EPR studies with 77Se-enriched (NiFeSe) hydrogenase of Desulfovibrio baculatus. Evidence for a selenium ligand to the active site nickel

The periplasmic hydrogenase containing equivalent amounts of nickel and selenium plus non-heme iron [NiFeSe) hydrogenase) has been purified from cells of the sulfate reducing bacterium Desulfovibrio baculatus (DSM 1748) grown on a lactate/sulfate medium containing natural Se isotopes and the nuclear isotope, 77Se. Both the 77Se-enriched and unenriched hydrogenases were shown to be free of other hydrogenases and characterized with regard to their Se contents. EPR studies of the reduced nickel signal generated by redox titrations of the enriched and unenriched (NiFeSe) hydrogenases demonstrated that the gx = 2.23 and gy = 2.17 resonances are appreciably broadened by the spin of the 77Se nucleus (I = 1/2). This observation demonstrates unambiguously that the unpaired electron is shared by the Ni and Se atoms and that Se serves as a ligand to the nickel redox center of the (NiFeSe) hydrogenase.     

The crystal structure of a reduced [NiFeSe] hydrogenase provides an image of the activated catalytic center.

BACKGROUND: [NiFeSe] hydrogenases are metalloenzymes that catalyze the reaction H2<-->2H+ + 2e-. They are generally heterodimeric, contain three iron-sulfur clusters in their small subunit and a nickel-iron-containing active site in their large subunit that includes a selenocysteine (SeCys) ligand. RESULTS: We report here the X-ray structure at 2.15 A resolution of the periplasmic [NiFeSe] hydrogenase from Desulfomicrobium baculatum in its reduced, active form. A comparison of active sites of the oxidized, as-prepared, Desulfovibrio gigas and the reduced D. baculatum hydrogenases shows that in the reduced enzyme the nickel-iron distance is 0.4 A shorter than in the oxidized enzyme. In addition, the putative oxo ligand, detected in the as-prepared D. gigas enzyme, is absent from the D. baculatum hydrogenase. We also observe higher-than-average temperature factors for both the active site nickel-selenocysteine ligand and the neighboring Glu18 residue, suggesting that both these moieties are involved in proton transfer between the active site and the molecular surface. Other differences between [NiFeSe] and [NiFe] hydrogenases are the presence of a third [4Fe4S] cluster replacing the [3Fe4S] cluster found in the D. gigas enzyme, and a putative iron center that substitutes the magnesium ion that has already been described at the C terminus of the large subunit of two [NiFe] hydrogenases. CONCLUSIONS: The heterolytic cleavage of molecular hydrogen seems to be mediated by the nickel center and the selenocysteine residue. Beside modifying the catalytic properties of the enzyme, the selenium ligand might protect the nickel atom from oxidation. We conclude that the putative oxo ligand is a signature of inactive 'unready' [NiFe] hydrogenases.     

The three classes of hydrogenases from sulfate-reducing bacteria of the genus Desulfovibrio

Three types of hydrogenases have been isolated from the sulfate-reducing bacteria of the genus Desulfovibrio. They differ in their subunit and metal compositions, physico-chemical characteristics, amino acid sequences, immunological reactivities, gene structures and their catalytic properties. Broadly, the hydrogenases can be considered as 'iron only' hydrogenases and nickel-containing hydrogenases. The iron-sulfur-containing hydrogenase ([Fe] hydrogenase) contains two ferredoxin-type (4Fe-4S) clusters and an atypical iron-sulfur center believed to be involved in the activation of H2. The [Fe] hydrogenase has the highest specific activity in the evolution and consumption of hydrogen and in the proton-deuterium exchange reaction and this enzyme is the most sensitive to CO and NO2-. It is not present in all species of Desulfovibrio. The nickel-(iron-sulfur)-containing hydrogenases [( NiFe] hydrogenases) possess two (4Fe-4S) centers and one (3Fe-xS) cluster in addition to nickel and have been found in all species of Desulfovibrio so far investigated. The redox active nickel is ligated by at least two cysteinyl thiolate residues and the [NiFe] hydrogenases are particularly resistant to inhibitors such as CO and NO2-. The genes encoding the large and small subunits of a periplasmic and a membrane-bound species of the [NiFe] hydrogenase have been cloned in Escherichia (E.) coli and sequenced. Their derived amino acid sequences exhibit a high degree of homology (70%); however, they show no obvious metal-binding sites or homology with the derived amino acid sequence of the [Fe] hydrogenase. The third class is represented by the nickel-(iron-sulfur)-selenium-containing hydrogenases [( NiFe-Se] hydrogenases) which contain nickel and selenium in equimolecular amounts plus (4Fe-4S) centers and are only found in some species of Desulfovibrio. The genes encoding the large and small subunits of the periplasmic hydrogenase from Desulfovibrio (D.) baculatus (DSM 1743) have been cloned in E. coli and sequenced. The derived amino acid sequence exhibits homology (40%) with the sequence of the [NiFe] hydrogenase and the carboxy-terminus of the gene for the large subunit contains a codon (TGA) for selenocysteine in a position homologous to a codon (TGC) for cysteine in the large subunit of the [NiFe] hydrogenase. EXAFS and EPR studies with the 77Se-enriched D. baculatus hydrogenase indicate that selenium is a ligand to nickel and suggest that the redox active nickel is ligated by at least two cysteinyl thiolate and one selenocysteine selenolate residues.(ABSTRACT TRUNCATED AT 400 WORDS)     

