Desulfovibrio desulfuricans iron hydrogenase: the structure shows unusual coordination to an active site Fe binuclear center.

BACKGROUND: Many microorganisms have the ability to either oxidize molecular hydrogen to generate reducing power or to produce hydrogen in order to remove low-potential electrons. These reactions are catalyzed by two unrelated enzymes: the Ni-Fe hydrogenases and the Fe-only hydrogenases. RESULTS: We report here the structure of the heterodimeric Fe-only hydrogenase from Desulfovibrio desulfuricans - the first for this class of enzymes. With the exception of a ferredoxin-like domain, the structure represents a novel protein fold. The so-called H cluster of the enzyme is composed of a typical [4Fe-4S] cubane bridged to a binuclear active site Fe center containing putative CO and CN ligands and one bridging 1, 3-propanedithiol molecule. The conformation of the subunits can be explained by the evolutionary changes that have transformed monomeric cytoplasmic enzymes into dimeric periplasmic enzymes. Plausible electron- and proton-transfer pathways and a putative channel for the access of hydrogen to the active site have been identified. CONCLUSIONS: The unrelated active sites of Ni-Fe and Fe-only hydrogenases have several common features: coordination of diatomic ligands to an Fe ion; a vacant coordination site on one of the metal ions representing a possible substrate-binding site; a thiolate-bridged binuclear center; and plausible proton- and electron-transfer pathways and substrate channels. The diatomic coordination to Fe ions makes them low spin and favors low redox states, which may be required for catalysis. Complex electron paramagnetic resonance signals typical of Fe-only hydrogenases arise from magnetic interactions between the [4Fe-4S] cluster and the active site binuclear center. The paucity of protein ligands to this center suggests that it was imported from the inorganic world as an already functional unit.

Isolation and characterization of a rubredoxin and two ferredoxins from Desulfovibrio africanus.

Rubredoxin and two distinct ferredoxins have been purified from Desulfovibrio africanus. The rubredoxin has a molecular weight of 6000 while the ferredoxins appear to be dimers of identical subunits of approximately 6000 to 7000 molecular weight. Rubredoxin contains one iron atom, no acid-labile sulfide and four cysteine residues per molecule. Its absorbance ratio A278/A490 is 2.23 and its amino acid composition is characterized by the absence of leucine and a preponderance of acidic amino acids. The two ferredoxins, designated I and II, are readily separated on DEAE-cellulose. The amino acid compositions of ferredoxins I and II show them to be different protein species; the greater number of acidic amino acid residues in ferredoxin I than in ferredoxin II appears to account for separation based on electronic charge. Both ferredoxins contain four iron atoms, four acid-labile residues per molecule. Spectra of the two ferredoxins differ from those of ferredoxins of other Desulfovibrio species by exhibiting a pronounced absorption peak at 283 nm consistent with an unusual high content of aromatic residues. The A385/A283 absorbance ratio of ferredoxins I and II are 0.56 and 0.62, respectively. The N-terminal sequencing data of the two ferredoxins clearly indicate that ferredoxins I and II are different protein species. However, the two proteins exhibit a high degree of homology.

Purification, characterization and biological activity of three forms of ferredoxin from the sulfate-reducing bacterium Desulfovibrio gigas.

Three forms of ferredoxin FdI, FdI', and FdII have been isolated from Desulfovibrio gigas, a sulfate reducer. They are separated by a combination of DEAE-cellulose and gel filtration chromatographic procedures. FdI and FdI' present a slight difference in isoelectric point which enables the separation of the two forms over DEAE-cellulose, while FdII is easily separated from the two other forms by gel filtration. The three forms have the same amino acid composition and are isolated in different aggregation states. Molecular weight determinations by gel filtration gave values of 18 000 for FdI and FdI' and 24 000 for FdII, whereas a value of 6000 is determined when dissociation is accomplished with sodium dodecyl sulfate. The electronic spectra are different and their ultraviolet-visible absorbance rations are 0.77, 0.87 and 0.68 respectively for FdI, FdI' and FdII. Despite these differences, the physiological activities of the three forms are similar as far as the reduction of sulfite by molecular hydrogen is concerned.

MCD and 1H-NMR spectroscopic studies of Desulfovibrio africanus ferredoxin I: revised amino-acid sequence and identification of secondary structure.

