A directional electron transfer regulator based on heme-chain architecture in the small tetraheme cytochrome c from Shewanella oneidensis

The macroscopic and microscopic redox potentials of the four hemes of the small tetraheme cytochrome c from Shewanella oneidensis were determined. The microscopic redox potentials show that the order of reduction is from hemes in the C-terminal domain (hemes 3 and 4) to the N-terminal domain (heme 1), demonstrating the polarization of the tetraheme chain during reduction. This makes heme 4 the most efficient electron delivery site. Furthermore, multi-step reduction of other redox centers through either heme 4 or heme 3 is shown to be possible. This has provided new insights into the two-electron reduction of the flavin in the homologous flavocytochrome c-fumarate reductase.     

The solution structure of a tetraheme cytochrome from Shewanella frigidimarina reveals a novel family structural motif

The bacteria belonging to the genus Shewanella are facultative anaerobes that utilize a variety of terminal electron acceptors which includes soluble and insoluble metal oxides. The tetraheme c-type cytochrome isolated during anaerobic growth of Shewanella frigidimarina NCIMB400 ( Sfc) contains 86 residues and is involved in the Fe(III) reduction pathways. Although the functional properties of Sfc redox centers are quite well described, no structures are available for this protein. In this work, we report the solution structure of the reduced form of Sfc. The overall fold is completely different from those of the tetraheme cytochromes c 3 and instead has similarities with the tetraheme cytochrome recently isolated from Shewanella oneidensis ( Soc). Comparison of the tetraheme cytochromes from Shewanella shows a considerable diversity in their primary structure and heme reduction potentials, yet they have highly conserved heme geometry, as is the case for the family of tetraheme cytochromes isolated from Desulfovibrio spp.     

Sulfate respiration in Desulfovibrio vulgaris Hildenborough. Structure of the 16-heme cytochrome c HmcA AT 2.5-A resolution and a view of its role in transmembrane electron transfer

The crystal structure of the high molecular mass cytochrome c HmcA from Desulfovibrio vulgaris Hildenborough is described. HmcA contains the unprecedented number of sixteen hemes c attached to a single polypeptide chain, is associated with a membrane-bound redox complex, and is involved in electron transfer from the periplasmic oxidation of hydrogen to the cytoplasmic reduction of sulfate. The structure of HmcA is organized into four tetraheme cytochrome c(3)-like domains, of which the first is incomplete and contains only three hemes, and the final two show great similarity to the nine-heme cytochrome c from Desulfovibrio desulfuricans. An isoleucine residue fills the vacant coordination space above the iron atom in the five-coordinated high-spin Heme 15. The characteristics of each of the tetraheme domains of HmcA, as well as its surface charge distribution, indicate this cytochrome has several similarities with the nine-heme cytochrome c and the Type II cytochrome c(3) molecules, in agreement with their similar genetic organization and mode of reactivity and further support an analogous physiological function for the three cytochromes. Based on the present structure, the possible electron transfer sites between HmcA and its redox partners (namely Type I cytochrome c(3) and other proteins of the Hmc complex), as well as its physiological role, are discussed.     

The tetraheme cytochrome CymA is required for anaerobic respiration with dimethyl sulfoxide and nitrite in Shewanella oneidensis

The tetraheme c-type cytochrome, CymA, from Shewanella oneidensis MR-1 has previously been shown to be required for respiration with Fe(III), nitrate, and fumarate [Myers, C. R., and Myers, J. M. (1997) J. Bacteriol. 179, 1143-1152]. It is located in the cytoplasmic membrane where the bulk of the protein is exposed to the periplasm, enabling it to transfer electrons to a series of redox partners. We have expressed and purified a soluble derivative of CymA (CymA(sol)) that lacks the N-terminal membrane anchor. We show here, by direct measurements of electron transfer between the purified proteins, that CymA(sol) efficiently reduces S. oneidensis fumarate reductase. This indicates that no further proteins are required for electron transfer between the quinone pool and fumarate if we assume direct reduction of CymA by quinols. By expressing CymA(sol) in a mutant lacking CymA, we have shown that this soluble form of the protein can complement the defect in fumarate respiration. We also demonstrate that CymA is essential for growth with DMSO (dimethyl sulfoxide) and for reduction of nitrite, implicating CymA in at least five different electron transfer pathways in Shewanella.     

