nitrogenase: standing at the crossroadsDouglas C Rees* and James B Howard†

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polypeptide fold characteristic of P-loop-containingnucleotide-binding proteins such as observed for ras,G-proteins and related proteins [17,18]. The MoFe-protein is an α2β2 heterotetramer, where the α and β sub-units exhibit similar polypeptide folds consisting of threedomains of the α/β-type, with some extra helices. Thisprotein contains two copies each of two different types ofunusual metallocenters (Figure 1): the FeMo-cofactor, thelikely substrate reduction site; and the P-cluster, which is

believed to participate in electron transfer from theFe-protein to the FeMo-cofactor. Each cluster containseight metals and associated sulfurs that are distinctivelyarranged in ways that have been neither observed in anyother enzymes nor modeled synthetically. These metallo-clusters are coordinated by ligands contributed bydifferent domains of the MoFe-protein that are present ina common core composed of a four-stranded, parallelβ-sheet flanked by α-helices (Figure 2); a similar structural

560 Mechanisms

Figure 1

Structural models for the nitrogenasemetalloclusters and coordinating ligands.(a) FeMo-cofactor. (b) The dithionite reducedPN form of the P-cluster. (c) The oxidized POX

state of the P-cluster. Protein Data Bank[57,58] coordinate sets 3MIN and 2MIN wereused for (a), (b), and (c), respectively. Allfigures in this review were prepared withMOLSCRIPT [59].

Cys β95 Cys β95

Cys α62 Cys α62

Cys α88Cys α88

Fe3Fe3

Cys β70Cys β70

homocitrate

Fe7

Fe3 Fe1 Fe1

Cys α275

Fe5

Fe7

Fe8

Fe7

Fe4Fe4

Fe8Fe5

Mo

Fe1

His α442

Fe2

Fe5

Fe2

Fe4

Fe6

Fe2

Fe6Fe6

Ser β188 Ser β188

Cys α154Cys α154

Cys β153 Cys β153

(a) (b) (c)

Current Opinion in Chemical Biology

Figure 2

Ribbon diagrams of a common domain coreobserved to coordinate complexmetalloclusters in the nitrogenaseMoFe-protein and iron-only hydrogenases.This core consists of a four-stranded, parallelβ-sheet surrounded by α-helices, with strandorder 2–1−3−4, going from top to bottom asdepicted in this figure. The metalloclustersinteract with the carboxy-terminal end of thesheet, near the ‘crossover’ position [60]between β strands 1 and 3. Cluster ligandsare positioned in the loop between strands 3and 4, and immediately after loop 4. (a) TheFeMo-cofactor and MoFe-protein α-subunitdomain 3 (residues α350 to α442). (b) TheFeMo-cofactor and MoFe-protein α-subunitdomain 2 (residues α222 to α297). (c) TheP-cluster and MoFe-protein β-subunitdomain 1 (residues β85 to β188). Similarinteractions are also observed between theP-cluster and α-subunit domain 2. (d) TheH-cluster and residues 224 to 381 of theiron-only hydrogenase. Residues 269 to334 loop away from this region and havebeen excluded. Protein Data Bank coordinatesets 3MIN and 1FEH were used for (a−c),and (d), respectively.

(a)

(c)

(b)

(d)

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Page 3: Nitrogenase: standing at the crossroads

organization is also observed in the coordination of theH-cluster at the active site of iron-only hydrogenases [19].

Kinetics and mechanism of substrate reductionAt the protein level, the basic mechanism of nitrogenaseinvolves four steps: first, formation of a complex betweenthe reduced Fe-protein with two bound ATP moleculesand the MoFe-protein; second, electron transfer betweenthe two proteins coupled to the hydrolysis of ATP; third,dissociation of the Fe-protein accompanied by re-reductionand exchange of ATP for ADP; and finally, repetition of thiscycle until sufficient numbers of electrons (and protons)have been accumulated so that available substrates can bereduced. The relative positions of the metalloclustersobserved in the structures of complexes of the nitrogenaseproteins [15] indicate that electron transfer from theFe-protein to the FeMo-cofactor proceeds through theP-clusters (Figure 3); the edge-to-edge distance of ~14 Å iscompatible with inter-protein electron transfer rates morerapid than the observed turnover time [20,21].

Thorneley and Lowe [22] have attempted to quantify thekinetically identifiable steps of the nitrogenase mechanism.Although modifications are necessary [5,23], especially withregard to the generality of dithionite as a reductant, signifi-cant features of the nitrogenase mechanism embodied inthis scheme can be stated: under saturating conditions ofFe-protein, dissociation of the Fe-protein−MoFe-proteincomplex represents the overall rate limiting step, with aturnover time of ~5 s–1; ATP hydrolysis only occurs in thecomplex of the two nitrogenase proteins; substrates onlybind to the reduced forms of MoFe-protein that are exclu-sively generated by Fe-protein and ATP; patterns ofcompetition between various substrates can be understoodto reflect binding to different oxidation states of MoFe-protein; and CO inhibits the reduction of all substratesexcept for proton reduction. It should be noted that thestates of MoFe-protein leading to substrate reduction havenot yet been produced either by coupling to other bio-chemical enzyme reducing systems or by electrochemicalreduction. Hence, the role of Fe-protein with ATP hydrol-ysis appears to be more than a simple electron donor and islikely to provide some larger conformational or structuralcontribution to the overall process.