Analysis and comparison of nucleotide sequences encoding the genes for [NiFe] and [NiFeSe] hydrogenases from Desulfovibrio gigas and Desulfovibrio baculatus.

The nucleotide sequences encoding the [NiFe] hydrogenase from Desulfovibrio gigas and the [NiFeSe] hydrogenase from Desulfovibrio baculatus (N.K. Menon, H.D. Peck, Jr., J. LeGall, and A.E. Przybyla, J. Bacteriol. 169:5401-5407, 1987; C. Li, H.D. Peck, Jr., J. LeGall, and A.E. Przybyla, DNA 6:539-551, 1987) were analyzed by the codon usage method of Staden and McLachlan. The reported reading frames were found to contain regions of low codon probability which are matched by more probable sequences in other frames. Renewed nucleotide sequencing showed the probable frames to be correct. The corrected sequences of the two small and large subunits share a significant degree of sequence homology. The small subunit, which contains 10 conserved cysteine residues, is likely to coordinate at least 2 iron-sulfur clusters, while the finding of a selenocysteine codon (TGA) near the 3' end of the [NiFeSe] large-subunit gene matched by a regular cysteine codon (TGC) in the [NiFe] large-subunit gene indicates the presence of some of the ligands to the active-site nickel in the large subunit.     

Hydrogenases in the "active" state: determination of g-matrix axes and electron spin distribution at the active site by 1H ENDOR spectroscopy

Hydrons and electrons are substrates for the enzyme hydrogenase, but cannot be observed in X-ray crystal structures. High-resolution 1H electron nuclear double resonance (ENDOR) spectroscopy offers a means to detect the distribution of protons and unpaired electrons. ENDOR spectra were recorded from frozen solutions of the nickel-iron hydrogenases of Desulfovibrio gigas and Desulfomicrobium baculatum, in the "active" state ("Ni-C" EPR signal) and analyzed by orientationally selective simulation methods. The experimental spectra were fitted using a structural model of the nickel-iron centre based on crystallographic results, allowing for differences in electron spin distribution as well as the spatial orientation of the g-matrix ( g-tensor), and anisotropic and isotropic hyperfine couplings of the protons nearest to the nickel ion. ENDOR signals, detected after complete deuterium exchange, were assigned to six protons of the cysteines bound to nickel. The assignment took advantage of the substitution of a selenium for a sulfur ligand, which occurs naturally between the [NiFeSe] and [NiFe] hydrogenases from Dm. baculatum and D. gigas, respectively, and was found to affect just two signals. The four signals with the largest hyperfine couplings, including isotropic contributions from 4.5 to 13.5 MHz, were assigned to the beta-methylene protons of the two terminal cysteine ligands, one of which is substituted by seleno-cysteine in [NiFeSe] hydrogenase. The electron spin is delocalized onto the nickel (50%) and its sulfur ligands, with a higher proportion on the terminal than the bridging ligands. The g-matrix was found to align with the active site in such a way that the g1- g2 plane is nearly coplanar (18.3 degrees) with the plane defined by nickel and three sulfur atoms, and the g2 axis deviates by 22.9 degrees from the vector between nickel and iron. Significantly for the reaction of the enzyme, direct evidence for the binding of hydrons at the active site was obtained by the detection of H/D-exchangeable ENDOR signals.     