Desulfovibrio africanus ferredoxin I was studied by magnetic circular dichroism and 1H-NMR spectroscopies. These showed the presence of histidine and tryptophan, in contrast to the previously reported amino-acid sequence (Bruschi and Hatchikian (1982) Biochimie 64, 503-507). This was redetermined and the revised sequence shown to contain both histidine and tryptophan, as well as four other corrections (Sery et al. (1994) Biochemistry, submitted). Electrospray mass spectrometry confirmed the mass of the ferredoxin was that given by the revised amino-acid sequence. The secondary structure of the ferredoxin I was investigated with two-dimensional 1H-NMR experiments and both alpha-helix and beta-sheet structure detected. The influence of the paramagnetism of the Fe4 S4 cluster on the NMR properties of the ferredoxin protons was investigated, by temperature-dependent experiments, and it was concluded that there is only a negligible dipolar contribution to resonance chemical shifts from this source. The significance of this for the determination of the three-dimensional structure of the ferredoxin by NMR is discussed.

Crystallization, preliminary X-ray study and crystal activity of the hydrogenase from Desulfovibrio gigas.

Hydrogenase (EC 1.12) from Desulfovibrio gigas is a dimeric enzyme (26 and 62 (X 10(3) Mr) that catalyzes the reversible oxidation of molecular hydrogen. Single crystals of hydrogenase have been produced using the hanging drop method, with either PEG (polyethylene glycol) 6000 or ammonium sulfate as precipitants at pH 6.5. X-ray examination of the crystals indicates that those obtained with ammonium sulfate are suitable for structure determination to at least 3.0 A resolution when synchrotron radiation Sources are used (1 A = 0.1 nm). The crystals are monoclinic, with space group C2, and cell dimensions a = 257.0 A, b = 184.7 A, c = 148.3 A and beta = 101.3 degrees, and contain between four and ten molecules per asymmetric unit. The enzyme can be reactivated within the crystals under reducing conditions without crystal damage.

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.

Cloning and sequencing of the locus encoding the large and small subunit genes of the periplasmic [NiFe]hydrogenase from Desulfovibrio fructosovorans.

The genetic locus encoding the periplasmic [NiFe]hydrogenase (Hyd) from Desulfovibrio fructosovorans was cloned and sequenced. The genes of this two-subunit enzyme have an operon organization in which the 0.94-kb gene encoding the small subunit precedes the 1.69-kb gene encoding the large subunit. A Shine-Dalgarno sequence is centered at -9 bp from the ATG of both subunits. The possible presence of another open reading frame downstream from the large-subunit-encoding gene is considered. The N-terminal sequence of the large 61-kDa subunit deduced from the nucleotide sequence is in perfect agreement with the results of the amino acid (aa) sequence determined by Edman degradation. A 50-aa leader peptide precedes the small 28-kDa subunit. The aa sequence of the enzyme shows nearly 65% homology with the [NiFe]Hyd aa sequence of Desulfovibrio gigas. Comparisons with a large range of Hyds from various bacterial species indicate the presence of highly conserved Cys residues, the implications of which are discussed from the point of view of nickel atom and cluster accommodation.

A pulsed EPR study of redox-dependent hyperfine interactions for the nickel centre of Desulfovibrio gigas hydrogenase.

The nickel centre of hydrogenase from Desulfovibrio gigas was studied by electron spin echo envelope modulation (ESEEM) spectroscopy in the oxidized, unready (Ni-A) and H2-reduced active (Ni-C) states, both in H2O and 2H2O solutions. Fourier transforms of the 3-pulse ESEEM, taken at 8.7 GHz, for Ni-A and Ni-C in H2O contained similar peaks with narrow linewidths at frequencies of 0.4, 1.2 and 1.6 MHz, and a broader peak centered at 4.5 MHz. At 11.6 GHz, the low frequency components showed small field-dependent shifts, while the high frequency component was shifted to 5.1 MHz. These results are consistent with the presence of 14N, possibly from imidazole, coupled to the nickel centre. In 2H2O, Ni-A was shown to be inaccessible for exchange with solvent deuterons. In contrast, Ni-C was accessible to solvent exchange, with a deuterium population being in close proximity to the metal ion. Thus, the nickel environment of the active protein is different from that in the oxidized or unready state. On illumination of Ni-C, although EPR changes are seen, 14N coupling remains, and for the 2H2O sample, deuterium coupling is also retained.