Beyond this kinetic analysis, a more detailed moleculardescription of the electron transfer processes has notbeen obtained. Missing from our picture of nitrogenaseduring turnover are the mechanistic details of such fun-damental processes as the relationship between ATPhydrolysis and inter-protein electron transfer; experimen-tal evidence concerning the path of electron transferbetween Fe-protein and MoFe-protein; and everyone’sfavorite question — where and how do substrates interactwith the FeMo-cofactor? Progress towards these goals isbeing realized through multiple approaches, includingthe use of mutant nitrogenases, alternate substrates,inhibitors, spectroscopic studies, model chemistry and

theoretical approaches. While space limitations precludea detailed discussion, a few selected examples serve toillustrate the potentials of these approaches as they arenow being applied to nitrogen fixation.

Mutants have been instrumental in many systems for dis-secting contributions of specific residues to the catalyticmechanism of enzymes. In the case of nitrogenase, site-directed mutagenesis studies have identified substitutionsof residues around the FeMo-cofactor (Figure 4) that havealtered substrate reduction properties. Among the bestcharacterized are replacements of residue His α195 [24,25],which donates a hydrogen bond to one of the bridging sul-furs of the cofactor. Replacement of this histidine with

Nitrogenase: standing at the crossroads Rees and Howard 561

Figure 3

A slice through the ADP•AlF4-stabilized nitrogenase complex [15] thatincludes the ADP•AlF4

–, [4Fe:4S] cluster, P-cluster, and FeMo-cofactor,illustrating the relationship between the nucleotide-binding and electrontransfer sites. Relative to the isolated Fe-protein, the change inconformation of the switch II region serves to reposition the Asp129sidechains for nucleotide hydrolysis and also moves the [4Fe:4S] clustercloser to the P-cluster. The linkage of these conformational changesthrough the switch II region provides the structural basis for the couplingof ATP hydrolysis and electron transfer by nitrogenase [55•]. This figurewas prepared from Protein Data Bank coordinate set 1N2C.

ADP

MgAlF

[4Fe:4S] cluster10 Å

Asp129

Switch II

MoFe-protein

Fe-protein

β-subunit α-subunit

FeMo-cofactor

P-cluster

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glutamine results in an enzyme that can still reduce acety-lene to ethylene yet cannot reduce dinitrogen. However,dinitrogen can bind to the enzyme and is an inhibitor ofacetylene reduction. These observations begin to separatethe components of the enzyme necessary for substratebinding from those necessary for electron and proton addi-tion to the substrate. This clearly shows that dinitrogenbinding to the FeMo-cofactor is necessary, but not suffi-cient, for catalysis. Other substitutions at this position resultin enzymes that catalyze a four-electron reduction of acety-lene to ethane, an activity that is not exhibited by thewild-type enzyme [26•]. An exciting recent development isthe application of microbial selection methods [27•] toidentify mutated forms of nitrogenase with altered sub-strate reduction properties. Through this approach, theGly α69Ser variant of the MoFe-protein was identified; itcan fix dinitrogen, but has significantly lower acetylenereduction efficiency.

Although it is commonly assumed that substrates andinhibitors are binding directly to a metallocluster, mostlikely the FeMo-cofactor, direct demonstration of this hasbeen notoriously difficult, and has still not been achievedfor N2. Hoffman, Hales and co-workers [28] have beenable to demonstrate that CO can bind to the FeMo-cofac-tor, a process that has also been monitored by stopped-flowIR spectroscopy [29]. More recently, the Seefeldt and

Hoffman groups [30] have identified electron paramagneticresonance (EPR) signals from bound CS2. Although thespecies represented by these signals account for only a fewpercent of the total FeMo-cofactor present, raising ques-tions about the mechanistic relevance of these states, theseobservations give hope that more detailed characterizationof intermediates will be possible using spectroscopicapproaches such as ENDOR and other EPR techniques,IR, resonance Raman and Mössbauer methods.

Model compounds provide the opportunity to evaluate thestructural and reactivity properties of relevant systems ingreater detail, and are becoming increasingly realistic in cap-turing aspects of the metalloclusters of the MoFe-protein, as detailed in recent contributions from the Holmgroup [31,32•]. Coucouvanis and co-workers [33•] havereported a series of MoFe3S3-containing clusters which indi-cate that metal−metal bonding interactions are present.These clusters undergo significant structural changesbetween oxidation states, suggesting that electron transferprocesses may result in making and breaking of metal–metalbonds that could drive mechanistically significant conforma-tional changes associated with substrate binding andreduction. In terms of reactivity, Sellmann et al. [34•] havedefined requirements of ferrous iron−sulfur compounds thatfacilitate reactivity towards hydrazine and related com-pounds, which have culminated in a mechanistic proposal

562 Mechanisms

Figure 4

View of the protein environment between theFeMo-cofactor and P-cluster of the MoFe-protein, including residues that have beenstudied by mutagenesis. Water moleculesnear the homocitrate are indicated by isolatedspheres. This figure was prepared fromProtein Data Bank coordinate set 3MIN.