Structure and mechanism of the flavocytochrome c fumarate reductase of Shewanella putrefaciens MR-1

Fumarate respiration is one of the most widespread types of anaerobic respiration. The soluble fumarate reductase of Shewanella putrefaciens MR-1 is a periplasmic tetraheme flavocytochrome c. The crystal structures of the enzyme were solved to 2.9 A for the uncomplexed form and to 2.8 A and 2.5 A for the fumarate and the succinate-bound protein, respectively. The structures reveal a flexible capping domain linked to the FAD-binding domain. A catalytic mechanism for fumarate reduction based on the structure of the complexed protein is proposed. The mechanism for the reverse reaction is a model for the homologous succinate dehydrogenase (complex II) of the respiratory chain. In flavocytochrome c fumarate reductase, all redox centers are in van der Waals contact with one another, thus providing an efficient conduit of electrons from the hemes via the FAD to fumarate.     

Role of the aromatic ring of Tyr43 in tetraheme cytochrome c(3) from Desulfovibrio vulgaris Miyazaki F

Tyrosine 43 is positioned parallel to the fifth heme axial ligand, His34, of heme 1 in the tetraheme cytochrome c(3). The replacement of tyrosine with leucine increased the redox potential of heme 1 by 44 and 35 mV at the first and last reduction steps, respectively; its effects on the other hemes are small. In contrast, the Y43F mutation hardly changed the potentials. It shows that the aromatic ring at this position contributes to lowering the redox potential of heme 1 locally, although this cannot be the major contribution to the extremely low redox potentials of cytochrome c(3). Furthermore, temperature-dependent line-width broadening in partially reduced samples established that the aromatic ring at position 43 participates in the control of the kinetics of intramolecular electron transfer. The rate of reduction of Y43L cytochrome c(3) by 5-deazariboflavin semiquinone under partially reduced conditions was significantly different from that of the wild type in the last stage of the reduction, supporting the involvement of Tyr43 in regulation of reduction kinetics. The mutation of Y43L, however, did not induce a significant change in the crystal structure.     

Redox-Bohr and other cooperativity effects in the nine-heme cytochrome C from Desulfovibrio desulfuricans ATCC 27774: crystallographic and modeling studies

The nine-heme cytochrome c is a monomeric multiheme cytochrome found in Desulfovibrio desulfuricans ATCC 27774. The polypeptide chain comprises 296 residues and wraps around nine hemes of type c. It is believed to take part in the periplasmic assembly of proteins involved in the mechanism of hydrogen cycling, receiving electrons from the tetraheme cytochrome c3. With the purpose of understanding the molecular basis of electron transfer processes in this cytochrome, we have determined the crystal structures of its oxidized and reduced forms at pH 7.5 and performed theoretical calculations of the binding equilibrium of protons and electrons in these structures. This integrated study allowed us to observe that the reduction process induced relevant conformational changes in several residues, as well as protonation changes in some protonatable residues. In particular, the surroundings of hemes I and IV constitute two areas of special interest. In addition, we were able to ascertain the groups involved in the redox-Bohr effect present in this cytochrome and the conformational changes that may underlie the redox-cooperativity effects on different hemes. Furthermore, the thermodynamic simulations provide evidence that the N- and C-terminal domains function in an independent manner, with the hemes belonging to the N-terminal domain showing, in general, a lower redox potential than those found in the C-terminal domain. In this way, electrons captured by the N-terminal domain could easily flow to the C-terminal domain, allowing the former to capture more electrons. A notable exception is heme IX, which has low redox potential and could serve as the exit path for electrons toward other proteins in the electron transfer pathway.     

Identification of a small tetraheme cytochrome c and a flavocytochrome c as two of the principal soluble cytochromes c in Shewanella oneidensis strain MR1.

