Crystal structure of the electron transfer complexrubredoxin–rubredoxin reductase ofPseudomonas aeruginosaGregor Hagelueken†, Lutz Wiehlmann‡, Thorsten M. Adams‡, Harald Kolmar§, Dirk W. Heinz¶, Burkhard Tummler‡,and Wolf-Dieter Schubert†�

†Molecular Host–Pathogen Interactions, ¶Division of Structural Biology, Helmholtz Centre for Infection Research, Inhoffenstrasse 7, D-38124 Braunschweig,Germany; ‡Klinische Forschergruppe OE 6711, Medizinische Hochschule Hannover, Carl Neuberg Strasse 1, D-30625 Hannover, Germany; and §ClemensSchoepf Institute for Organic Chemistry and Biochemistry, Darmstadt University of Technology, Petersenstrasse 22, D-64287 Darmstadt, Germany

Edited by Johann Deisenhofer, University of Texas Southwestern Medical Center, Dallas, TX, and approved June 6, 2007 (received for review March 29, 2007)

Crude oil spills represent a major ecological threat because of thechemical inertness of the constituent n-alkanes. The Gram-negative bacterium Pseudomonas aeruginosa is one of the fewbacterial species able to metabolize such compounds. Threechromosomal genes, rubB, rubA1, and rubA2 coding for anNAD(P)H:rubredoxin reductase (RdxR) and two rubredoxins (Rdxs)are indispensable for this ability. They constitute an electrontransport (ET) pathway that shuttles reducing equivalents fromcarbon metabolism to the membrane-bound alkane hydroxylasesAlkB1 and AlkB2. The RdxR–Rdx system also is crucial as part of theoxidative stress response in archaea or anaerobic bacteria. Theredox couple has been analyzed in detail as a model system for ETprocesses. We have solved the structure of RdxR of P. aeruginosaboth alone and in complex with Rdx, without the need for cross-linking, and both structures were refined at 2.40- and 2.45-Åresolution, respectively. RdxR consists of two cofactor-bindingdomains and a C-terminal domain essential for the specific recog-nition of Rdx. Only a small number of direct interactions governmutual recognition of RdxR and Rdx, corroborating the transientnature of the complex. The shortest distance between the redoxcenters is observed to be 6.2 Å.

The ubiquitous, Gram-negative bacterium Pseudomonas aerugi-nosa is metabolically highly versatile, allowing it to survive

numerous specialized ecological niches in addition to soil andaquatic environments. This versatility allows P. aeruginosa to beboth an opportunistic pathogen, chronically colonizing the respi-ratory tract of cystic fibrosis patients or causing acute infections inopen wounds (1, 2), and a valuable ally by degrading ecologicalpollutants such as detergents (3, 4) or n-alkanes (5, 6). Mineral-ization of n-alkanes inter alia from crude oil spills involves five genes(7). The membrane-bound alkane hydroxylases AlkB1 and AlkB2oxidize terminal carbons of chemically inert n-alkanes, allowingfurther oxidation and degradation. Electrons for this initial reactionderive from carbon metabolism relayed through an electron trans-port (ET) chain involving FAD-dependent NAD(P)H:rubredoxinreductase (RdxR) RubB and two AlkG2-type (see below) rubre-doxins (Rdxs) RubA2 and RubA1 (7–9), encoded by the genecluster rubB (PA5349), rubA2 (PA5350), and rubA1 (PA5351).Whereas alkB1 and alkB2 expression is strictly n-alkane-dependent,RubB/RubA1/RubA2 are constitutively produced (8), indicating amore general but as yet unidentified role for this ET chain in P.aeruginosa (7).

Rdxs are small (�6 kDa), redox-active iron–sulfur proteins foundin anaerobic or microaerophilic archaea and bacteria (10). A centraliron, coordinated by four cysteines, constitutes the redox-active sitealternating between �2 and �3 oxidation states. Rdxs are crucialfor oxidative stress responses in anaerobic organisms by rapidlytransferring metabolic reducing equivalents to superoxide reduc-tases or rubredoxin:oxygen oxidoreductases to reduce oxygen orreactive oxygen species (11, 12). The link to general metabolism isprovided by NAD(P)H and NAD(P)H:RdxRs (11, 13, 14). RdxRs

are either heterodimeric (class 1, RdxR from Desulfovibrio gigas)and bind the cofactors FAD and FMN (15), or single-chain proteins(class 2, RubB) exclusively binding FAD. Related enzymes occur inaerobic bacteria and eukaryotes, and include both electron shuttlingenzymes and redox enzymes such as glutathione reductase (GR)(16). For simplicity, the gene products of PA5349, PA5350, andPA5351 are henceforth referred to as RdxR and Rdx, respectively.

Several RdxR-like enzymes and many Rdxs have been exten-sively studied both structurally and biophysically (17–30). Here, wepresent the crystal structure of a dedicated RdxR from P. aerugi-nosa strain PAO1 at 2.40-Å resolution. The interaction of RdxR andRdx, which we describe at 2.45-Å resolution, has not been analyzedstructurally so far, possibly because of the assumption of a highlytransient and hence structurally inaccessible ET complex. Thesestructures of RdxR and Rdx in a functional complex provide astructural basis to understand the ET processes of these much-studied proteins.

