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.     

Reactivity of [Fe] and [Ni-Fe-Se] hydrogenases with their oxido-reduction partner: the tetraheme cytochrome c3.

In order to understand the electron transfer mechanisms for the [Fe] and [Ni-Fe] hydrogenases, a kinetic study of cytochrome c3 reduction has been undertaken. Cyclic voltammetry and controlled-potential amperometry techniques have been used to investigate the intermolecular electron-transfer reaction between cytochrome c3 and [Fe] hydrogenase from Desulfovibrio vulgaris Hildenborough. Electron-transfer cross-reactions between [Fe] or [Ni-Fe-Se] hydrogenase and cytochrome c3 from Desulfovibrio vulgaris Hildenborough or Desulfovibrio desulfuricans Norway have been studied. Some structural implications are considered from these experimental data.     

Further characterization of the [Fe]-hydrogenase from Desulfovibrio desulfuricans ATCC 7757.

The properties of the periplasmic hydrogenase from Desulfovibrio desulfuricans ATCC 7757, previously reported to be a single-subunit protein [Glick, B. R., Martin, W. G., and Martin, S. M. (1980) Can. J. Microbiol. 26, 1214-1223] were reinvestigated. The pure enzyme exhibited a molecular mass of 53.5 kDa as measured by analytical ultracentrifugation and was found to comprise two different subunits of 42.5 kDa and 11 kDa, with serine and alanine as N-terminal residues, respectively. The N-terminal amino acid sequences of its large and small subunits, determined up to 25 residues, were identical to those of the Desulfovibrio vulgaris Hildenborough [Fe]-hydrogenase. D. desulfuricans ATCC 7757 hydrogenase was free of nickel and contained 14.0 atoms of iron and 14.4 atoms of acid-labile sulfur/molecule and had E400, 52.5 mM-1.cm-1. The purified hydrogenase showed a specific activity of 62 kU/mg of protein in the H2-uptake assay, and the H2-uptake activity was higher than H2-evolution activity. The enzyme isolated under aerobic conditions required incubation under reducing conditions to express its maximum activity both in the H2-uptake and 2H2/1H2 exchange reaction. The ratio of the activity of activated to as-isolated hydrogenase was approximately 3. EPR studies allowed the identification of two ferredoxin-type [4Fe-4S]1+ clusters in hydrogenase samples reduced by hydrogen. In addition, an atypical cluster exhibiting a rhombic signal (g values 2.10, 2.038, 1.994) assigned to the H2-activating site in other [Fe]-hydrogenases was detected in partially reduced samples. Molecular properties, EPR spectroscopy, catalytic activities with different substrates and sensitivity to hydrogenase inhibitors indicated that D. desulfuricans ATCC 7757 periplasmic hydrogenase is a [Fe]-hydrogenase, similar in most respects to the well characterized [Fe]-hydrogenase from D. vulgaris Hildenborough.     

The interaction of polymeric viologens with hydrogenases from Desulfovibrio desulfuricans and Clostridium pasteurianum.

The interaction between hydrogenases from either Desulfovibrio desulfuricans or Clostridium pasteurianum and electron donors methyl viologen or polymeric viologens was examined. Extracts from each organism contained a single gel electophoretic band of active hydrogenase. The hydrogenase of D. desulfuricans was much more stable than that of Cl. pasteurianum. With methyl viologen apparent Km and Vm values were 0.5 mM and 0.62 mumole H2/min per milligram protein for the Cl. pasteurianum and 0.7 and 6.2 mumole H2/min per milligram protein, respectively, for the D. desulfuricans enzyme. The hydrogenases bound the polymeric viologens more tightly than methyl viologen, more so for the enzyme of D. desulfuricans than for Cl. pasteurianum. Maximal rate of hydrogen production was less with the polymeric than with methyl viologen. The results suggest that the D. desulfuricans enzyme in conjunction wiion than that from Cl. pasteurianum.     

Extraction of periplasmic hydrogenase from Desulfovibrio vulgaris (Hildenborough)

Abstract Periplasmic hydrogenase from Desulfovibrio vulgaris (Hildenborough) was extracted according to the method of van der Westen [8] and the effect of trace minerals on the extractability of this enzyme was investigated. The final growth yields in the presence or absence of trace minerals were the same; however, the growth was much faster and the amount of periplasmic hydrogenase extracted was significantly lower in the presence of trace minerals. Polyacrylamide gel electrophoresis showed the presence of 2 hydrogenases in D. vulgaris , one soluble and the other possibly membrane-bound.     