Influence of the protein structure surrounding the active site on the catalytic activity of [NiFeSe] hydrogenases

A combined experimental and theoretical study of the catalytic activity of a [NiFeSe] hydrogenase has been performed by H/D exchange mass spectrometry and molecular dynamics simulations. Hydrogenases are enzymes that catalyze the heterolytic cleavage or production of H₂. The [NiFeSe] hydrogenases belong to a subgroup of the [NiFe] enzymes in which a selenocysteine is a ligand of the nickel atom in the active site instead of cysteine. The aim of this research is to determine how much the specific catalytic properties of this hydrogenase are influenced by the replacement of a sulfur by selenium in the coordination of the bimetallic active site versus the changes in the protein structure surrounding the active site. The pH dependence of the D₂/H⁺ exchange activity and the high isotope effect observed in the Michaelis constant for the dihydrogen substrate and in the single exchange/double exchange ratio suggest that a “cage effect” due to the protein structure surrounding the active site is modulating the enzymatic catalysis. This “cage effect” is supported by molecular dynamics simulations of the diffusion of H₂ and D₂ from the outside to the inside of the protein, which show different accumulation of these substrates in a cavity next to the active site.     

Structural features of [NiFeSe] and [NiFe] hydrogenases determining their different properties: a computational approach

Hydrogenases are metalloenzymes that catalyze the reversible reaction \textH₂ \leftrightarrows 2\textH ⁺ + 2\texte ^- {\text{H}}_{2} \leftrightarrows 2{\text{H}}^{ + } + 2{\text{e}}^{ - } , being potentially useful in H₂ production or oxidation. [NiFeSe] hydrogenases are a particularly interesting subgroup of the [NiFe] class that exhibit tolerance to O₂ inhibition and produce more H₂ than standard [NiFe] hydrogenases. However, the molecular determinants responsible for these properties remain unknown. Hydrophobic pathways for H₂ diffusion have been identified in [NiFe] hydrogenases, as have proton transfer pathways, but they have never been studied in [NiFeSe] hydrogenases. Our aim was, for the first time, to characterize the H₂ and proton pathways in a [NiFeSe] hydrogenase and compare them with those in a standard [NiFe] hydrogenase. We performed molecular dynamics simulations of H₂ diffusion in the [NiFeSe] hydrogenase from Desulfomicrobium baculatum and extended previous simulations of the [NiFe] hydrogenase from Desulfovibrio gigas (Teixeira et al. in Biophys J 91:2035–2045, 2006). The comparison showed that H₂ density near the active site is much higher in [NiFeSe] hydrogenase, which appears to have an alternative route for the access of H₂ to the active site. We have also determined a possible proton transfer pathway in the [NiFeSe] hydrogenase from D. baculatum using continuum electrostatics and Monte Carlo simulation and compared it with the proton pathway we found in the [NiFe] hydrogenase from D. gigas (Teixeira et al. in Proteins 70:1010–1022, 2008). The residues constituting both proton transfer pathways are considerably different, although in the same region of the protein. These results support the hypothesis that some of the special properties of [NiFeSe] hydrogenases could be related to differences in the H₂ and proton pathways.     

Cloning and sequencing of a [NiFe] hydrogenase operon from Desulfovibrio vulgaris Miyazaki F

A hydrogenase operon was cloned from chromosomal DNA isolated from Desulfovibrio vulgaris Miyazaki F with the use of probes derived from the genes encoding [NiFe] hydrogenase from Desulfovibrio vulgaris Hildenborough. The nucleic acid sequence of the cloned DNA indicates this hydrogenase to be a two-subunit enzyme: the gene for the small subunit (267 residues; molecular mass = 28763 Da) precedes that for the large subunit (566 residues; molecular mass = 62495 Da), as in other [NiFe] and [NiFeSe] hydrogenase operons. The amino acid sequences of the small and large subunits of the Miyazaki hydrogenase share 80% homology with those of the [NiFe] hydrogenase from Desulfovibrio gigas. Fourteen cysteine residues, ten in the small and four in the large subunit, which are thought to co-ordinate the iron-sulphur clusters and the active-site nickel in [NiFe] hydrogenases, are found to be conserved in the Miyazaki hydrogenase. The subunit molecular masses and amino acid composition derived from the gene sequence are very similar to the data reported for the periplasmic, membrane-bound hydrogenase isolated by Yagi and coworkers, suggesting that this hydrogenase belongs to the general class of [NiFe] hydrogenases, despite its low nickel content and apparently anomalous spectral properties.     