ResultsRdxR Is Indispensable for n-Alkane Oxidation in P. aeruginosa. Amutant of P. aeruginosa with an isogenic rdxR transposon insertionis unable to grow on hexadecane [supporting information (SI) Fig.5], confirming earlier reports that RdxR is essential for the utili-zation of n-alkanes as sole carbon source by different Pseudomonasstrains (7, 31). This effect is completely reverted by supplying theRdxR gene on plasmid pUCP20 (SI Fig. 5). Interestingly, bothRdxR and the Rdxs rubA1 and rubA2 from P. aeruginosa cansubstitute for their counterparts in Pseudomonas putida GPo1 (7).

Structure Determination and Refinement. Yellow, FAD-boundRdxR crystals (space group P6122) diffract x-rays to 2.40 Å. Thestructure was solved by molecular replacement using nitrite reduc-tase from Pyrococcus furiosus [Protein Data Bank (PDB) ID code1XHC; 26% sequence identity] as a search model. The final modelof RdxR, comprising residues 4–384, refines to a final R factor of16.6% (Rfree, 20.4%) (Table 1).

Author contributions: G.H., L.W., T.M.A., H.K., D.W.H., B.T., and W.-D.S. designed research;G.H. and T.M.A. performed research; G.H. and W.-D.S. analyzed data; and G.H. and W.-D.S.wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Abbreviations: ET, electron transport; Rdx, rubredoxin; RdxR, rubredoxin reductase; GR,glutathione reductase; mAIF, murine apoptosis inducing factor; BphA4, bacterial ferre-doxin reductase; PdxR, putidaredoxin reductase; TrxR, thioredoxin reductase.

Data deposition: The atomic coordinates and structure factors have been deposited in theProtein Data Bank, www.pdb.org (PDB ID codes 2v3a and 2v3b).

�To whom correspondence should be addressed. E-mail: wolf-dieter.schubert@helmholtz-hzi.de.

This article contains supporting information online at www.pnas.org/cgi/content/full/0702919104/DC1.

© 2007 by The National Academy of Sciences of the USA

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Orange-red crystals of the RdxR–Rdx complex, produced bymixing oxidized RdxR and oxidized Rdx in a molar ratio of 1:1.2,belong to the orthorhombic space group P2221 and diffract x-raysto 2.30-Å resolution. The structure was solved by molecular re-placement using the refined structure of RdxR and the Rdx fromP. furiosus (32) (PDB ID code 1BQ8; sequence identity, 54%). Thecomplex includes residues 4–384 of RdxR and residues 1–53 (of55) of Rdx, and refines to an R factor of 18.1% (Rfree, 24.6%)(Table 1).

Structure of RdxR. Structurally, RdxR comprises an NAD(P)H-binding domain, an FAD-binding domain, and a C-terminal Rdx-binding domain (Fig. 1 A and B). The NAD(P)H-binding domain,encompassing residues 111–240, is inserted into the FAD-bindingdomain through two extended loops, �G/�5 and �N/�8, dividing

the latter into an N-terminal (residues 1–110) and a C-terminal(residues 241–313) subdomain. The Rdx-binding domain consists ofa five-stranded mixed �-sheet (�R-V), topped by the C-terminal�-helix �10.

The fold of RdxR is related to those of GR (33), murine apoptosisinducing factor (mAIF) (20), the ferredoxin reductase componentof biphenyl dioxygenase [bacterial ferredoxin reductase (BphA4)](19, 34), and putidaredoxin reductase (PdxR) (35) (see below).Although a sequence alignment of these proteins reveals severalinsertions and deletions in peripheral regions (SI Fig. 6), thecofactor-binding domains are structurally conserved resulting inrmsd values of RdxR relative to mAIF, BphA4, and PdxR of 2.2,2.9, and 3.7 Å, respectively. Compared with the 384 residues ofRdxR, BphA4 (408), PdxR (422), and mature mAIF (489) areconsiderably longer. In BphA4 and PdxR, these additional residuesmostly constitute two �-helices forming a C-terminal three-helixbundle. The C terminus of mAIF is even longer because of theinsertion of a putative protein–protein interaction loop (20, 36).

FAD- and NAD(P)H-Binding Sites. FAD is recognized by RdxRthrough numerous interactions including water-mediated and di-rect hydrogen bonds, a salt bridge from the AMP-phosphate toLys-45, and hydrophobic interactions mainly to the aromatic aden-osine and isoalloxazine moieties (Fig. 1C). The extended confor-mation of FAD is similar to that observed in GR, PdxR, BphA4,and mAIF. Whereas the xylene ring of the isoalloxazine moiety isburied in a hydrophobic pocket and the si-side is shielded fromsolvent, the re-side faces the NAD(P)H-binding cavity (Fig. 1 A andC; SI Fig. 7). The atoms N5 and O4 of FAD are part of an extensivehydrogen bonding network to a salt-bridged glutamate–lysine pair(Glu-159/Lys-320) (Fig. 1C; SI Fig. 7 and below). Strikingly, Lys-320functionally replaces a lysine located near the N terminus (approx-imately at position 50) in all other GR-fold enzymes (SI Fig. 6). Inaddition, RdxR lacks a tryptophan-lid shielding the pteridine-ringof FAD from the solvent in PdxR, BphA4, and mAIF (SI Fig. 6).