Nonaheme cytochrome c, a new physiological electron acceptor for [Ni,Fe] hydrogenase in the sulfate-reducing bacterium Desulfovibrio desulfuricans Essex: primary sequence, molecular parameters, and redox properties

A nonaheme cytochrome c was purified to homogeneity from the soluble and the membrane fractions of the sulfate-reducing bacterium Desulfovibrio desulfuricans Essex. The gene encoding for the protein was cloned and sequenced. The primary structure of the multiheme protein was highly homologous to that of the nonaheme cytochrome c from D. desulfuricans ATCC 27774 and to that of the 16-heme HmcA protein from Desulfovibrio vulgaris Hildenborough. The analysis of the sequence downstream of the gene encoding for the nonaheme cytochrome c from D. desulfuricans Essex revealed an open reading frame encoding for an HmcB homologue. This operon structure indicated the presence of an Hmc complex in D. desulfuricans Essex, with the nonaheme cytochrome c replacing the 16-heme HmcA protein found in D. vulgaris. The molecular and spectroscopic parameters of nonaheme cytochrome c from D. desulfuricans Essex in the oxidized and reduced states were analyzed. Upon reduction, the pI of the protein changed significantly from 8.25 to 5.0 when going from the Fe(III) to the Fe(II) state. Such redox-induced changes in pI have not been reported for cytochromes thus far; most likely they are the result of a conformational rearrangement of the protein structure, which was confirmed by CD spectroscopy. The reactivity of the nonaheme cytochrome c toward [Ni,Fe] hydrogenase was compared with that of the tetraheme cytochrome c(3); both the cytochrome c(3) and the periplasmic [Ni,Fe] hydrogenase originated from D. desulfuricans Essex. The nonaheme protein displayed an affinity and reactivity toward [Ni,Fe] hydrogenase [K(M) = 20.5 +/- 0.9 microM; v(max) = 660 +/- 20 nmol of reduced cytochrome min(-1) (nmol of hydrogenase)(-1)] similar to that of cytochrome c(3) [K(M) = 12.6 +/- 0.7 microM; v(max) = 790 +/- 30 nmol of reduced cytochrome min(-1) (nmol of hydrogenase)(-1)]. This shows that nonaheme cytochrome c is a competent physiological electron acceptor for [Ni,Fe] hydrogenase.     

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.     

EPR and redox properties of Desulfovibrio vulgaris Miyazaki hydrogenase: comparison with the Ni-Fe enzyme from Desulfovibrio gigas.

We have carried out a detailed redox titration monitored by EPR on the hydrogenase from Desulfovibrio vulgaris Miyazaki. Typical 3Fe and nickel signals have been observed, which are very similar to those given by Desulfovibrio gigas hydrogenase in all the characteristic redox states of the enzyme. This confirms that D. vulgaris Miyazaki hydrogenase is a Ni-Fe enzyme closely related to that from D. gigas, as was recently proposed on the basis of sequence comparisons (Deckers, H.M., Wilson, F.R. and Voordouw, G. (1990) J. Gen. Microb. 136, 2021-2028).     

Purification and characterization of ferredoxin from Desulfovibrio vulgaris Miyazaki

Two ferredoxins, Fd I and Fd II, were isolated and purified from Desulfovibrio vulgaris Miyazaki. The major component, Fd I, is an iron-sulfur protein of Mr 12,000, composed of two identical subunits. The absorption spectra of Fd I and Fd II have a broad absorption shoulder near 400 nm characteristic of iron-sulfur proteins. The purity index, A400/A280, of Fd I is 0.69, and its millimolar absorption coefficient at 400 nm is 3.73 per Fe. It contains two redox centers with discrete redox behaviors. The amino acid composition and the N-terminal sequence of Fd I are similar to those of Fd III of Desulfovibrio africanus Benghazi and Fd II of Desulfovibrio desulfuricans Norway. Fd I does not serve as an electron carrier for the hydrogenase of D. vulgaris Miyazaki, but it serves as a carrier for pyruvate dehydrogenase of this bacterium. The evolution of H2 from pyruvate was observed by a reconstructed system containing purified hydrogenase, cytochrome C3, Fd I, partially purified pyruvate dehydrogenase, and CoA. The H2-sulfite reducing system can be reconstructed from the purified hydrogenase, cytochrome C3, Fd I and desulfoviridin (sulfite reductase), but the reaction rate is very slow compared to that of the crude extract at the same molar ratio of the components.     

Site-directed mutagenesis of tetraheme cytochrome c3. Modification of oxidoreduction potentials after heme axial ligand replacement.