Two abundant, low-redox-potential cytochromes c were purified from the facultative anaerobe Shewanella oneidensis strain MR1 grown anaerobically with fumarate. The small cytochrome was completely sequenced, and the genes coding for both proteins were cloned and sequenced. The small cytochrome c contains 91 residues and four heme binding sites. It is most similar to the cytochromes c from Shewanella frigidimarina (formerly Shewanella putrefaciens) NCIMB400 and the unclassified bacterial strain H1R (64 and 55% identity, respectively). The amount of the small tetraheme cytochrome is regulated by anaerobiosis, but not by fumarate. The larger of the two low-potential cytochromes contains tetraheme and flavin domains and is regulated by anaerobiosis and by fumarate and thus most nearly corresponds to the flavocytochrome c-fumarate reductase previously characterized from S. frigidimarina to which it is 59% identical. However, the genetic context of the cytochrome genes is not the same for the two Shewanella species, and they are not located in multicistronic operons. The small cytochrome c and the cytochrome domain of the flavocytochrome c are also homologous, showing 34% identity. Structural comparison shows that the Shewanella tetraheme cytochromes are not related to the Desulfovibrio cytochromes c(3) but define a new folding motif for small multiheme cytochromes c.     

Identification of 42 possible cytochrome C genes in the Shewanella oneidensis genome and characterization of six soluble cytochromes

Through pattern matching of the cytochrome c heme-binding site (CXXCH) against the genome sequence of Shewanella oneidensis MR-1, we identified 42 possible cytochrome c genes (27 of which should be soluble) out of a total of 4758. However, we found only six soluble cytochromes c in extracts of S. oneidensis grown under several different conditions: (1) a small tetraheme cytochrome c, (2) a tetraheme flavocytochrome c-fumarate reductase, (3) a diheme cytochrome c4, (4) a monoheme cytochrome c5, (5) a monoheme cytochrome c', and (6) a diheme bacterial cytochrome c peroxidase. These cytochromes were identified either through N-terminal or complete amino acid sequence determination combined with mass spectroscopy. All six cytochromes were about 10-fold more abundant when cells were grown at low than at high aeration, whereas the flavocytochrome c-fumarate reductase was specifically induced by anaerobic growth on fumarate. When adjusted for the different heme content, the monoheme cytochrome c5 is as abundant as are the small tetraheme cytochrome and the tetraheme fumarate reductase. Published results on regulation of cytochromes from DNA microarrays and 2D-PAGE differ somewhat from our results, emphasizing the importance of multifaceted analyses in proteomics.     

Ferredoxin electron transfer site on cytochrome c3. Structural hypothesis of an intramolecular electron transfer pathway within a tetra-heme cytochrome.

To specify electron exchanges involving Desulfovibrio desulfuricans Norway tetra-heme cytochrome c3, the chemical modification of arginine 73 residue, was performed. Biochemical and biophysical studies have shown that the modified cytochrome retains its ability to both interact and act as an electron carrier with its redox partners, ferredoxin and hydrogenase. Moreover, the chemical modification effects on the cytochrome c3 1H NMR spectrum were similar to that induced by the presence of ferredoxin. This suggests that arginine 73 is localized on the cytochrome c3 ferredoxin interacting site. The identification of heme 4, the closest heme to arginine 73, as the ferredoxin interacting heme helps us to hypothesize about the role of the three other hemes in the molecule. A structural hypothesis for an intramolecular electron transfer pathway, involving hemes 4, 3 and 1, is proposed on the basis of the crystal structures of D. vulgaris Miyazaki and D. desulfuricans Norway cytochromes c3. The unique role of some structural features (alpha helix, aromatic residues) intervening between the heme groups, is proposed.     

Full assignment of heme redox potentials of cytochrome c3 of D. vulgaris Miyazaki F by 1H-NMR.

Site-specific heme assignment of the 1H-NMR spectrum of cytochrome c3 of D. vulgaris Miyazaki F, a tetraheme protein, was established. The major reduction of the heme turned out to take place in the order of hemes I, III, IV and II (numbering in the crystal structure). The hemes with the smallest and greatest solvent accessibility were reduced at the highest and lowest potentials on average, respectively. A cooperative interheme interaction was attributed to a pair of the closest hemes, namely, hemes III and IV. This assignment can provide the physiochemical basis for the elucidation of electron transfer of this protein.     