Neither of our crystal structures includes NAD(P)H bound toRdxR. However, FAD-based superpositioning of NAD(P)H-bound GR (1GRB; ref. 16) or BphA4 (1F3P; ref. 19) on RdxR,clearly indicates the NAD(P)H-binding pocket to be conserved inRdxR. Minor torsion angle adjustments of residues along the lengthof the modeled cofactor suffice to allow NADH being placed intothe binding pocket. Similar conformational adjustments have beenobserved to occur on NAD(P)� binding in both GR and BphA4(19, 37). In the modeled complex, the nicotinamide ring ofNAD(P)H stacks on the isoalloxazine ring of FAD at a distance of

Table 1. Crystallographic data

Measurement RdxR–Rdx RdxR

Data statisticsSpace group P2221 P6122Unit cell length, Å

a 61.1 119.6b 97.1 119.6c 81.3 158.1

Wavelength, Å 1.741 0.981Resolution range, Å 50.0–2.3 50.0–2.4Mosaicity, ° 1.4 0.5Completeness, % 90.6 (86.2) 98.1 (82.9)Redundancy 3.8 (3.4) 3.6 (2.8)Unique reflections 19,054 25,303Wilson B factor, Å2 41.0 38.7I/�(I) 12.1 (3.0) 11.8 (2.7)Rmerge, % 10.1 (40.2) 7.3 (34.0)

Refinement statisticsResolution range, Å 50.0–2.45 50.0–2.40R/Rfree, % 18.1/24.6 16.6/20.4Average B factor of protein atoms, Å2 40.5 30.2rmsd bonds, Å 0.01 0.02rmsd angles, ° 1.5 1.6Ramachandran plot, %

Favored 97.4 97.6Allowed 100 100

Values in parentheses refer to the shell of highest resolution.

Fig. 1. Structure of RdxR. (A) Cartoon representation of uncomplexed RdxR using a green (N terminus) to orange (C terminus) color gradient. FAD and NADH(modeled) are shown as ball-and-stick models in orange and translucent gray. (B) Schematic representation of the RdxR secondary structure: circles, triangles,and lines indicate �-helices, �-strands, and loops, respectively. (C) Schematic view of the FAD-binding site. Hydrogen bonds and salt bridges are indicated bydotted lines, and hydrophobic interactions are indicated by gray arcs. 2Fo � Fc electron density for FAD (blue mesh) is contoured at 1.0�. Color coding: C, gray;N, blue; O, red; S, yellow; P, orange; Fe, green. Covalent bonds are yellow for RdxR and red for Rdx.

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�3 Å, suitable for hydride transfer. The positions of atoms NN7 andNO7 of the modeled NAD(P)H are occupied by two water mole-cules in the NAD(P)H-free structure of RdxR, further corrobo-rating the inferred model (SI Fig. 7A). Distinct conformations ofTyr-288 observed in RdxR and RdxR–Rdx presumably representconformations adopted in the absence or presence of NAD(P)H,respectively. The latter conformation places the side chain ofTyr-288 opposite the NO2ONC2 bond of the nicotinamide-riboseindicating that bound NAD(P)H would be further stabilized by anO–H–� interaction (SI Fig. 7A).

Dimerization of RdxR. Despite distinct crystal packing arrangementsfor RdxR and RdxR–Rdx, RdxR forms identical dimers in bothstructures. The observed dimer, burying a surface area of 3,260 Å2

(38), involves the convex surface of each monomer. Dimers havealso been reported for GR, PdxR, BphA4, and mAIF (19, 20, 23,33). In GR and similar S-S oxidoreductases, dimerization is essen-tial as residues from both monomers create the active-site pocket(16, 37). The functional relevance of dimerization observed inelectron transferases (PdxR, BphA4, and RdxR), however, cur-rently remains unclear (23). In RdxR, dimerization restricts accessto the NAD(P)H binding pocket and results in a steric clashbetween the modeled adenine moiety of NAD(P)H and �-helix �8�of the neighboring molecule (SI Fig. 8). Moreover, numerous watermolecules at the dimer interface and an unfavorable Arg-242–Arg-242� contact all support a weak RdxR–RdxR� interaction. We thusassume that RdxR dimers form at high protein concentrations usedduring crystallization, rather than being functionally relevant.

Structure of the RdxR–Rdx Complex. We have cocrystallized RdxRwith RubA2 (PA5350), an AlkG2-type (see below) Rdx from P.aeruginosa. As in other Rdxs, the redox-active Fe3� (confirmed byx-ray anomalous scattering) (Fig. 2A) of RubA2, is tetrahedrallycoordinated by four cysteines (Figs. 1C and 2A). Crystal structuresof several Rdx have been discussed in detail (17, 27–30, 32, 39).RubA2 most closely resembles the Rdx of P. furiosus (1BQ8; rmsd,0.88 Å) (32) (SI Fig. 9).

As expected, the electron transfer complex between RdxR andRdx is a 1:1 complex of the two proteins (Fig. 2A). Rdx binds to theconcave side of RdxR, interacting with each of its three domains.The accessible surface area buried on formation of the complex isa mere �1,120 Å (38). This comparatively small interface (40)covers 17% and 3% of the total surface areas of Rdx and RdxR,respectively.

The shortest direct distance between redox centers of bothmolecules (FAD-N3 to Cys-9Rdx-S�) is 6.2 Å, significantly below the15-Å limit proposed for physiologically relevant ET reactions (41).A well resolved, bridging water molecule subdivides this distanceinto two shorter segments of 2.8 and 5.7 Å, potentially affectingboth likelihood and speed of electron transfer between the redoxpartners (Fig. 1C) (42–44).