Conversion of the central [4Fe-4S] cluster into a [3Fe-4S] cluster leads to reduced hydrogen-uptake activity of the F420-reducing hydrogenase of Methanococcus voltae.

As in many other hydrogenases, the small subunit of the F420-reducing hydrogenase of Methanococcus voltae contains three iron-sulfur clusters. The arrangement of the three [4Fe-4S] clusters corresponds to the arrangement of [Fe-S] clusters in the [NiFeSe] hydrogenase of Desulfomicrobium baculatum. Many other hydrogenases contain two [4Fe-4S] clusters and one [3Fe-4S] cluster with a relatively high redox potential, which is located in the central position between a proximal and a distal [4Fe-4S] cluster. We have investigated the role of the central [4Fe-4S] cluster in M. voltae with regard to its effect on the enzyme activity and its spectroscopic properties. Using site-directed mutagenesis, we constructed a strain in which one cysteine ligand of the central [4Fe-4S] cluster was replaced by proline. The mutant protein was purified, and the [4Fe-4S] to [3Fe-4S] cluster conversion was confirmed by EPR spectroscopy. The conversion resulted in an increase in the redox potential of the [3Fe-4S] cluster by about 400 mV. The [NiFe] active site was not affected significantly by the mutation as assessed by the unchanged Ni EPR spectrum. The specific activity of the mutated enzyme did not show any significant differences with the artificial electron acceptor benzyl viologen, but its specific activity with the natural electron acceptor F420 decreased tenfold.     

Distribution of Hydrogenase Genes in Desulfovibrio spp. and Their Use in Identification of Species from the Oil Field Environment

The distribution of genes for [Fe], [NiFe], and [NiFeSe] hydrogenases was determined for 22 Desulfovibrio species. The genes for [NiFe] hydrogenase were present in all species, whereas those for the [Fe] and [NiFeSe] hydrogenases had a more limited distribution. Sulfate-reducing bacteria from 16S rRNA groups other than the genus Desulfovibrio (R. Devereux, M. Delaney, F. Widdel, and D. A. Stahl, J. Bacteriol. 171:6689-6695, 1989) did not react with the [NiFe] hydrogenase gene probe, which could be used to identify different Desulfovibrio species in oil field samples following growth on lactate-sulfate medium.     

Function of periplasmic hydrogenases in the sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough

The sulfate-reducing bacterium Desulfovibrio vulgaris Hildenborough possesses four periplasmic hydrogenases to facilitate the oxidation of molecular hydrogen. These include an [Fe] hydrogenase, an [NiFeSe] hydrogenase, and two [NiFe] hydrogenases encoded by the hyd, hys, hyn1, and hyn2 genes, respectively. In order to understand their cellular functions, we have compared the growth rates of existing (hyd and hyn1) and newly constructed (hys and hyn-1 hyd) mutants to those of the wild type in defined media in which lactate or hydrogen at either 5 or 50% (vol/vol) was used as the sole electron donor for sulfate reduction. Only strains missing the [Fe] hydrogenase were significantly affected during growth with lactate or with 50% (vol/vol) hydrogen as the sole electron donor. When the cells were grown at low (5% [vol/vol]) hydrogen concentrations, those missing the [NiFeSe] hydrogenase suffered the greatest impairment. The growth rate data correlated strongly with gene expression results obtained from microarray hybridizations and real-time PCR using mRNA extracted from cells grown under the three conditions. Expression of the hys genes followed the order 5% hydrogen>50% hydrogen>lactate, whereas expression of the hyd genes followed the reverse order. These results suggest that growth with lactate and 50% hydrogen is associated with high intracellular hydrogen concentrations, which are best captured by the higher activity, lower affinity [Fe] hydrogenase. In contrast, growth with 5% hydrogen is associated with a low intracellular hydrogen concentration, requiring the lower activity, higher affinity [NiFeSe] hydrogenase.     