Isolation, amino acid analysis and N-terminal sequence determination of the two subunits of the nickel-containing hydrogenase of Desulfovibrio gigas.

The two subunits of the nickel-iron hydrogenase from Desulfovibrio gigas have been purified by preparative sodium dodecyl sulfate polyacrylamide gel electrophoresis and their amino acid compositions have been determined. The N-terminal sequences for 15 residues of the large subunit (Mr 62,000) and 25 residues of the small subunit (Mr 26,000), respectively, were established. The occurrence of several cysteine residues in the small subunit is discussed in relation with their possible role in the binding of the redox centers of the enzyme.

Hydrogenase: a hydrogen-metabolizing enzyme. What do the crystal structures tell us about its mode of action?

Hydrogenases are proteins which metabolize the most simple of chemical compounds, molecular hydrogen, according to the reaction H2<-->2H+ + 2e-. These enzymes are found in many microorganisms of great biotechnological interest such as methanogenic, acetogenic, nitrogen fixing, photosynthetic or sulfate-reducing bacteria. The X-ray structure of a dimeric [NiFe] hydrogenase together with a wealth of biophysical, biochemical and genetic studies have revealed that the large subunit contains the bimetallic [Ni-Fe] active site, with biologically uncommon CO and CN ligands to the iron, whereas the small subunit contains three iron-sulfur cluster. During catalysis, the nickel atom is most likely responsible for a base-assisted heterolytic cleavage of the hydrogen molecule whereas the iron atom could be redox active. Specific channels are probably required for the transfer of the chemical reaction partners (H2, H+ and e-) between the active site, deeply buried inside the protein, and the molecular surface. The generation of a functional enzyme, including the assembly of the complex catalytic center, requires maturation and involves a large number of auxiliary proteins which have been partly characterized by molecular biology.

Characterization of the nickel-iron periplasmic hydrogenase from Desulfovibrio fructosovorans.

The periplasmic hydrogenase from Desulfovibrio fructosovorans grown on fructose/sulfate medium was purified to homogeneity. It exhibits a molecular mass of 88 kDa and is composed of two different subunits of 60 kDa and 28.5 kDa. The absorption spectrum of the enzyme is characteristic of an iron-sulfur protein and its absorption coefficients at 400 and 280 nm are 50 and 180 mM-1 cm-1, respectively. D. fructosovorans hydrogenase contains 11 +/- 1 iron atoms, 0.9 +/- 0.15 nickel atom and 12 +/- 1 acid-labile sulfur atoms/molecule but does not contain selenium. The amino acid composition of the protein and of its subunits, as well as the N-terminal sequences of the small and large subunits, have been determined. The cysteine residues of the protein are distributed between the large (9 residues) and the small subunits (11 residues). Electron spin resonance (ESR) properties of the enzyme are consistent with the presence of nickel(III), [3Fe-4S] and [4Fe-4S] clusters. The hydrogenase of D. fructosovorans isolated under aerobic conditions required an incubation with hydrogen or other reductants in order to express its full catalytic activity. H2 uptake and H2 evolution activities doubled after a 3-h incubation under reducing conditions. Comparison with the (NiFe) hydrogenase from D. gigas shows great structural similarities between the two proteins. However, there are significant differences between the catalytic properties of the two enzymes which can be related to the respective state of their nickel atom. ESR showed a higher proportion of the Ni-B species (g = 2.33, 2.16, 2.01) which can be related to a more facile conversion to the ready state. The periplasmic location of the enzyme and the presence of hydrogenase activity in other cellular compartments are discussed in relation to the ability of D. fructosovorans to participate actively in interspecies hydrogen transfer.

Energetics of growth of a defined mixed culture of Desulfovibrio vulgaris and Methanosarcina barkeri: maintenance energy coefficient of the sulfate-reducing organism in the absence and presence of its partner.