Arg α96

Gly α69

Homocitrate

Arg α359

Cys α275

His α442

His α195

Tyr β98

Gln α191

Cys α62

Cys β95

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that involves structural rearrangements of the FeMo-cofactorin the activated state. To date, structural alterations in theprotein-bound metal centers have only been observed in theP-cluster; however, the possibility that the FeMo-cofactormay isomerize in more highly reduced states, upon ligandbinding, or outside of the protein, cannot be dismissed.Recently, Verma and Lee [35•] have reported a series of imi-doiron (III) cubanes that share surprising structuralsimilarities to iron−sulfur clusters, and that may serve to illu-minate aspects of the interaction between nitrogencompounds and the FeMo-cofactor.

Although theoretical studies of systems as complex as thenitrogenase metallocenters might seem daunting, given theexperimental difficulties in studying the nitrogenase mecha-nism, theoretical and computational approaches could wellprovide key insights into the behavior of this system.Density functional calculations now have the capability oftreating systems of this size, and several reports haveappeared addressing the binding modes and affinity of N2and reduced intermediates to the models of the FeMo-cofac-tor [36–38]. Although a consistent mechanistic picture hasyet to emerge, the ability to energetically evaluate differentsequences of substrate binding, proton transfer and electrontransfer steps should be very useful in defining the mecha-nism of substrate reduction by nitrogenase, particularly forthe characterization of possible intermediates.

Redox states of the nitrogenase metalloclustersA useful starting point for analyzing the nitrogenase mech-anism is to identify the redox states accessible to thevarious protein component metalloclusters. The simplestand best characterized center is the [4Fe:4S] cluster of theFe-protein, but even here there have been rather astonish-ing developments. Traditionally, the [4Fe:4S] cluster wasbelieved to undergo a one electron redox cycle betweenthe [4Fe:4S]2+ and [4Fe:4S]1+ states. Although the latter isEPR active, spin quantitation of this reduced form wascomplicated by the existence of multiple spin states. Theability of Fe-protein to both serve as a one-electron donorand to bind two ATPs supported the idea that both ATPsare hydrolyzed during inter-protein electron transfer toyield an overall ATP/e− ratio of 2. More recently, an all-fer-rous [4Fe:4S]0 form of the Fe-protein cluster has beenprepared [39,40,41•], which is the first known example ofthis oxidation state for a [4Fe:4S] cluster, either in proteinsor model compounds. In addition, Fe-protein is the onlyknown example of a native, protein-bound [4Fe:4S] clus-ter that can exist in more than two oxidation states. Whilethe ability of Fe-protein to potentially serve as a two-elec-tron donor is attractive (because all known substrates ofnitrogenase are reduced by multiples of two electrons), themechanistic relevance of the all-ferrous form of theFe-protein remains to be definitively established, althoughan ATP/e− ratio near 1 with this state has been reported[42•]. Because the [4Fe:4S]1+ state of Fe-protein is clearlycompetent for substrate reduction as a one-electron donor,if the all-ferrous protein can function mechanistically as a

two-electron donor, this would be yet another uniqueproperty for nitrogenase.

Although the Fe-protein redox properties are unusual,they are relatively well described compared with the com-plex behavior of the clusters in the MoFe-protein. Asisolated in the presence of dithionite, the FeMo-cofactorexists in the so-called MN or semi-reduced state that ischaracterized by a distinctive S = 3/2 EPR spectra. For thisstate, a plausible assignment is (7Fe:9S:Mo)+, in whichformal oxidation states can be assigned as Mo4+, nine S2–,six ferrous Fe2+ and one ferric Fe3+ ([43]; see Update).Whereas one-electron reduced and oxidized forms of theFeMo-cofactor have been observed, more highly reducedor oxidized forms have not yet been detected. As isolatedin the presence of dithionite, the protein P-clusters are inthe PN state, where, based upon Mössbauer spectroscopy,all eight irons are assigned to the ferrous state [44], so thatthe overall charge is [8Fe:7S]2+ and the clusters are EPRsilent. A variety of oxidized forms of the P-cluster, depend-ing on the mode of oxidation, have been identified, ofwhich the best characterized is the two-electron oxidizedform, POX, corresponding to [8Fe:7S]4+ with a g = 12 signaldetected by parallel mode EPR [45,46]. Two, structurallydistinct, forms of the P-cluster have been identified byX-ray crystallography that have been suggested to corre-spond to the PN and POX states, although definitiveassignments have not been made [13]. Indeed, one of thechallenges in the crystallographic analyses is to maintainthe protein in defined redox states during crystallizationand X-ray data collection [47]. One-electron oxidized [48],as well as states more oxidized than POX, have also beenobserved spectroscopically, although the higher oxidationstates have been more difficult to study because thesestates are often formed irreversibly.