The nature of the axial ligands of a heme group is an important factor in maintaining the oxidation-reduction potential of a c-type cytochrome. Cytochrome c3 from Desulfovibrio vulgaris Hildenborough contains four bis-histidinyl coordinated hemes with low oxidation-reduction potentials. Site-directed mutagenesis was used to generate a mutant in which histidine 70, the sixth axial ligand of heme 4, has been replaced by a methionine. The mutant protein was expressed in Desulfovibrio desulfuricans G200 at a level similar to the wild type cytochrome. A model for the three-dimensional structure of D. vulgaris Hildenborough cytochrome c3 was generated on the basis of the crystal structure of D. vulgaris Miyazaki cytochrome c3 in order to investigate the effects of the H70M mutation. The model, together with NMR data, suggested that methionine 70 has effectively replaced histidine 70 as the sixth axial ligand of heme 4 without significant alteration of the structure. A large increase of at least 200 mV of one of the four oxidation-reduction potentials was observed by electrochemistry and is interpreted in terms of structure/potential relationships.     

The hmc operon of Desulfovibrio vulgaris subsp. vulgaris Hildenborough encodes a potential transmembrane redox protein complex.

The nucleotide sequence of the hmc operon from Desulfovibrio vulgaris subsp. vulgaris Hildenborough indicated the presence of eight open reading frames, encoding proteins Orf1 to Orf6, Rrf1, and Rrf2. Orf1 is the periplasmic, high-molecular-weight cytochrome (Hmc) containing 16 c-type hemes and described before (W. B. R. Pollock, M. Loutfi, M. Bruschi, B. J. Rapp-Giles, J. D. Wall, and G. Voordouw, J. Bacteriol. 173:220-228, 1991). Orf2 is a transmembrane redox protein with four iron-sulfur clusters, as indicated by its similarity to DmsB from Escherichia coli. Orf3, Orf4, and Orf5 are all highly hydrophobic, integral membrane proteins with similarities to subunits of NADH dehydrogenase or cytochrome c reductase. Orf6 is a cytoplasmic redox protein containing two iron-sulfur clusters, as indicated by its similarity to the ferredoxin domain of [Fe] hydrogenase from Desulfovibrio species. Rrf1 belongs to the family of response regulator proteins, while the function of Rrf2 cannot be derived from the gene sequence. The expression of individual genes in E. coli with the T7 system confirmed the open reading frames for Orf2, Orf6, and Rrf1. Deletion of 0.4 kb upstream from orf1 abolished the expression of Hmc in D. desulfuricans G200, indicating this region to contain the hmc operon promoter. The expression of two truncated hmc genes in D. desulfuricans G200 resulted in stable periplasmic c-type cytochromes, confirming the domain structure of Hmc. We propose that Hmc and Orf2 to Orf6 form a transmembrane protein complex that allows electron flow from the periplasmic hydrogenases to the cytoplasmic enzymes that catalyze the reduction of sulfate. The domain structure of Hmc may be required to allow interaction with multiple hydrogenases.     

Functional expression of Desulfovibrio vulgaris Hildenborough cytochrome c3 in Desulfovibrio desulfuricans G200 after conjugational gene transfer from Escherichia coli.

Plasmid pJRDC800-1, containing the cyc gene encoding cytochrome c3 from Desulfovibrio vulgaris subsp. vulgaris Hildenborough, was transferred by conjugation from Escherichia coli DH5 alpha to Desulfovibrio desulfuricans G200. The G200 strain produced an acidic cytochrome c3 (pI = 5.8), which could be readily separated from the Hildenborough cytochrome c3 (pI = 10.5). The latter was indistinguishable from cytochrome c3 produced by D. vulgaris subsp. vulgaris Hildenborough with respect to a number of chemical and physical criteria.     

Hydrogen metabolism in Desulfovibrio desulfuricans strain New Jersey (NCIMB 8313)--comparative study with D. vulgaris and D. gigas species

This article aims to study hydrogen production/consumption in Desulfovibrio (D.) desulfuricans strain New Jersey, a sulfate reducer isolated from a medium undergoing active biocorrosion and to compare its hydrogen metabolism with two other Desulfovibrio species, D. gigas and D. vulgaris Hildenborough. Hydrogen production was followed during the growth of these three bacterial species under different growth conditions: no limitation of sulfate and lactate, sulfate limitation, lactate limitation, pyruvate/sulfate medium and in the presence of molybdate. Hydrogen production/consumption by D. desulfuricans shows a behavior similar to that of D. gigas but a different one from that of D. vulgaris, which produces higher quantities of hydrogen on lactate/sulfate medium. The three species are able to increase the hydrogen production when the sulfate became limiting. Moreover, in a pyruvate/sulfate medium hydrogen production was lower than on lactate/sulfate medium. Hydrogen production by D. desulfuricans in presence of molybdate is extremely high. Hydrogenases are key enzymes on production/consumption of hydrogen in sulfate reducing organisms. The specific activity, number and cellular localization of hydrogenases vary within the three Desulfovibrio species used in this work, which could explain the differences observed on hydrogen utilization.     