The maintenance energy coefficient of Desulfovibrio vulgaris was studied by using a chemostat, with Methanosarcina barkeri or sulfate as the electron acceptor; lithium lactate or sodium pyruvate served as the electron donor. The experiments showed that the growth energetics of D. vulgaris or M. barkeri were greatly affected by maintenance energy coefficients. When D. vulgaris grew on lactate or pyruvate medium with sulfate, these coefficients reached 4.40 and 2.80 mM g-1 h-1, respectively; on lactate medium in the presence of M. barkeri the same coefficient reached a value of 2.90 mM g-1 h-1. Results also showed that the increase of the value of the maintenance energy coefficient corresponded to a decrease of the biomass produced. D. vulgaris maximal growth yield values calculated by use of the Pirt equation were slightly higher with M. barkeri (maximal growth yield, 10 g/mol) than with sulfate (maximal growth yield, 7.5 g/mol). This finding could be interpreted by reference to the ATP-generating reactions involved in D. vulgaris growth in the presence of sulfate or M. barkeri.

Crystal structure of the ferredoxin I from Desulfovibrio africanus at 2.3 A resolution.

The crystal structure of the ferredoxin I from the sulfate-reducing bacterium Desulfovibrio africanus (DaFdI) has been solved and refined by X-ray diffraction. The crystals are orthorhombic with a = 96.6 A, b = 58.1 A, and c = 20.7 A, space group P2(1)2(1)2, and two ferredoxin molecules per asymmetric unit. The initial electron density map has been obtained by combining phasing by molecular replacement methods, anomalous scattering, and noncrystallographic averaging. The final crystallographic R factor is 0.182 with 10-2.3 A resolution data. In parallel, the amino acid sequence was redetermined. This showed that DaFdI contains 64 residues (instead of 61) including one free cysteine, one histidine, and one tryptophan in the C-terminal part of the molecule. The current molecular model includes the two molecules of the asymmetric unit, 67 water molecules, and one sulfate ion. The DaFdI overall folding very closely resembles that of ferredoxins of known structure. Comparisons with the single cluster ferredoxins from Desulfovibrio gigas and Bacillus thermoproteolyticus show that the presence or the absence of a disulfide bridge does not significantly affect the folding of the other half of the molecule, including the characteristic alpha-helix of the single cluster ferreddoxins. Like other ferredoxins or analogs, the [4Fe-4S] iron--sulfur cluster presents, at 2.3 A resolution, a cubane-like geometry. By contrast, its immediate environment is different as it includes, besides the four cysteic sulfur ligands, the sulfur atom of the free cysteine. This sulfur atom, which is buried within the protein, is in van der Waals contact with one labile sulfur of the cluster and one liganded cysteic sulfur. The association of a [4Fe-4S] cluster with one free cysteic sulfur is similar to that previously found in both X-ray structures of Azotobacter vinelandii and Peptococcus aerogenes [Stout, C. D. (1989) J. Mol. Biol. 205, 545-555; Backes, G., et al. (1991) J. Am. Chem. Soc. 113, 2055-2064]. Chemical sequence analysis suggests that this characteristic [4Fe-4S] cluster sulfur environment is widely distributed among ferredoxins.

Purification and characterization of cytochrome c3, ferredoxin, and rubredoxin isolated from Desulfovibrio desulfuricans Norway.

Different electron carriers of the non-desulfoviridin-containing, sulfate-reducing bacterium Desulfovibrio desulfuricans (Norway strain) have been studied. Two nonheme iron proteins, ferredoxin and rubredoxin, have been purified. This ferredoxin contains four atoms of non-heme iron and acid-labile sulfur and six residues of cysteine per molecule. Its amino acid composition suggests that it is homologous with the other Desulfovibrio ferredoxins. The rubredoxin is also an acidic protein of 6,000 molecular weight and contains one atom of iron and four cysteine residues per molecule. The amino acid composition and molecular weight of the cytochrome c3 from D. desulfuricans (strain Norway 4) are reported. Its spectral properties are very similar to those of the other cytochromes c3 (molecular weight, 13,000) of Desulfovibrio and show that it contains four hemes per molecule. This cytochrome has a very low redox potential and acts as a carrier in the coupling of hydrogenase and thiosulfate reductase in extracts of Desulfovibrio gigas and Desulfovibrio desulfuricans (Norway strain) in contrast to D. gigas cytochrome c3 (molecular weight, 13,000). A comparison of the activities of the cytochrome c3 (molecular weight, 13,000) of D. gigas and that of D. desulfuricans in this reaction suggests that these homologous proteins can have different specificity in the electron transfer chain of these bacteria.