Because of the complex spectroscopic properties of thesemetallocenters, identification and quantifying of clusteroxidation states in nitrogenase can be challenging. For themost part, low temperature magnetic spectroscopic meth-ods on rapid freeze-quenched trapped states have beenuseful in attempts to assign oxidation states and electrontransfer events. At the temperatures of enzyme turnover,however, only the visible spectra, with all the caveats asso-ciated with the weak, broad and ambiguously assignedtransitions, have been used to follow the reactions. As anexample, Fe-protein with a deletion of residue Leu127(L127∆) can form a tight, inactive complex with the MoFe-protein in the absence of nucleotide [49]. In the presenceof dithionite, the individual L127∆ Fe-protein and theMoFe-protein both exhibit their characteristic EPR sig-nals. When the two proteins are mixed, however, theFe-protein EPR signal disappears, indicating that this cen-ter becomes oxidized, while the EPR signal of theFeMo-cofactor remains, indicating that it is still in the MN

state [50]. Because the P-clusters have not been observedto become more reduced than the as-isolated PN state, andassuming that nitrogenase is really not a black hole for

Nitrogenase: standing at the crossroads Rees and Howard 563

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electrons, then it is likely that either there has been somenon-catalytic formation of product (such as H2 formation[51•]) or that the spectroscopic signatures of new oxidationstates have not been recognized. When these experimentsare repeated starting with MoFe-protein in the POX state,EPR signals from the P+1 state are observed [50]. Thus,the relevance of the various states that are more oxidizedthan PN or MN are very much open to question becausethe enzyme system is not carrying out reductive chemistry.For example, there are a number of chemical and biologi-cal reductants for POX, yet only reduced Fe-protein withATP hydrolysis is an effective reductant for MN, presum-ably via PN. These studies indicate the complexities andchallenges even for seemingly straightforward processessuch as following the electrons.

Role of nucleotide In addition to ‘Where does dinitrogen bind and how is itreduced?’ the other major mechanistic question for nitro-genase is ‘What is the role of ATP?’. Biochemically, thelatter question arguably has the greater significancebecause of the striking parallels between the involvementof nucleotide hydrolysis in the nitrogenase mechanism andin a variety of signal transduction processes epitomized byras p21 and G-proteins. These parallels are most stronglycaptured in the structural homologies between proteins inthese pathways. Given the requirement for fixed nitrogenearly in the development of life, it is plausible that this par-ticular structural motif originated with nitrogen fixation, ashas been suggested from sequence analyses [52].

Because ATP hydrolysis of this magnitude is not requiredfor dinitrogen reduction to be thermodynamically favor-able [53], this requirement must reflect an involvement inthe kinetic mechanism. Possibilities include conversion ofthe hydrolysis of ATP into lowering the redox potential ofthe metallocenters (i.e. the production of ‘super-reducingelectrons’) or the utilization of ATP as a timing mechanismfor driving a series of conformational changes. In all likeli-hood, the role of ATP probably reflects both of thesepossibilities, and undoubtedly others.

Nucleotide binding clearly influences the electrochemicalproperties of Fe-protein, as binding of ATP or ADP stabi-lizes the oxidized state and lowers the redox potential by~100 mV [54]. Because both ATP and ADP have compara-ble effects, it is not clear why nucleotide hydrolysis wouldbe required to generate lower potential electrons. Fe-pro-tein becomes an even better reductant when complexed toMoFe-protein, although much of this may be due to com-plex formation rather than nucleotide binding, since theL127∆ complex also has a lowered redox potential in theabsence of nucleotide [50]. Given the inability of donorsother than Fe-protein to function in substrate reduction, itdoes not seem that driving force, as defined by the redoxpotential, is a kinetic or thermodynamic limitation. Nor iselectrochemical accessibility of the metalloclusters likelyto be a problem, because electrons can be transferred

between the MoFe-protein clusters and exogenous redoxgroups, at least for some oxidation states [50].

The similarities between Fe-protein and nucleotideswitch proteins suggests that the role of nucleotide hydrol-ysis may be required to drive a series of conformationalchanges that mediate the interactions between Fe-proteinand MoFe-protein (see [1,55•]). One of the regions ofgreatest conformational variability in switch proteinsbetween the NTP and NDP states involves the so-calledswitch II region. In the case of Fe-protein, the switch IIregion includes Asp125, Gly128, Asp129 and Cys132.Consequently, this region contains residues that interactwith both the nucleotide and the cluster. Analysis of thenitrogenase complex stabilized by ADP•AlF4

− [15] sub-stantiated these interactions (Figure 3), and demonstratedthat the conformation of switch II that is required for ATPhydrolysis is coupled to a repositioning of the [4Fe:4S]cluster such that the cluster is in closer proximity to theMoFe-protein, which should facilitate efficient inter-pro-tein electron transfer. As the nucleotides convert betweenthe ATP, transition state, ADP+Pi, ADP, etc. -bound states,the conformation of the switch II region will change, nec-essarily modulating the electron transfer properties of thenitrogenase proteins and coupling the hydrolysis of ATP toquasi-unidirectional electron transfer from the Fe-proteinto the MoFe-protein [55•].