Electrostatic surface potentials for both Rdx and RdxR, calcu-lated by using Adaptive Poisson–Boltzmann Solver (45), revealsignificant charge complementarity (see below). The interface isdominated by H-bonded interactions centered about the axisrunning from FAD-N3 to Rdx-Fe3�. These interactions includedirect hydrogen bonds between residues Met-290RdxR–Cys-42Rdx,Arg-297RdxR–Gly-43Rdx, Ser-44RdxR–Val-8Rdx, and Ser-44RdxR–Val-7Rdx, as well as water-mediated hydrogen bonds between residuesThr-318RdxR–Asp-41Rdx and Pro-316RdxR–Asp-41Rdx (Fig. 2B).Three salt bridges within the interface connecting residues Glu-21Rdx–Lys-377RdxR, Arg-21RdxR–Asp-48Rdx, and Asp-41Rdx–Lys-372RdxR are solvent exposed and partially disordered, indicatingweak contributions to recognition. This matches the observationthat the affinity of P. putida GPO1 (or Pseudomonas oleovorans)RdxR–Rdx is independent of ionic strength (27). Finally, themethylene groups of Glu-21Rdx contribute a van der Waals inter-action to Leu-373RdxR. Overall, the high charge complementarity of

the two molecules, coupled to a small interaction surface andrelatively few directed interactions, ensure a specific yet transientinteraction between RdxR and Rdx.

Binding of Native and Ni2�-Substituted Rdx to RdxR. Purification ofRdx via Ni-nitrilotriacetic acid affinity chromatography results in a�1:1 mixture of Ni2�- and Fe3�-substituted Rdx separable by anionexchange chromatography. To test whether RdxR–Rdx affinity isinfluenced by the metal content and oxidation state of Rdx, wedetermined the KD of RdxFe3� and RdxNi2� for Rdx (27). Nonlinearregression of the RdxFe binding data (Fig. 2C) indicates a KD of 3 �1 �M, comparable with the KD of 1 � 0.1 �M for P. putida GPO1RdxR–Rdx (27). Despite a diminished effect on FAD fluorescenceby RdxNi2� (lower equilibrium �F515) (Fig. 2C), the KD of 5 � 2 �Mof RdxR–RdxNi is comparable with that of RdxR–RdxFe.

DiscussionRelationship of FAD-Dependent Reductases. Phylogenetic analysis(46) of several structurally characterized, FAD-dependent

Fig. 2. Structure of the Rdx–RdxR complex. (A) Cartoon representation. RdxRcolors are as in Fig. 1A, and Rdx is in dark red. FAD, modeled NADH (translu-cent), and cysteine residues of the iron-binding site are shown in ball-and-stickmode, and Fe3� is shown as a green sphere. Anomalous-difference electrondensity (red) contoured at 6.0� documents the presence of the Fe3�. Aminoacid exchanges between RubA1 and RubA2 are indicated as spheres in Rdx(conservative, orange; nonconservative, blue). (B) Interactions surroundingthe redox-active site of the complex. Interacting residues are shown as ball-and-stick models (C of RdxR, yellow, and of Rdx, red-brown). FAD is shown inball-and-stick mode. (C) Binding curves for the interaction of RdxR to Fe3�

(red)- and Ni2� (green)-substituted Rdx.

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NAD(P)H oxidoreductases, indicates GR-fold enzymes to be evo-lutionarily quite distant both from AdxR- and FNR-type enzymes(Fig. 3A). The insertion of the NAD(P)H-binding domain into theFAD-binding domain is distinct for the three branches, indicatingindependent gene insertion events (47, 48). The GR-branch itselfcomprises two phylogenetically and functionally distinct sub-branches, namely electron transferases and disulfide oxidoreduc-tases (Fig. 3A). Thioredoxin reductase (TrxR) (22, 49–51) occupiesan intermediate position by mechanistically resembling disul-fide oxidoreductases but functionally belonging to the electrontransferases.

Several distinguishing features of RdxR underline its evolution-ary distance from other GR-branch electron transferases such asthe mitochondrial apoptosis inducing factor, prokaryotic PdxR, andBphA4. Most importantly, C-terminal Lys-320 of RdxR function-ally replaces a conserved N-terminal lysine, implicated in FADbinding and hydride transfer (19, 20, 23, 37). The pteridine moietyof FAD, shielded from the solvent by a Trp-lid in other FdxRs, isexposed at the FAD-N3 atom in RdxR. Finally, the C-terminaldomains of RdxRs and FdxRs share only marginal sequence andstructural similarity (SI Fig. 6), reflecting their divergent substratespecificities.

RdxR Provides a Preformed Redox Scaffold for Rdx. Interestingly,although TrxR and RdxR share a similar fold, the enzymes employdivergent catalytic strategies. In TrxR, domains rotate relative toeach other so that both NADPH and the catalytic disulfide interactwith the same side of the flavin cofactor (49, 50). In RdxR, bycontrast, substrate- and cofactor-binding sites lie on opposite sidesof FAD (as in GR), eliminating the need for major conformationalchanges and allowing for a simpler electron transport pathway.Correspondingly, the structure of RdxR and, in particular, theFAD-binding domain remains largely unperturbed by Rdx com-plexation (Fig. 3B). rmsd values for C� atoms are 0.42 Å for RdxRand 0.22 Å for the FAD-binding domain. Small yet significantconformational changes (52) do, however, occur both in theNAD(P)H-binding domain (presumably because NAD(P)H is lack-ing) and in the loops connecting both cofactor binding domains.Similarly, complex formation induces the RdxR C-terminal domainto rotate away slightly from the Rdx-binding site resolving anunfavorably close contact between Leu-373RdxR and Glu-22Rdx. Theuncomplexed conformation of RdxR thus represents a preformedRdx binding site, avoiding time-consuming conformational changesthat gate ET reactions in other complexes (53).