Microcalorimetric studies of the growth of sulfate-reducing bacteria: energetics of Desulfovibrio vulgaris growth.

The metabolism of Desulfovibrio vulgaris Hildenborough grown on medium containing lactate or pyruvate plus a high concentration of sulfate (36 mM) was studied. Molecular growth yields were 6.7 +/- 1.3 and 10.1 +/- 1.7 g/mol for lactate and pyruvate, respectively. Under conditions in which the energy source was the sole growth-limiting factor, we observed the formation of 0.5 mol of hydrogen per mol of lactate and 0.1 mol of hydrogen per mol of pyruvate. The determination of metabolic end products revealed that D. vulgaris produced, in addition to normal end products (acetic acid, carbon dioxide, hydrogen sulfide) and molecular hydrogen, 2 and 5% of ethanol per mol of lactate and pyruvate, respectively. Power-time curves of growth of D. vulgaris on lactate and pyruvate were obtained, by the microcalorimetric Tian-Calvet apparatus. The enthalpies (delta Hmet) associated with the oxidation of these substrates and calculated from growth thermograms were -36.36 +/- 5 and -70.22 +/- 3 kJ/mol of lactate and pyruvate, respectively. These experimental values were in agreement with the homologous values assessed from the theoretical equations of D. vulgaris metabolism of both lactate and pyruvate. The hydrogen production by this sulfate reducer constitutes an efficient regulatory system of electrons, from energy source through the pathway of sulfate reduction. This hydrogen value may thus facilitate interactions between this strain and other environmental microflora, especially metagenic bacteria.

Microcalorimetric studies of the growth of sulfate-reducing bacteria: comparison of the growth parameters of some Desulfovibrio species.

We performed a comparative study of the growth energetics of some species of Desulfovibrio by measuring microcalorimetric and molar growth yield values. Lactate and pyruvate were used as energy sources for sulfate reduction. On lactate-sulfate media Desulfovibrio desulfuricans Norway, Desulfovibrio gigas, and Desulfovibrio africanus exhibited molar growth yields of 4.1 +/- 0.6, 3.7 +/- 1.7, and 1.8 +/- 0.1 g/mol, respectively, whereas on pyruvate-sulfate media the molar growth yields were higher (8.5 +/- 0.8, 7.7 +/- 1.6, and 3.5 +/- 0.5 g/mol, respectively). Thus, we found that D. africanus was the least efficient species in converting energy into cell material. The uncoupling of energy in this strain was obvious since its catabolic activities were high compared with those of the two other strains. The enthalpy changes associated with lactate and pyruvate metabolism were -49 +/- 0.7 and -70.2 +/- 6.0 jK/mol, respectively, for D. desulfuricans, -76.6 +/- 1.8 and -91.2 +/- 1.1 kJ/mol, respectively, for D. gigas, and -78.8 +/- 7.2 and -88.0 +/- 6.2 kJ/mol, respectively, for D. africanus. D. gigas and D. africanus produced only acetate, CO2 and hydrogen sulfide as metabolic end products. In addition to these normal end products, D. desulfuricans Norway produced a small amount of butanol. This butanol production was interpreted as reflecting a regulatory system of electron flow during the catabolism of both substrates. Such metabolism was comparable to that reported for D. vulgaris, which lost part of the reducing power of its energy sources through hydrogen evolution.

Crystallization and preliminary crystallographic analysis of the pyruvate-ferredoxin oxidoreductase from Desulfovibrio africanus.

For the first time, crystals of a pyruvate-ferredoxin oxidoreductase (PFOR) suitable for X-ray analysis have been obtained. This enzyme catalyzes, in anaerobic organisms, the crucial energy-yielding reaction of pyruvate decarboxylation to acetylCoA. Polyethylene glycol and divalent metal cations have been used to crystallize the PFOR from the sulfate-reducing bacterium Desulfovibrio africanus. Two different orthorhombic (P212121 ) crystal forms have been grown with unit-cell dimensions a = 86.1, b = 146.7, c = 212.5 A and a = 84.8, b = 144.9, c = 203.0 A. Both crystals diffract to 2.3 A resolution using synchrotron radiation.