ConclusionsThis review has focused on how nitrogenase works; otherimportant questions include ‘How and when did nitroge-nase arise in biological systems?’ (see Update), ‘How isnitrogenase made biosynthetically?’, ‘How can model sys-tems based on nitrogenase fix nitrogen?’, and ‘Isnitrogenase the only way to enzymatically fix dinitrogen?’.On the latter issue, a very exciting recent development[56] is the initial characterization of a new type of nitroge-nase distinct from the ‘conventional’ system. Rationalityagain not withstanding, as far as the mechanism of the con-ventional nitrogenase is concerned, there is a distinctfeeling that we’re standing at the crossroads; through acombination of inspiration, hard work, luck, and perhaps afew deals, the remaining secrets may finally be revealed.

UpdateYoo et al. [61•] have recently reported a Mössbauer study ofthe MoFe-protein selectively enriched with 57Fe-contain-ing FeMo-cofactor which suggests that the MN state is bestdescribed as [7Fe:9S:Mo]–1, with formal oxidation statesassigned as four ferrous Fe+2, three ferric Fe+3, nine S–2 andone Mo+4. This state is reduced by two electrons from theENDOR-derived description discussed in [43]. A het-erodimeric form of the Fe-protein, in which Asp39 issubstituted by Asn39 in one of the two subunits, does notsupport substrate reduction by the MoFe-protein, althoughMgATP hydrolysis and MgATP-dependent primary elec-tron transfer to the MoFe-protein were reported [62•].Recent biochemical studies [63•] have confirmed some

564 Mechanisms

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general mechanistic parallels between nitrogenase and thelight-independent protochlorophyllide reductase involvedin bacteriochlorophyll biosynthesis, which complementpreviously noted sequence similarities between these twosystems. These findings suggest the possibility that (bacte-rio)chlorophyll biosynthesis associated with photosynthesismay be evolutionarily related to nitrogen fixation.

AcknowledgementsDiscussions with members of our groups and nitrogenase colleagues aregreatly appreciated. Research in the authors' laboratory was supported byUnited States Public Health Service grant GM45162 (DCR and JBH) andNational Science Foundation MCB9513512 (JBH).

References and recommended readingPapers of particular interest, published within the annual period of review,have been highlighted as:

• of special interest••of outstanding interest

1. Howard JB, Rees DC: Nitrogenase: a nucleotide-dependentmolecular switch. Annu Rev Biochem 1994, 63:235-264.

2. Burgess BK, Lowe DJ: Mechanism of molybdenum nitrogenase.Chem Rev 1996, 96:2983-3011.

3. Howard JB, Rees DC: Structural basis of biological nitrogenfixation. Chem Rev 1996, 96:2965-2982.

4. Seefeldt LC, Dean DR: Role of nucleotides in nitrogenasecatalysis. Acc Chem Res 1997, 30:260-266.

5. Smith BE: Structure, function and biosynthesis of themetallosulfur clusters in nitrogenases. Adv Inorg Chem 1999,47:159-218.

6. Simpson FB, Burris RH: A nitrogen pressure of 50 atmospheresdoes not prevent evolution of hydrogen by nitrogenase. Science1984, 224:1095-1096.

7. Eady RR: Structure-function relationships of alternativenitrogenases. Chem Rev 1996, 96:3013-3030.

8. Georgiadis MM, Komiya H, Chakrabarti P, Woo D, Kornuc JJ,Rees DC: Crystallographic structure of the nitrogenase ironprotein from Azotobacter vinelandii. Science 1992,257:1653-1659.

9. Schlessman JL, Woo D, Joshua-Tor L, Howard JB, Rees DC:Conformational variability in structures of the nitrogenase ironproteins from Azotobacter vinelandii and Clostridiumpasteurianum. J Mol Biol 1998, 280:669-685.

10. Jang SB, Seefeldt LC, Peters JW: Modulating the midpointpotential of the [4Fe-4S] cluster of the nitrogenase Fe protein.Biochemistry 2000, 39:641-648.

11. Kim J, Rees DC: Crystallographic structure and functionalimplications of the nitrogenase molybdenum-iron protein fromAzotobacter vinelandii. Nature 1992, 360:553-560.

12. Bolin JT, Campobasso N, Muchmore SW, Morgan TV, Mortenson LE:The structure and environment of the metal clusters in thenitrogenase MoFe protein from Clostridium pasteurianum.In Molybdenum Enzymes, Cofactors and Model Systems. ACSSymposium Series No. 535. Edited by Stiefel EI, Coucouvanis D,Newton WE. Washington, DC: American Chemical Society;1993:186-195.