A dynamic periplasmic electron transfer network enables respiratory flexibility beyond a thermodynamic regulatory regime

Microorganisms show an astonishing versatility in energy metabolism. They can use a variety of different catabolic electron acceptors, but they use them according to a thermodynamic hierarchy, which is determined by the redox potential of the available electron acceptors. This hierarchy is reflected by a regulatory machinery that leads to the production of respiratory chains in dependence of the availability of the corresponding electron acceptors. In this study, we showed that the γ-proteobacterium Shewanella oneidensis produces several functional electron transfer chains simultaneously. Furthermore, these chains are interconnected, most likely with the aid of c-type cytochromes. The cytochrome pool of a single S. oneidensis cell consists of ca. 700 000 hemes, which are reduced in the absence on an electron acceptor, but can be reoxidized in the presence of a variety of electron acceptors, irrespective of prior growth conditions. The small tetraheme cytochrome (STC) and the soluble heme and flavin containing fumarate reductase FccA have overlapping activity and appear to be important for this electron transfer network. Double deletion mutants showed either delayed growth or no growth with ferric iron, nitrate, dimethyl sulfoxide or fumarate as electron acceptor. We propose that an electron transfer machinery that is produced irrespective of a thermodynamic hierarchy not only enables the organism to quickly release catabolic electrons to a variety of environmental electron acceptors, but also offers a fitness benefit in redox-stratified environments.     

Calculated coupling of transmembrane electron and proton transfer in dihemic quinol:fumarate reductase

The quinol:fumarate reductase of Wolinella succinogenes binds a low- and a high-potential heme b group in its transmembrane subunit C. Both hemes are part of the electron transport chain between the two catalytic sites of this redox enzyme. The oxidation-reduction midpoint potentials of the hemes are well established but their assignment in the structure has not yet been determined. By simulating redox titrations, using continuum electrostatics calculations, it was possible to achieve an unequivocal assignment of the low- and high-potential hemes to the distal and proximal positions in the structure, respectively. Prominent features governing the differences in midpoint potential between the two hemes are the higher loss of reaction field energy for the proximal heme and the stronger destabilization of the oxidized form of the proximal heme due to several buried Arg and Lys residues. According to the so-called "E-pathway hypothesis", quinol:fumarate reductase has previously been postulated to exhibit a novel coupling of transmembrane electron and proton transfer. Simulation of heme b reduction indicates that the protonation state of the conserved residue Glu C180, predicted to play a key role in this process, indeed depends on the redox state of the hemes. This result clearly supports the E-pathway hypothesis.     

Reduction kinetics of the four hemes of cytochrome c3 from Desulfovibrio vulgaris by flash photolysis.

The reduction of the tetraheme cytochrome c3 (from Desulfovibrio vulgaris, strains Miyazaki F and Hildenbourough) by flavin semiquinone and reduced methyl viologen follows a monophasic kinetic profile, even though the four hemes do not have equivalent reduction potentials. Rate constants for reduction of the individual hemes are obtained subsequent to incrementally reducing the cytochrome by phototitration. The dependence of each rate constant on the reduction potential difference between the heme and the reductant can be described by outer sphere electron transfer theroy. Thus, the very low reduction potentials of the cytochrome c3 hemes compensate for the very large solvent accessibility of the hemes. The relative rate constants for electron transfer to the four hemes of cytochrome c3 are consistent with the assignments of reduction potential to hemes previously made by Park et al. (Park, J.-S., Kano, K., Niki, S. and Akutsu, H. (1991) FEBS Lett. 285, 149-151) using NMR techniques. The ionic strength dependence of the observed rate constant for reduction by the methyl viologen radical cation indicates that ionic strength substantially alters the structure and/or the heme reduction potentials of the cytochrome. This result is confirmed by reduction with a neutral flavin species (5-deazariboflavin semiquinone) in which the reactivity of the highest potential heme decreases and the reactivity of the lowest potential heme increases at high (500 mM) ionic strength, and by the sensitivity of heme methyl resonances to ionic strength as observed by 1H-NMR. These unusual ionic strength-dependent effects may be due to a combination of structural changes in the cytochrome and alterations of the electrostatic fields at elevated ionic strengths.     

Time-resolved electron transfer at the donor side of Rhodopseudomonas viridis photosynthetic reaction centers in whole cells

We have studied the electron transfer reactions from the tetraheme cytochrome of Rhodopseudomonas viridis to the oxidized primary donor in whole cells with a new high sensitivity spectrophotometer. In this apparatus the monochromatic detecting flashes are provided by a YAG pumped Optical Parametric Oscillator, allowing a 10 ns time resolution. When four hemes are reduced the observed electron transfer reaction sequence is the following: first the low-potential c552 heme (the number refers to the maximum absorption wavelength in the alpha-band region) is oxidized with a half time of 130 ns, in agreement with previous reports of measurements performed with purified reaction centers. Then, the electron hole is transferred to the low potential c554 heme with a half time of 2.6 µs. When only the two high potential hemes are reduced the observed electron transfer sequence is the following: oxidation of the high potential c559 heme in the hundreds of ns time range (410 ns), reduction of this heme by the high potential c556 heme in the µs time range (2.7 µs). This confirms the first steps of electron transfer observed in isolated reaction centers. However, in the microsecond time domain, the overall amount of oxidized hemes increases suggesting that, in vivo, the equilibrium constant between the P⁺/P and the c559^ox/c559^red couples is significantly lower than expected from the difference in their midpoint potentials.     