The [NiFeSe] hydrogenase from Desulfovibrio vulgaris Hildenborough is a bacterial lipoprotein lacking a typical lipoprotein signal peptide

Desulfovibrio vulgaris Hildenborough has a membrane-bound [NiFeSe] hydrogenase whose mode of membrane association was unknown since it is constituted by two hydrophilic subunits. This work shows that this hydrogenase is a bacterial lipoprotein bound to the membrane by lipidic groups found at the N-terminus of the large subunit, which is unusual since it is missing the typical lipoprotein signal peptide. Nevertheless, the large subunit has a conserved four residue lipobox and its synthesis is sensitive to the signal peptidase II inhibitor globomycin. The D. vulgaris [NiFeSe] hydrogenase is the first example of a bacterial lipoprotein translocated through the Tat pathway.     

The iron-sulfur centers of the soluble [NiFeSe] hydrogenase, from Desulfovibrio baculatus (DSM 1743). EPR and M?ssbauer characterization

The soluble (cytoplasmic plus periplasmic) Ni/Fe-S/Se-containing hydrogenase from Desulfovibrio baculatus (DSM 1743) was purified from cells grown in an 57Fe-enriched medium, and its iron-sulfur centers were extensively characterized by M?ssbauer and EPR spectroscopies. The data analysis excludes the presence of a [3Fe-4S] center, either in the native (as isolated) or in the hydrogen-reduced states. In the native state, the non-heme iron atoms are arranged as two diamagnetic [4Fe-4S]2+ centers. Upon reduction, these two centers exhibit distinct and unusual M?ssbauer spectroscopic parameters. The centers were found to have similar mid-point potentials (approximately -315 mV) as determined by oxidation-reduction titratins followed by EPR.     

On the novel H2-activating iron-sulfur center of the "Fe-only" hydrogenases

The two hydrogenases (I and II) of the anaerobic N2-fixing bacterium Clostridium pasteurianum (Cp) and the hydrogenases of the anaerobes Megasphaera elsdenii (Me) and Desulfovibrio vulgaris (strain Hildenborough, Dv), contain iron-sulfur clusters but not nickel. They are the most active hydrogenases known. All four enzymes in their reduced states give rise to EPR signals typical of [4Fe-4S]1+ clusters but exhibit novel EPR signals in their oxidized states. For example, Cp hydrogenase I exhibits a sharp rhombic EPR signal when oxidized under mild conditions but the enzyme is inactivated by over-oxidation and then exhibits an axial EPR signal. A similar axial signal is observed from mildly oxidized hydrogenase I after treatment with CO. EPR, M?ssbauer and ENDOR spectroscopy indicate that the EPR signals from the oxidized enzyme and its CO derivative arise from a novel spin-coupled Fe center. Low temperature magnetic circular dichroism (MCD) studies reveal that an EPR-silent Fe-S cluster with S greater than 1/2 is also present in oxidized hydrogenase I. From a study of all spectroscopic properties of Cp, Dv, and Me hydrogenases, it is concluded that the H2-activating site of all four is a novel Fe-S cluster with S greater than 0 and integer, which in the oxidized state is exchange-coupled to a S = 1/2 species. The data are most consistent with the S = 1/2 species being a low spin Fe(III) center. The H2-activating site is susceptible to oxidative rearrangements to yield both active and inactive states of the enzyme. We discuss the possible implications of these finding to methods of enzyme oxidation and purification procedures currently used for hydrogenases.     

Overexpression, Isolation, and Spectroscopic Characterization of the Bidirectional [NiFe] Hydrogenase from Synechocystis sp. PCC 6803