13. Peters JW, Stowell MHB, Soltis SM, Finnegan MG, Johnson MK,Rees DC: Redox-dependent structural changes in the nitrogenaseP-cluster. Biochemistry 1997, 36:1181-1187.

14. Mayer SM, Lawson DM, Gormal CA, Roe SM, Smith BE: New• insights into structure-function relationships in nitrogenase: a

1.6 Å resolution X-ray crystallographic study of Klebsiellapneumoniae MoFe-protein. J Mol Biol 1999, 292:871-891.

Details the structure of the MoFe-protein and associated metalloclusters atthe highest published resolution.

15. Schindelin H, Kisker C, Schlessman JL, Howard JB, Rees DC:Structure of ADP-AlF4

– stabilized nitrogenase complex and itsimplications for signal transduction. Nature 1997, 387:370-376.

16. Rees DC, Schindelin H, Kisker C, Schlessman JL, Peters JW,Seefeldt LC, Howard JB: Complex structures of nitrogenase.In Biological Nitrogen Fixation for the 21st Century. Edited byElmerich C, Kondorosi A, Newton WE. Dordrecht: Kluwer AcademicPublishers; 1998:11-16.

17. Kjeldgaard M, Nyborg J, Clark BFC: The GTP binding motif:variations on a theme. FASEB J 1996, 10:1347-1368.

18. Sprang SR: G protein mechanisms: insights from structuralanalysis. Annu Rev Biochem 1997, 66:639-678.

19. Peters JW: Structure and mechanism of iron-only hydrogenases.Curr Opin Struct Biol 1999, 9:670-676.

20. Gray HB, Winkler JR: Electron transfer in proteins. Annu RevBiochem 1996, 65:537-561.

21. Page CC, Moser CC, Chen X, Dutton PL: Natural engineeringprinciples of electron tunneling in biological oxidation-reduction.Nature 1999, 402:47-52.

22. Thorneley RNF, Lowe DJ: Kinetics and mechanism of thenitrogenase enzyme system. In Molybdenum Enzymes. Edited bySpiro TG. New York: John Wiley and Sons Inc.; 1985:221-284.

23. Duyvis MG, Wassink H, Haaker H: Nitrogenase of Azotobactervinelandii: kinetic analysis of the Fe protein redox cycle.Biochemistry 1998, 37:17345-17354.

24. Scott DJ, May HD, Newton WE, Brigle KE, Dean DR: Role for thenitrogenase MoFe protein αα-subunit in FeMo-cofactor binding andcatalysis. Nature 1990, 343:188-190.

25. Kim C-H, Newton WE, Dean DR: Role of the MoFe proteinαα-subunit histidine-195 residue in FeMo-cofactor binding andnitrogenase catalysis. Biochemistry 1995, 34:2798-2808.

26. Fisher K, Dilworth MJ, Kim C-H, Newton WE: Azotobacter vinelandii• nitrogenases containing altered MoFe proteins with substitutions

in the FeMo-cofactor environment: effects on the catalyzedreduction of acetylene and ethylene. Biochemistry 2000,39:2970-2979.

The substrate reduction properties of altered MoFe-proteins with sub-stituents at positions α195 or α191 are characterized and interpreted interms of the interactions of acetylene and ethylene with the MoFe-protein.

27. Christiansen J, Cash VL, Seefeldt LC, Dean DR: Isolation and• characterization of an acetylene resistant nitrogenase. J Biol

Chem 2000, 275:11459-11464. Describes the application of a genetic strategy to isolate mutant nitrogenas-es that can fix nitrogen in the presence of acetylene, which normally inhibitsthis process. The Gly α69Ser mutant identified in this screen apparently onlyhas the low-affinity binding site for acetylene to which acetylene and nitro-gen can bind competitively and with approximately the same affinity. It is sug-gested that the face of the FeMo-cofactor capped by Val α70 is likely torepresent the high-affinity acetylene-binding site.

28. Christie PD, Lee H-I, Cameron LM, Hales BJ, Orme-Johnson WH,Hoffman BM: Identification of the CO-binding cluster innitrogenase MoFe protein by ENDOR of 57Fe isotopomers. J AmChem Soc 1996, 118:8707-8709.

29. George SJ, Ashby GA, Wharton CW, Thorneley RNF: Time-resolvedbinding of carbon monoxide to nitrogenase monitored bystopped-flow infrared spectroscopy. J Am Chem Soc 1997,119:6450-6451.

30. Ryle MJ, Lee H-I, Seefeldt LC, Hoffman BM: Nitrogenasereduction of carbon disulfide: freeze-quench EPR andENDOR evidence for three sequential intermediates withcluster-bound carbon moieties. Biochemistry 2000,39:1114-1119.