Crystal structure of the oxidised and reduced acidic cytochrome c3from Desulfovibrio africanus.

Unique among sulphate-reducing bacteria, Desulfovibrio africanus has two periplasmic tetraheme cytochromes c3, one with an acidic isoelectric point which exhibits an unusually low reactivity towards hydrogenase, and another with a basic isoelectric point which shows the usual cytochrome c3reactivity. The crystal structure of the oxidised acidic cytochrome c3of Desulfovibrio africanus (Dva.a) was solved by the multiple anomalous diffraction (MAD) method and refined to 1.6 A resolution. Its structure clearly belongs to the same family as the other known cytochromes c3, but with weak parentage with those of the Desulfovibrio genus and slightly closer to the cytochromes c3of Desulfomicrobium norvegicum. In Dva.a, one edge of heme I is completely exposed to the solvent and surrounded by a negatively charged protein surface. Heme I thus seems to play an important role in electron exchange, in addition to heme III or heme IV which are the electron exchange ports in the other cytochromes c3. The function of Dva.a and the nature of its redox partners in the cell are thus very likely different.By alignment of the seven known 3D structures including Dva.a, it is shown that the structure which is most conserved in all cytochromes c3is the four-heme cluster itself. There is no conserved continuous protein structure which could explain the remarkable invariance of the four-heme cluster. On the contrary, the proximity of the heme edges is such that they interact directly by hydrophobic and van der Waals contacts. This direct interaction, which always involves a pyrrole CA-CB side-chain and its bound protein cysteine Sgammaatom, is probably the main origin of the four-heme cluster stability. The same kind of interaction is found in the chaining of the hemes in other multihemic redox proteins.The crystal structure of reduced Dva. a was solved at 1.9 A resolution. The comparison of the oxidised and reduced structures reveals changes in the positions of water molecules and polar residues which probably result from changes in the protonation state of amino acids and heme propionates. Water molecules are found closer to the hemes and to the iron atoms in the reduced than in the oxidised state. A global movement of a chain fragment in the vicinity of hemes III and IV is observed which result very likely from the electrostatic reorganization of the polypeptide chain induced by reduction.     

Insight into molecular stability and physiological properties of the diheme cytochrome CYC41 from the acidophilic bacterium Acidithiobacillus ferrooxidans

The cyc1 gene encoding the soluble dihemic cytochrome c CYC(41) from Acidithiobacillus ferrooxidans, an acidophilic organism, has been cloned and expressed in Escherichia coli as the host organism. The cytochrome was successfully produced and folded only in fermentative conditions: this allowed us to determine the molecular basis of the heme insertion at extreme pH. Point mutations at two sequence positions (E121 and Y63) were introduced near the two hemes in order to assign individual redox potentials to the hemes and to identify the interaction sites with the redox partners, rusticyanin and cytochrome oxidase. Characterization of mutants E121A, Y63A, and Y63F CYC(41) with biochemical and biophysical techniques were carried out. Substitution of tyrosine 63 by phenylalanine alters the environment of heme B. This result indicates that heme B has the lower redox potential. Interaction studies with the two physiological partners indicate that CYC(41) functions as an electron wire between RCy and cytochrome oxidase. A specific glutamate residue (E121) located near heme A is directly involved in the interaction with RCy. A docking analysis of CYC(41), RCy, and cytochrome oxidase allowed us to propose a model for the complex in agreement with our experimental data.     

Cytochrome c3 from Desulfovibrio gigas: crystal structure at 1.8 A resolution and evidence for a specific calcium-binding site.