The bidirectional [NiFe] hydrogenase of the cyanobacterium Synechocystis sp. PCC 6803 was purified to apparent homogeneity by a single affinity chromatography step using a Synechocystis mutant with a Strep-tag II fused to the C terminus of HoxF. To increase the yield of purified enzyme and to test its overexpression capacity in Synechocystis the psbAII promoter was inserted upstream of the hoxE gene. In addition, the accessory genes (hypF, C, D, E, A, and B) from Nostoc sp. PCC 7120 were expressed under control of the psbAII promoter. The respective strains show higher hydrogenase activities compared with the wild type. For the first time a Fourier transform infrared (FTIR) spectroscopic characterization of a [NiFe] hydrogenase from an oxygenic phototroph is presented, revealing that two cyanides and one carbon monoxide coordinate the iron of the active site. At least four different redox states of the active site were detected during the reversible activation/inactivation. Although these states appear similar to those observed in standard [NiFe] hydrogenases, no paramagnetic nickel state could be detected in the fully oxidized and reduced forms. Electron paramagnetic resonance spectroscopy confirms the presence of several iron-sulfur clusters after reductive activation. One [4Fe4S]⁺ and at least one [2Fe2S]⁺ cluster could be identified. Catalytic amounts of NADH or NADPH are sufficient to activate the reaction of this enzyme with hydrogen.     

Hydrogenases in Desulfovibrio vulgaris Hildenborough: structural and physiologic characterisation of the membrane-bound [NiFeSe] hydrogenase

The genome of Desulfovibrio vulgaris Hildenborough (DvH) encodes for six hydrogenases (Hases), making it an interesting organism to study the role of these proteins in sulphate respiration. In this work we address the role of the [NiFeSe] Hase, found to be the major Hase associated with the cytoplasmic membrane. The purified enzyme displays interesting catalytic properties, such as a very high H₂ production activity, which is dependent on the presence of phospholipids or detergent, and resistance to oxygen inactivation since it is isolated aerobically in a Ni(II) oxidation state. Evidence was obtained that the [NiFeSe] Hase is post-translationally modified to include a hydrophobic group bound to the N-terminal, which is responsible for its membrane association. Cleavage of this group originates a soluble, less active form of the enzyme. Sequence analysis shows that [NiFeSe] Hases from Desulfovibrionacae form a separate family from the [NiFe] enzymes of these organisms, and are more closely related to [NiFe] Hases from more distant bacterial species that have a medial [4Fe4S]^2+/1+ cluster, but not a selenocysteine. The interaction of the [NiFeSe] Hase with periplasmic cytochromes was investigated and is similar to the [NiFe]₁ Hase, with the Type I cytochrome c ₃ as the preferred electron acceptor. A model of the DvH [NiFeSe] Hase was generated based on the structure of the Desulfomicrobium baculatum enzyme. The structures of the two [NiFeSe] Hases are compared with the structures of [NiFe] Hases, to evaluate the consensual structural differences between the two families. Several conserved residues close to the redox centres were identified, which may be relevant to the higher activity displayed by [NiFeSe] Hases. Electronic Supplementary Material Supplementary material is available for this article at     

Hydrogenases in sulfate-reducing bacteria function as chromium reductase

The ability of sulfate-reducing bacteria (SRB) to reduce chromate VI has been studied for possible application to the decontamination of polluted environments. Metal reduction can be achieved both chemically, by H₂S produced by the bacteria, and enzymatically, by polyhemic cytochromes c₃. We demonstrate that, in addition to low potential polyheme c-type cytochromes, the ability to reduce chromate is widespread among [Fe], [NiFe], and [NiFeSe] hydrogenases isolated from SRB of the genera Desulfovibrio and Desulfomicrobium. Among them, the [Fe] hydrogenase from Desulfovibrio vulgaris strain Hildenborough reduces Cr(VI) with the highest rate. Both [Fe] and [NiFeSe] enzymes exhibit the same K_m towards Cr(VI), suggesting that Cr(VI) reduction rates are directly correlated with hydrogen consumption rates. Electron paramagnetic resonance spectroscopy enabled us to probe the oxidation by Cr(VI) of the various metal centers in both [NiFe] and [Fe] hydrogenases. These experiments showed that Cr(VI) is reduced to paramagnetic Cr(III), and revealed inhibition of the enzyme at high Cr(VI) concentrations. The significant decrease of both hydrogenase and Cr(VI)-reductase activities in a mutant lacking [Fe] hydrogenase demonstrated the involvement of this enzyme in Cr(VI) reduction in vivo. Experiments with [3Fe-4S] ferredoxin from Desulfovibrio gigas demonstrated that the low redox [Fe-S] (non-heme iron) clusters are involved in the mechanism of metal reduction by hydrogenases.     