RdxR Discriminates Between Two Types of Rdx. Sequence alignmentsindicate that Rdxs involved in alkane oxidation fall into two classes,denoted AlkG1- and AlkG2-type Rdxs (9). RubA2 and RubA1 ofP. aeruginosa PAO1 are both AlkG2-type Rdxs, are encoded byneighboring genes, and are 80% identical by amino acid sequence.Several exchanged residues cluster around the Fe3�/2�-binding site(Fig. 2A) potentially affecting the Fe-center redox potential (26).Some substitutions affect the molecular surface involved in targetrecognition, implying that RubA2 and RubA1 may interact withdistinct electron acceptors.

Other n-alkane using bacteria encode two distinct Rdxs, ofAlkG1- and AlkG2-type. Only the latter transfers electrons fromRdxR to alkane hydroxylases (9). In AlkG1-type Rdxs, an addi-tional arginine is inserted immediately downstream of the secondmetal-binding CXXCG motif. Our complex structure indicates thatthis results in an unfavorably close contact to the positively chargedmolecular surface of RdxR, explaining why AlkG1-type Rdxs areunable to productively interact with RdxR (9, 27).

Electron Transfer from RdxR to Rdx. Electron transfer fromNAD(P)H to Rdx involves a reductive and an oxidative step withrespect to RdxR. NAD(P)H binding initiates the reductive halfreaction, involving hydride transfer from NAD(P)H-C4 to FAD-N5. The resulting blue charge transfer complex between FADH�

and NAD� is detectable as a broad absorption peak between 500and 800 nm after bleaching FAD (data not shown). During theoxidative half reaction, two electrons are sequentially transferredfrom FADH to two Rdx molecules (25), resulting in a flavinsemiquinone reaction intermediate that has, however, not beenobserved spectroscopically for RdxR (25).

The electron transfer reactions alter the protonation state ofFAD-N5. First, a hydride is transferred to N5 from NAD(P)H,whereas the loss of the second electron to Rdx necessitates theremoval of a proton. A Glu/Lys pair (Glu-159/Lys-320 in RdxR),involved in an intricate interaction network with FAD-N5 (Fig. 1C;SI Fig. 7A) and functionally conserved in ETases or replaced by ahistidine-bound ‘‘central water’’ (SI Fig. 7B) in AdxR (48), wasthought to be crucial for the hydrogen transfer reactions (19, 20, 23,37, 48). Replacing these residues in mAIF, however, results in lossof FAD (20), whereas adding FAD increases activity beyondwild-type levels (20). This residue pair thus appears primarily to berequired for FAD binding in RdxR-type enzymes rather than forhydride transfer.

Implications for Interprotein ET. According to the Marcus theory(54–57), ET kinetics depend on the edge-to-edge distance of the

Fig. 3. Phylogenetic topology of structurally characterized enzymes functionally and/or structurally related to RdxR. (A) FNR, plant-type ferredoxin reductase;AdxR, adrenodoxin reductase; AIF, apoptosis inducing factor; LpdR, lipoamide reductase; TptR, trypanothione reductase. Sequence identity to RdxR is indicatedby percentage. (B) Superposition of uncomplexed (gray) and complexed RdxR (colored as in Fig. 1A). Cofactors are shown as ball-and-stick models. For clarity,the position of Rdx is indicated by a brown sphere. (C) Comparison of the Rdx (red) and Trx (black) binding sites of RdxR and TrxR. For clarity, only the molecularsurface of RdxR is shown. �G0 optimized ET rates (42) between FAD and each point of the RdxR surface are indicated by a color gradient (red, high ET rate; green,medium; blue, low). (Inset) Close-up view of the cofactors involved.

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redox cofactors involved, the driving force of the reaction (�G0),and the reorganization energy (�) (55). At a given distance, ETrates will be highest for activationless reactions where �G0 � ��.For this optimal case, the distance of 6.2 Å between the two redoxcenters in the RdxR–Rdx complex, would allow transfer rates in thepicosecond range (1011 s�1) (41). Assuming the redox potentials forRdxR and Rdx of P. aeruginosa to be similar to those of P. putida(25), indicates a �G0 of around �0.25 eV. Combined with � � 1 eV,typical for intermolecular ET (41), and a packing density of 0.64[calculated by using the ET rates package (41)], this reduces the ETrate to 109 s�1 (or 108 s�1 for � � 1.5 eV) (41). However, note thatRdxR successively transfers single electrons to two distinct Rdxmolecules. �G0 is therefore presumably different for both reactions(25), leading to differing ET rates for each of the reactions. Thus,although the crystal structure of RdxR–Rdx allows the maximaltheoretical rate of electron transfer to be estimated, parameterssuch as �G0 and � would need to be determined experimentally toreveal the true ET rate between RdxR and Rdx. Nevertheless, theET rate for RdxR–Rdx is probably faster than typical turnover ratesof ET proteins (41), implying that diffusional steps such as Rdx- andNADH-binding may be rate limiting. The binding site is thus underevolutionary pressure to optimize the interaction. �G0-optimizedET rates, calculated for all points on the surface of RdxR (ref. 41;Fig. 3C), demonstrate that the binding site is optimized to allowdocking of Rdx at a pronounced tunneling hot spot of RdxR.Interestingly, structurally unrelated Trx binds to a distinct, secondhot spot located on the opposite side of the FAD cofactor in therelated structure of thioredoxin reductase (Fig. 3C), although theprocess of electron transfer in that system is more complex than inRdxR–Rdx (22).