31. Huang JS, Holm RH: Synthesis, identification, and reactivityproperties of symmetrical MoFe3S4 double cubanes with Fe-S-Feand Fe-O-Fe bridges. Inorg Chem 1998, 37:2247-2254.

32. Osterloh F, Sanakis Y, Staples RJ, Münck E, Holm RH: A• molybdenum-iron-sulfur cluster containing structural elements

relevant to the P-cluster of nitrogenase. Angew Chem Intl Ed1999, 38:2066-2070.

A cluster with a Mo6Fe20S30 core has been prepared and characterized thatcontains a Mo2Fe6S9 fragment that resembles the arrangement of metalsand sulfur in the PN form of the P-cluster.

33. Han J, Beck K, Ockwig N, Coucouvanis D: Synthetic analogs for the• MoFe3S3 subunit of the nitrogenase cofoactor: structural

features associated with the total number of valence electronsand the possible role of M–M and multiple M-S bonding in the

Nitrogenase: standing at the crossroads Rees and Howard 565

Page 8: Nitrogenase: standing at the crossroads

function of nitrogenase. J Am Chem Soc 1999,121:10448-10449.

Implications of electron count and metal−metal bonding for the structure andfunction of the clusters such as the FeMo-cofactor are discussed, based onsynthesis and characterization of MoFe3S3-containing clusters.

34. Sellmann D, Utz J, Blum N, Heinemann FW: On the function of• nitrogenase FeMo cofactors and competitive catalysts: chemical

principles, structural blue-prints and the relevance of iron sulfurcomplexes for N2 fixation. Coord Chem Rev 1999,190-192:607-627.

Considerations are discussed for the development of an iron−sulfur-com-plex-based system to achieve the catalytic reduction of dinitrogen.

35. Verma AK, Lee SC: Reductive cleavage of the N–N bond: synthesis• of imidoiron(III) cubanes. J Am Chem Soc 1999,

121:10838-10839.The synthesis of imidoiron(III) cubanes are described that exhibit structuralparallels to iron−sulfur clusters and involve both iron—nitrogen bonding andreductive cleavage of N−N bonds, which may be relevant to the mechanismof nitrogenase.

36. Dance I: Understanding structure and reactivity of newfundamental inorganic molecules: metal sulfides,metallocarbohedrenes, and nitrogenase. Chem Commun1998:523-530.

37. Siegbahn PEM, Westerberg J, Svensson M, Crabtree RH: Nitrogenfixation by nitrogenases: a quantum chemistry study. J PhysChem B 1998, 102:1615-1623.

38. Rod TH, Hammer B, Nørskov JK: Nitrogen adsorption andhydrogenation on a MoFe6S9 complex. Phys Rev Letters 1999,82:4054-4057.

39. Watt GD, Reddy KRN: Formation of an all ferrous Fe4S4 cluster inthe iron protein-component of Azotobacter vinelandii nitrogenase.J Inorg Biochem 1994, 53:281-294.

40. Angove HC, Yoo SJ, Burgess BK, Münck E: Mössbauer and EPRevidence for an all-ferrous Fe4S4 cluster with S = 4 in theFe protein of nitrogenase. J Am Chem Soc 1997, 119:8730-8731.

41. Yoo SJ, Angove HC, Burgess BK, Hendrich MP, Münck E:• Mössbauer and integer-spin EPR studies and spin-coupling

analysis of the [4Fe-4S]0 cluster of the Fe protein fromAzotobacter vinelandii nitrogenase. J Am Chem Soc 1999,121:2534-2545.

Detailed characterization of the electronic and spectroscopic properties ofthe all-ferrous form of the Fe-protein.

42. Erickson JA, Nyborg AC, Johnson JL, Truscott SM, Gunn A,• Nordmeyer FR, Watt GD: Enhanced efficiency of ATP hydrolysis

during nitrogenase catalysis utilizing reductants that form theall-ferrous redox state of the Fe protein. Biochemistry 1999,38:14279-14285.

Evaluation of the amount of ATP hydrolyzed per electron transferred by theall-ferrous Fe-protein suggests that ratios of 1 may be achieved, which wouldbe twice as efficient in terms of ATP consumption as generally believed.

43. Lee H-I, Hales BJ, Hoffman BM: Metal-ion valencies of the FeMocofactor in CO-inhibited and resting state nitrogenase by 57FeQ-band ENDOR. J Am Chem Soc 1997, 119:11395-11400.

44. McLean PA, Papaefthymiou V, Orme-Johnson WH, Münck E: Isotopichybrids of nitrogenase: Mössbauer study of MoFe protein withselective Fe57 enrichment of the P-cluster. J Biol Chem 1987,262:12900-12903.

45. Surerus KK, Hendrich MP, Christie PD, Rottgardt D, Orme-Johnson WH,Münck E: Mössbauer and integer-spin EPR of the oxidized P-clustersof nitrogenase: POX is a non-Kramers system with a nearlydegenerate ground doublet. J Am Chem Soc 1992, 114:8579-8590.