Analysis of the active center of hydrogenase from Desulfovibrio vulgaris Miyazaki by magnetic measurements

Hydrogenase [hydrogen: ferricytochrome c3 oxidoreductase, EC 1.12.2.1] solubilized and purified from the particulate fraction of Desulfovibrio vulgaris Miyazaki F (IAM 12604) contains 8 iron and 8 labile sulfide ions in one molecule which is composed of two unequal subunits (Mr: 60,000 + 29,000). It does not contain nickel atoms. The EPR (electron paramagnetic resonance) spectrum has an isotropic signal at g = 2.017 which is independent of the temperature. The peak-to-peak width of the signal is about 20 G. The signal intensity is nearly equivalent to 1 unpaired electron per molecule. No other signals can be detected in the field range between 2,240 and 4,240 G (which corresponds to g-values between 2.91 and 1.54). Ferricyanide has only a little effect on the shape and intensity of the EPR signal. The hydrogenase reduced under H2 is EPR silent. The M?ssbauer spectrum has no hyperfine splitting at 4K. The isomer shift and quadrupole splitting at 77K are 0.38 and 0.87 mm/s, respectively. Based on these magnetic measurements, the structure of the active center of hydrogenase was suggested to be [4Fe-4S]3+ + [4Fe-4S]2+.     

Inhibition of Desulfovibrio gigas hydrogenase with copper salts and other metal ions

The effect of several transition metals on the activity of Desulfovibrio gigas hydrogenase has been studied. Co(II) and Ni(II) at a concentration of 1 mM did not modify the activity of the enzyme; nor did they affect the pattern of activation/deactivation. Cu(II) inhibited the active hydrogenase, prepared by treatment with hydrogen, but had little effect on the 'unready' enzyme unless a reductant such as ascorbate was present, in which case inactivation took place either in air or under argon. Hg(II) also inactivated the enzyme irreversible in the 'unready' state without the requirement for reductants. The reaction of H2 uptake with methyl viologen was much more sensitive to inhibition than the H2/tritium exchange activity. EPR spectra of this preparation showed that the rates of decline were [3Fe-4S] signal greater than H2-uptake activity greater than Ni-A signal. Similar results were obtained when the protein was treated with Hg(II). The results demonstrate that the [3Fe-4S] cluster is not essential for H2-uptake activity with methyl viologen, but the integrity of [4Fe-4S] clusters is probably necessary to catalyze the reduction of methyl viologen with hydrogen. D. gigas hydrogenase was found to be highly resistant to digestion by proteases.     

[NiFe] hydrogenase from Desulfovibrio desulfuricans ATCC 27774: gene sequencing, three-dimensional structure determination and refinement at 1.8 Å and modelling studies of its interaction with the tetrahaem cytochrome c 3

The primary and three-dimensional structures of a [NiFe] hydrogenase isolated from D. desulfitricans ATCC 27774 were determined, by nucleotide analysis and single-crystal X-ray crystallography. The three-dimensional structural model was refined to R=0.167 and Rfree=0.223 using data to 1.8 A resolution. Two unique structural features are observed: the [4Fe-4S] cluster nearest the [NiFe] centre has been modified [4Fe-3S-3O] by loss of one sulfur atom and inclusion of three oxygen atoms; a three-fold disorder was observed for Cys536 which binds to the nickel atom in the [NiFe] centre. Also, the bridging sulfur atom that caps the active site was found to have partial occupancy, thus corresponding to a partly activated enzyme. These structural features may have biological relevance. In particular, the two less-populated rotamers of Cys536 may be involved in the activation process of the enzyme, as well as in the catalytic cycle. Molecular modelling studies were carried out on the interaction between this [NiFe] hydrogenase and its physiological partner, the tetrahaem cytochrome c3 from the same organism. The lowest energy docking solutions were found to correspond to an interaction between the haem IV region in tetrahaem cytochrome c3 with the distal [4Fe-4S] cluster in [NiFe] hydrogenase. This interaction should correspond to efficient electron transfer and be physiologically relevant, given the proximity of the two redox centres and the fact that electron transfer decay coupling calculations show high coupling values and a short electron transfer pathway. On the other hand, other docking solutions have been found that, despite showing low electron transfer efficiency, may give clues on possible proton transfer mechanisms between the two molecules.