Electrostatics are important for the formation and kinetics ofredox complexes (40). Comparing the charge distribution at themolecular surface of RdxR–Rdx and related complexes indicatescharges to be complementary for each redox pair (Fig. 4). Therespective dipole moments (bioportal.weizmann.ac.il/dipol/) inter-sect at acute angles (37° for FNR/Fdx, 47° for TrxR/Trx) allowingfor favorable steering of the incoming reaction partner (Fig. 4) (58).Thus, despite evolutionary pressure to conserve substrate andcofactor interactions (59), ET couples have diverged with respect toelectrostatic forces and guidance of their particular substrate.

The pronounced electrostatic homing system, the docking of Rdxprecisely at the tunneling hot spot of RdxR, and the absence of anyappreciable conformational changes during Rdx binding wouldappear to suggest complex formation to proceed by a ‘‘simple-’’rather than a ‘‘gated-’’ or ‘‘dynamic-docking’’ mechanism (60).

Concluding Remarks. Biological one-electron redox reactions gen-erally depend on an electron distribution system consisting of anFAD-dependent NAD(P)H-oxidoreductase combined with a smalliron-binding protein such as ferredoxin, cytochrome, or rubre-doxin. Small size and differing redox potentials of the iron-bindingproteins ensure that a multitude of electron acceptors can besupplied by a comparatively small number of electron carriers. Ouranalyses indicate the RdxR–Rdx system to be a structurally andfunctionally distinct electron shuttling system. Features of thecomplex such as the preformed interaction surface, the pronouncedelectrostatic homing system, a short tunneling distance, as well asthe weak dissociation constant imply the complex to be optimizedfor rapid transport of reducing equivalents to the actual place ofreduction. Clearly, the RdxR–Rdx system is crucial to P. aeruginosato grow on n-alkanes. However, employing such a sophisticatedredox chain for this single purpose appears disproportionate, im-plying additional but as-yet-unidentified roles of the RdxR–Rdxcouple in P. aeruginosa.

MethodsCloning, Protein Expression, and Purification. The genes PA5349(RdxR) and PA5350 (Rdx) of P. aeruginosa PAO1 were amplifiedfrom genomic DNA using following PCR primers: RdxR (PA5349),5�-GCGCTCTAGATAACGAGGGCAAAAAATGAGCGA-GCGTGCGCCCCTGGTA-3�, 5�-GCGCAGATCTAGCCAT-GAGGCCGGGTAACTCTTTG-3�; and Rdx (PA5350),5�-GACGGCCATATGCGCAAGTGGCAATGCGTGGTC-3�,5�-GCGCAGATCTGGCGATCTCGATCATCTCGAA-3�.

The NdeI/BglII digestion products were cloned into pBBR22bII,a derivative of pBBR22b (61), resulting in His6 fusions of the targetproteins. Proteins were produced in Escherichia coli Tuner cells(Novagen, Madison, WI) in LB medium and 37 �g/ml chloram-phenicol. At an OD600 of 1.0 (37°C), protein expression was inducedby 0.5 mM isopropyl-�-D-thiogalactoside and continued overnightat 20°C. Cells were centrifuged, resuspended, and lysed by Frenchpress, and cell debris was removed by centrifugation. Purificationinvolved Ni-nitrilotriacetic acid affinity chromatography (Qiagen,Hilden, Germany), anion exchange chromatography (MonoQ; GEHealthcare, Chalfont St. Giles, U.K.), and gel filtration (Superdex75; GE Healthcare). Ni2�- (because of Ni-nitrilotriacetic acid) andFe3�-binding Rdx were separated during ion-exchange chromatog-raphy. Proteins were dialyzed against 100 mM NaCl, 50 mMTris�HCl (pH 8). A 5 mM concentration of �-mercaptoethanol wasadded to RdxR, to prevent cysteine oxidation. Proteins were storedat 4°C at concentrations of 8.5 mg/ml (RdxR) and 30 mg/ml (Rdx).

Alkane Oxidation. P. aeruginosa strain TBCF10839 wild type, theisogenic RdxR-transposon mutant (62), and the RdxR-comple-mented strain were grown on E2 minimal agar. The plates wereplaced in a sealed container, and hexadecane was supplied as thesole carbon source. Growth was monitored for 3–5 days at 30°C.

Equilibrium Binding Studies. A Nanodrop ND-3300 fluorospectrom-eter was used to monitor Rdx-induced changes in FAD fluores-cence. The intensity of blue light-emitting diode (�max � 470nm)-induced FAD fluorescence was monitored at 515 nm. Aconcentration of 12 �M for RdxR was used throughout, whereasthat of Rdx varied between 0 and 140 �M. A short optical pathway(�1 mm) largely eliminates inner filter effects. Measurements intriplicate were performed in 50 mM Tris�HCl (pH 8) and50 mM NaCl. Dissociation constants (KD) were determined asdescribed (27).

Fig. 4. Electrostatics of interprotein ET reactions. RdxR–Rdx (A) is comparedwith other ET complexes: TrxR–Trx (B) (50) and FNR–Fdx (C) (34). Complexes areshown in cartoon representation with the reductase in yellow and the sub-strate in red. FADs are shown in ball-and-stick mode, and dipole moments areshown as dumbbells. Electrostatic surface potentials are mapped onto van derWaals surfaces (red, negative; blue, positive; white, neutral) in open-bookmode.