Crystals of the tetraheme cytochrome c3 from sulfate-reducing bacteria Desulfovibrio gigas (Dg) (MW 13 kDa, 111 residues, four heme groups) were obtained and X-ray diffraction data collected to 1.8 A resolution. The structure was solved by the method of molecular replacement and the resulting model refined to a conventional R-factor of 14.9%. The three-dimensional structure shows many similarities to other known crystal structures of tetraheme c3 cytochromes, but it also shows some remarkable differences. In particular, the location of the aromatic residues around the heme groups, which may play a fundamental role in the electron transfer processes of the molecule, are well conserved in the cases of hemes I, III, and IV. However, heme II has an aromatic environment that is completely different to that found in other related cytochromes c3. Another unusual feature is the presence of a Ca2+ ion coordinated by oxygen atoms supplied by the protein within a loop near the N-terminus. It is speculated that this loop may be stabilized by the presence of this Ca2+ ion, may contribute to heme-redox perturbation, and might even be involved in the specificity of recognition with its redox partner.     

Molecular basis for redox-Bohr and cooperative effects in cytochrome c3 from Desulfovibrio desulfuricans ATCC 27774: crystallographic and modeling studies of oxidized and reduced high-resolution structures at pH 7.6

The tetraheme cytochrome c3 is a small metalloprotein with ca. 13,000 Da found in sulfate-reducing bacteria, which is believed to act as a partner of hydrogenase. The three-dimensional structure of the oxidized and reduced forms of cytochrome c3 from Desulfovibrio desulfuricans ATCC 27774 at pH 7.6 were determined using high-resolution X-ray crystallography and were compared with the previously determined oxidized form at pH 4.0. Theoretical calculations were performed with both structures, using continuum electrostatic calculations and Monte Carlo sampling of protonation and redox states, in order to understand the molecular basis of the redox-Bohr and cooperativity effects related to the coupled transfer of electrons and protons. We were able to identify groups that showed redox-linked conformational changes. In particular, Glu61, His76, and propionate D of heme II showed important contributions to the redox-cooperativity, whereas His76, propionate A of heme I, and propionate D of heme IV were the key residues for the redox-Bohr effect. Upon reduction, an important movement of the backbone region surrounding hemes I and II was also identified, that, together with a few redox-linked conformational changes in side-chain residues, results in a significant decrease in the solvent accessibility of hemes I and II.     

Membrane tetraheme cytochrome c(m552) of the ammonia-oxidizing nitrosomonas europaea: a ubiquinone reductase

Cytochrome c(m552) (cyt c(m552)) from the ammonia-oxidizing Nitrosomonas europaea is encoded by the cycB gene, which is preceded in a gene cluster by three genes encoding proteins involved in the oxidation of hydroxylamine: hao, hydroxylamine oxidoreductase; orf2, a putative membrane protein; cycA, cyt c(554). By amino acid sequence alignment of the core tetraheme domain, cyt c(m552) belongs to the NapC/TorC family of tetra- or pentaheme cytochrome c species involved in electron transport from membrane quinols to a variety of periplasmic electron shuttles leading to terminal reductases. However, cyt c(m552) is thought to reduce quinone with electrons originating from HAO. In this work, the tetrahemic 27 kDa cyt c(m552) from N. europaea was purified after extraction from membranes using Triton X-100 with subsequent exchange into n-dodecyl beta-d-maltoside. The cytochrome had a propensity to form strong SDS-resistant dimers likely mediated by a conserved GXXXG motif present in the putative transmembrane segment. Optical spectra of the ferric protein contained a broad ligand-metal charge transfer band at approximately 625 nm indicative of a high-spin heme. Mossbauer spectroscopy of the reduced (57)Fe-enriched protein revealed the presence of high-spin and low-spin hemes in a 1:3 ratio. Multimode EPR spectroscopy of the native state showed signals from an electronically interacting high-spin/low-spin pair of hemes. Upon partial reduction, a typical high-spin heme EPR signal was observed. No EPR signals were observed from the other two low-spin hemes, indicating an electronic interaction between these hemes as well. UV-vis absorption data indicate that CO (ferrous enzyme) or CN(-) (ferric or ferrous enzyme) bound to more than one and possibly all hemes. Other anionic ligands did not bind. The four ferrous hemes of the cytochrome were rapidly oxidized in the presence of oxygen. Comparative modeling, based on the crystal structure and conserved residues of the homologous NrfH protein from Desulfovibrio of cyt c(m552), predicted some structural elements, including a Met-ligated high-spin heme in a quinone-binding pocket, and likely axial ligands to all four hemes.