Energetics of Growth of a Defined Mixed Culture of Desulfovibrio vulgaris and Methanosarcina barkeri: Interspecies Hydrogen Transfer in Batch and Continuous Cultures.

Interspecies hydrogen transfer was studied in Desulfovibrio vulgaris-Methanosarcina barkeri mixed cultures. Experiments were performed under batch and continuous growth culture conditions. Lactate or pyruvate was used as an energy source. In batch culture and after 30 days of simultaneous incubation, these organisms were found to yield 1.5 mol of methane and 1.5 mol of carbon dioxide per mol of lactate fermented. When M. barkeri served as the hydrogen acceptor, growth yields of D. vulgaris were higher compared with those obtained on pyruvate without any electron acceptor other than protons. In continuous culture, all of the carbon derived from the oxidation of lactate was recovered as methane and carbon dioxide, provided the dilution rate was minimal. Increasing the dilution rate induced a gradual accumulation of acetate, causing acetate metabolism to cease at above mu = 0.05 h. Under these conditions all of the methane produced originated from carbon dioxide. The growth yields of D. vulgaris were measured when sulfate or M. barkeri was the electron acceptor. Two key observations resulted from the present study. First, although sulfate was substituted by M. barkeri, metabolism of D. vulgaris was only slightly modified. The coculture-fermented lactate produced equimolar quantities of carbon dioxide and methane. Second, acetogenesis and methane formation from acetate were completely separable.

Molecular study and partial characterization of iron-only hydrogenase in Desulfovibrio fructosovorans.

An iron-only hydrogenase was partially purified and characterized from Desulfovibrio fructosovorans wild-type strain. The enzyme exhibits a molecular mass of 56 kDa and is composed of two distinct subunits HydA and HydB (46 and 13 kDa, respectively). The N-terminal amino acid sequences of the two subunits of the enzyme were determined with the aim of designing degenerate oligonucleotides. Direct and inverse polymerase chain reaction techniques were used to clone the hydrogenase encoding genes. A 9-nucleotide region located 75 bp upstream from the translational start codon of the D. fructosovorans hydA gene was found to be highly conserved. The analysis of the deduced amino acid sequence of these genes showed the presence of a signal sequence located in the small subunit, exhibiting the consensus sequence which is likely to be involved in the specific export mechanism of hydrogenases. Two ferredoxin-like motives involved in the coordination of [4Fe-4S] clusters were identified in the N-terminal domain of the large subunit. The amino acid sequence of the [Fe] hydrogenase from D. fructosovorans was compared with the amino acid sequences from eight other hydrogenases (cytoplasmic and periplasmic). These enzymes share an overall 18% identity and 28% similarity. The identity reached 73% and 69% when the D. fructosovorans hydrogenase sequence was compared with the hydrogenase sequences from Desulfovibrio vulgaris Hildenborough and Desulfovibrio vulgaris oxamicus Monticello, respectively.

Structural model of the Fe-hydrogenase/cytochrome c553 complex combining transverse relaxation-optimized spectroscopy experiments and soft docking calculations.

Fe-hydrogenase is a 54-kDa iron-sulfur enzyme essential for hydrogen cycling in sulfate-reducing bacteria. The x-ray structure of Desulfovibrio desulfuricans Fe-hydrogenase has recently been solved, but structural information on the recognition of its redox partners is essential to understand the structure-function relationships of the enzyme. In the present work, we have obtained a structural model of the complex of Fe-hydrogenase with its redox partner, the cytochrome c(553), combining docking calculations and NMR experiments. The putative models of the complex demonstrate that the small subunit of the hydrogenase has an important role in the complex formation with the redox partner; 50% of the interacting site on the hydrogenase involves the small subunit. The closest contact between the redox centers is observed between Cys-38, a ligand of the distal cluster of the hydrogenase and Cys-10, a ligand of the heme in the cytochrome. The electron pathway from the distal cluster of the Fe-hydrogenase to the heme of cytochrome c(553) was investigated using the software Greenpath and indicates that the observed cysteine/cysteine contact has an essential role. The spatial arrangement of the residues on the interface of the complex is very similar to that already described in the ferredoxin-cytochrome c(553) complex, which therefore, is a very good model for the interacting domain of the Fe-hydrogenase-cytochrome c(553).