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Crystallization. Hexagonal RdxR crystals were grown at 20°C bysitting-drop vapor diffusion with equal volumes (0.1 �l) of protein(8.5 mg/ml) and reservoir solution [5% PEG 1000, 40% PEG 300,0.1 M Tris�HCl (pH 7)]. Crystals were flash frozen in liquid nitrogenwithout further cryoprotection.

RdxR–Rdx complex crystals were also grown at 20°C bysitting-drop vapor diffusion. RdxR at 10 mg/ml was mixed witha 1.2 molar excess of Rdx and diluted to 8.5 mg/ml. Equalvolumes (0.1 �l) of protein mixture and reservoir solution (0.2M KF, 20% PEG 3350) were used. Microseeding with severelyintergrown initial crystals yielded plate-shaped orthorhombiccrystals of the complex. Mother liquor supplemented with 25%PEG 400 was used for cryoprotection.

Structure Determination. X-ray diffraction data for RdxR werecollected at � � 0.92 Å at beamline BL1 (BerlinerElektronenspeicherring-Gesellschaft fur Synchrotronstrahlung,Berlin, Germany), for RdxR–Rdx at � � 1.7 Å at beamline

BW7A (European Molecular Biology Laboratory, Hamburg,Germany) using MarCCD detectors (MarResearch, Norder-stedt, Germany). HKL2000 (63) was used for data processing,PHASER (64) for molecular replacement, CNS (65) and CCP4suites (66) for structure refinement, and COOT (67) for modelbuilding, structural analysis, and validation. Figures were pre-pared by using PYMOL. Crystallographic statistics are listed inTable 1.

We thank Drs. M. Groves (European Molecular Biology Laboratory)and U. Muller (Berliner Elektronenspeicherring-Gesellschaft fur Syn-chrotronstrahlung) for support during data collection. Synchrotron beamtime at beamlines BW7A (Deutsches Elektronen Synchrotron, Ham-burg, Germany) and BL1 (Berliner Elektronenspeicherring-Gesellschaftfur Synchrotronstrahlung, Berlin, Germany) is gratefully acknowledged.This study was supported by the Deutsche ForschungsgemeinschaftSCHU1365/1-2 (to W.-D.S.) and by the Fonds der Chemischen Industrie(D.W.H.).

1. Eberl L, Tummler B (2004) Int J Med Microbiol 294:123–131.2. Pruitt BA, Jr, McManus AT, Kim SH, Goodwin CW (1998) World J Surg

22:135–145.3. Hagelueken G, Adams TM, Wiehlmann L, Widow U, Kolmar H, Tummler B,

Heinz DW, Schubert WD (2006) Proc Natl Acad Sci USA 103:7631–7636.4. Hsu YC (1963) Nature 200:1091–1092.5. Vandecasteele JP, Blanchet D, Tassin JP, Bonamy AM, Guerrillot L (1983)

Acta Biotechnol 3:339–344.6. Abalos A, Vinas M, Sabate J, Manresa MA, Solanas AM (2004) Biodegradation

15:249–260.7. Smits TH, Witholt B, van Beilen JB (2003) Antonie van Leeuwenhoek 84:193–

200.8. Marin MM, Yuste L, Rojo F (2003) J Bacteriol 185:3232–3237.9. van Beilen JB, Neuenschwander M, Smits TH, Roth C, Balada SB, Witholt B

(2002) J Bacteriol 184:1722–1732.10. Lovenberg W, Sobel BE (1965) Proc Natl Acad Sci USA 54:193–199.11. Frazao C, Silva G, Gomes CM, Matias P, Coelho R, Sieker L, Macedo S, Liu

MY, Oliveira S, Teixeira M, et al. (2000) Nat Struct Biol 7:1041–1045.12. Fareleira P, Santos BS, Antonio C, Moradas-Ferreira P, LeGall J, Xavier AV,

Santos H (2003) Microbiology 149:1513–1522.13. Grunden AM, Jenney FE, Jr, Ma K, Ji M, Weinberg MV, Adams MW (2005)

Appl Environ Microbiol 71:1522–1530.14. Ma K, Adams MW (1999) J Bacteriol 181:5530–5533.15. Chen L, Liu MY, LeGall J, Fareleira P, Santos H, Xavier AV (1993) Eur

J Biochem 216:443–448.16. Karplus PA, Schulz GE (1987) J Mol Biol 195:701–729.17. Frey M, Sieker L, Payan F, Haser R, Bruschi M, Pepe G, LeGall J (1987) J Mol

Biol 197:525–541.18. Mattevi A, Obmolova G, Kalk KH, van Berkel WJ, Hol WG (1993) J Mol Biol

230:1200–1215.19. Senda T, Yamada T, Sakurai N, Kubota M, Nishizaki T, Masai E, Fukuda M,

Mitsuidagger Y (2000) J Mol Biol 304:397–410.20. Mate MJ, Ortiz-Lombardia M, Boitel B, Haouz A, Tello D, Susin SA,

Penninger J, Kroemer G, Alzari PM (2002) Nat Struct Biol 9:442–446.21. Hunter WN, Bailey S, Habash J, Harrop SJ, Helliwell JR, Aboagye-Kwarteng