46. Pierik J, Wassink H, Haaker H, Hagen R: Redox properties and EPRspectroscopy of the P clusters of Azotobacter vinelandii MoFeprotein. Eur J Biochem 1993, 212:51-61.

47. Schindelin H, Kisker C, Rees DC: The molybdenum-cofactor: acrystallographic perspective. J Biol Inorg Chem 1997, 2:773-781.

48. Tittsworth RC, Hales BJ: Detection of EPR signals assigned to the1-equiv-oxidized P-clusters of the nitrogenase MoFe-protein fromAzotobacter vinelandii. J Am Chem Soc 1993, 115:9763-9767.

49. Lanzilotta WN, Fisher K, Seefeldt LC: Evidence for electron transferfrom the nitrogenase iron protein to the molybdenum-iron proteinwithout MgATP hydrolysis: characterization of a tightprotein–protein complex. Biochemistry 1996, 35:7188-7196.

50. Lanzilotta WN, Seefeldt LC: Changes in the midpoint potentials ofthe nitrogenase metal centers as a result of iron proteinmolybdenum-iron protein complex-formation. Biochemistry 1997,36:12976-12983.

51. Yousafzai FK, Eady RR: MgATP-independent hydrogen evolution• catalyzed by nitrogenase: an explanation for the missing

electron(s) in the MgADP-AlF4 transition state complex.Biochem J 1999, 339:511-515.

A careful analysis of the electron balance during formation of theADP•AlF4

– stabilized nitrogenase complex, prepared with MgADP andAlF4

– indicates that a very slow rate of H+ reduction can be supported inthe absence of MgATP.

52. Koonin EV: A superfamily of ATPases with diverse functionscontaining either classical or deviant ATP-binding motif. J Mol Biol1993, 229:1165-1174.

53. Alberty RA: Thermodynamics of nitrogenase reactions. J BiolChem 1994, 269:7099-7102.

54. Watt GD, Wang Z-C, Knotts RR: Redox reactions of and nucleotidebinding to the iron protein of Azotobacter vinelandii. Biochemistry1986, 25:8156-8162.

55. Rees DC, Howard JB: Structural bioenergetics and energy• transduction mechanisms. J Mol Biol 1999, 293:343-350.General considerations of energy transduction mechanisms are discussed,emphasizing nitrogenase and the general family of the nucleotide switch proteins.

56. Ribbe M, Gadkari D, Meyer O: N2 fixation by Streptomycesthermoautotrophicus involves a molybdenum-dinitrogenase and amanganese-superoxide oxidoreductase that couple N2 reductionto the oxidation of superoxide produced from O2 by amolybdenum CO dehydrogenase. J Biol Chem 1997,272:26627-26633.

57. Sussman J, Lin D, Jiang J, Manning N, Prilusky J, Ritter O, Abola E:Protein Data Bank (PDB): database of three-dimensionalstructural information of biological macromolecules. ActaCrystallogr D 1998, 54:1078-1084.

58. Berman HM, Westbrook J, Feng Z, Gilliland G, Bhat TN, Weissig H,Shindyalov IN, Bourne PE: The Protein Data Bank. Nucleic AcidsRes 2000, 28:235-242.

59. Kraulis PJ: MOLSCRIPT — a program to produce both detailed andschematic plots of protein structures. J Appl Cryst 1991,24:946-950.

60. Brändén C-I: Relation between structure and function of αα//ββproteins. Q Rev Biophys 1980, 13:317-338.

61. Yoo SJ, Angove HC, Papaefthymiou V, Burgess BK, Münck E:• Mössbauer study of the MoFe protein of nitrogenase from

Azotobacter vinelandii using selective 57Fe enrichment of theM-centers. J Am Chem Soc 2000, 122:4926-4936.

A Mössbauer analysis of the iron sites in the FeMo-cofactor of the nitroge-nase MoFe-protein is described, including proposals for the formal oxidationstates in different redox forms of this cofactor.

62. Chan JM, Wu W, Dean DR, Seefeldt LC: Construction and• characterization of a heterodimeric iron protein: defining roles for

adenosine triphosphate in nitrogenase catalysis. Biochemistry2000, 39:7221-7228.

The construction and characterization of a heterodimeric form of theFe-protein is described, in which one of the two Fe-protein subunits con-tains an amino acid substitution that prevents ATP hydrolysis when presentin both subunits.

63. Fujita Y, Bauer CE: Reconstitution of light-independent• protochlorophyllide reductase from purified BchL and BchN-BchB

subunits. J Biol Chem 2000, 275:23583-23588. Previously noted sequence similarities between the nitrogenase Fe-proteinand MoFe-protein subunits, and the products of the BchL, BchN and BchBgenes of Rhodobacter capsulatus, respectively, are reflected in mechanisticparallels involving ATP-dependent electron transfer reactions. These obser-vations suggest that there may be evolutionary relationships linking the basicprocesses of photosynthesis and nitrogen fixation.

566 Mechanisms