T, Smith K, Fairlamb AH (1992) J Mol Biol 227:322–333.22. Williams CH, Jr (1995) FASEB J 9:1267–1276.23. Sevrioukova IF, Li H, Poulos TL (2004) J Mol Biol 336:889–902.24. Lee HJ, Lian LY, Scrutton NS (1996) Biochem Soc Trans 24:447S.25. Lee HJ, Basran J, Scrutton NS (1998) Biochemistry 37:15513–15522.26. Kummerle R, Zhuang-Jackson H, Gaillard J, Moulis JM (1997) Biochemistry

36:15983–15991.27. Perry A, Tambyrajah W, Grossmann JG, Lian LY, Scrutton NS (2004)

Biochemistry 43:3167–3182.28. Lin IJ, Gebel EB, Machonkin TE, Westler WM, Markley JL (2005) Proc Natl

Acad Sci USA 102:14581–14586.29. Bonisch H, Schmidt CL, Bianco P, Ladenstein R (2005) Acta Crystallogr D

61:990–1004.30. Chen CJ, Lin YH, Huang YC, Liu MY (2006) Biochem Biophys Res Commun

349:79–90.31. Peterson JA, Coon MJ (1968) J Biol Chem 243:329–334.32. Bau R, Rees DC, Kurtz DM, Scott RA, Huang H, Adams MWW, Eidsness MK

(1998) J Biol Inorg Chem 3:484–493.

33. Schulz GE, Schirmer RH, Sachsenheimer W, Pai EF (1978) Nature 273:120–124.

34. Kurisu G, Kusunoki M, Katoh E, Yamazaki T, Teshima K, Onda Y, Kimata-Ariga Y, Hase T (2001) Nat Struct Biol 8:117–121.

35. Kuznetsov VY, Blair E, Farmer PJ, Poulos TL, Pifferitti A, Sevrioukova IF(2005) J Biol Chem 280:16135–16142.

36. Cande C, Cecconi F, Dessen P, Kroemer G (2002) J Cell Sci 115:4727–4734.37. Pai EF, Schulz GE (1983) J Biol Chem 258:1752–1757.38. Krissinel E, Henrick K (2005) in CompLife 2005, ed Berthold MR (Springer,

Heidelberg), pp 163–174.39. Kurihara K, Tanaka I, Chatake T, Adams MW, Jenney FE, Jr, Moiseeva N, Bau

R, Niimura N (2004) Proc Natl Acad Sci USA 101:11215–11220.40. Crowley PB, Carrondo MA (2004) Proteins 55:603–612.41. Page CC, Moser CC, Chen X, Dutton PL (1999) Nature 402:47–52.42. van Amsterdam IM, Ubbink M, Einsle O, Messerschmidt A, Merli A, Cavazzini

D, Rossi GL, Canters GW (2002) Nat Struct Biol 9:48–52.43. Beratan DN, Betts JN, Onuchic JN (1991) Science 252:1285–1288.44. Lin J, Balabin IA, Beratan DN (2005) Science 310:1311–1313.45. Baker NA, Sept D, Joseph S, Holst MJ, McCammon JA (2001) Proc Natl Acad

Sci USA 98:10037–10041.46. Kumar S, Tamura K, Nei M (2004) Brief Bioinform 5:150–163.47. Schulz GE (1980) J Mol Biol 138:335–347.48. Ziegler GA, Schulz GE (2000) Biochemistry 39:10986–10995.49. Waksman G, Krishna TS, Williams CH, Jr, Kuriyan J (1994) J Mol Biol

236:800–816.50. Lennon BW, Williams CH, Jr, Ludwig ML (2000) Science 289:1190–1194.51. Mustacich D, Powis G (2000) Biochem J 346:1–8.52. Schneider TR (2000) Acta Crystallogr D 56:714–721.53. Leys D, Basran J, Talfournier F, Sutcliffe MJ, Scrutton NS (2003) Nat Struct

Biol 10:219–225.54. Marcus RA (1956) J Chem Phys 24:966–978.55. Moser CC, Keske JM, Warncke K, Farid RS, Dutton PL (1992) Nature

355:796–802.56. Marcus RA, Sutin N (1985) Biochim Biophys Acta 811:265–322.57. Gray HB, Winkler JR (1996) Annu Rev Biochem 65:537–561.58. De Pascalis AR, Jelesarov I, Ackermann F, Koppenol WH, Hirasawa M, Knaff

DB, Bosshard HR (1993) Protein Sci 2:1126–1135.59. Page CC, Moser CC, Dutton PL (2003) Curr Opin Chem Biol 7:551–556.60. Liang ZX, Kurnikov IV, Nocek JM, Mauk AG, Beratan DN, Hoffman BM

(2004) J Am Chem Soc 126:2785–2798.61. Rosenau F, Jager KE (2004) in Enzyme Functionality: Design, Engineering and

Screening, ed Svendsen A (Dekker, New York), pp 617–631.62. Wiehlmann L, Munder A, Adams T, Juhas M, Kolmar H, Salunkhe P, Tummler

B (2007) Int J Med Microbiol, in press.63. Otwinowski Z, Minor W (1997) Methods Enzymol 276:307–326.64. McCoy AJ, Grosse-Kunstleve RW, Storoni LC, Read RJ (2005) Acta Crystal-

logr D 61:458–464.65. Brunger AT, Adams PD, Clore GM, DeLano WL, Gros P, Grosse-Kunstleve

RW, Jiang JS, Kuszewski J, Nilges M, Pannu NS, et al. (1998) Acta CrystallogrD 54:905–921.

66. CCP4 (1994) Acta Crystallogr D 50:760–763.67. Emsley P, Cowtan K (2004) Acta Crystallogr D 60:2126–2132.

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