Selenium is involved in regulation of periplasmic hydrogenase gene expression in Desulfovibrio vulgaris Hildenborough

Desulfovibrio vulgaris Hildenborough is a good model organism to study hydrogen metabolism in sulfate-reducing bacteria. Hydrogen is a key compound for these organisms, since it is one of their major energy sources in natural habitats and also an intermediate in the energy metabolism. The D. vulgaris Hildenborough genome codes for six different hydrogenases, but only three of them, the periplasmic-facing [FeFe], [FeNi]1, and [FeNiSe] hydrogenases, are usually detected. In this work, we studied the synthesis of each of these enzymes in response to different electron donors and acceptors for growth as well as in response to the availability of Ni and Se. The formation of the three hydrogenases was not very strongly affected by the electron donors or acceptors used, but the highest levels were observed after growth with hydrogen as electron donor and lowest with thiosulfate as electron acceptor. The major effect observed was with inclusion of Se in the growth medium, which led to a strong repression of the [FeFe] and [NiFe]1 hydrogenases and a strong increase in the [NiFeSe] hydrogenase that is not detected in the absence of Se. Ni also led to increased formation of the [NiFe]1 hydrogenase, except for growth with H2, where its synthesis is very high even without Ni added to the medium. Growth with H2 results in a strong increase in the soluble forms of the [NiFe]1 and [NiFeSe] hydrogenases. This study is an important contribution to understanding why D. vulgaris Hildenborough has three periplasmic hydrogenases. It supports their similar physiological role in H2 oxidation and reveals that element availability has a strong influence in their relative expression.     

Uranium reduction by Desulfovibrio desulfuricans strain G20 and a cytochrome c3 mutant

Previous in vitro experiments with Desulfovibrio vulgaris strain Hildenborough demonstrated that extracts containing hydrogenase and cytochrome c3 could reduce uranium(VI) to uranium(IV) with hydrogen as the electron donor. To test the involvement of these proteins in vivo, a cytochrome c3 mutant of D. desulfuricans strain G20 was assayed and found to be able to reduce U(VI) with lactate or pyruvate as the electron donor at rates about one-half of those of the wild type. With electrons from hydrogen, the rate was more severely impaired. Cytochrome c3 appears to be a part of the in vivo electron pathway to U(VI), but additional pathways from organic donors can apparently bypass this protein.     

Desulfovibrio vulgaris Hildenborough HydE and HydG interact with the HydA subunit of the [FeFe] hydrogenase

HydE, HydF, and HydG participate in the synthesis of the complex di-iron center of [FeFe] hydrogenases. The hydE, hydF, hydG, hydA, and hydB genes of Desulfovibrio vulgaris Hildenborough were cloned and His-tag pull-down assays were used to study the potential interaction between HydE, HydF, and HydG with the HydA and HydB protein subunits of the D. vulgaris [FeFe] hydrogenase. Interaction of HydE and HydG with HydA was demonstrated. HydF did not interact with HydA, and none of the accessory proteins appeared to interact with HydB. This suggests that specific protein-protein interactions may be required during [FeFe] cluster synthesis and/or insertion.     

Activation and deactivation of the membrane-bound hydrogenase from Desulfovibrio desulfuricans, Norway strain

The hydrogenase from D. desulfuricans, when isolated in air, had a low activity in the hydrogen-methyl viologen reductase assay, and no activity in the hydrogen-methylene blue reductase assay. The activity increased markedly during incubation under hydrogen. This process is interpreted in terms of conversion of the enzyme from a relatively inactive Unready state to the Active state. Oxidation by dichloro-indophenol caused conversion to a state in which the hydrogen-uptake activity to methyl viologen was preserved, but hydrogen-methylene blue activity was not. This form is termed the Ready state. This behaviour resembles that of the hydrogenase of Desulfovibrio gigas and thus may be a widespread property of this class of hydrogenases. The electron-spin-resonance spectra of the D. desulfuricans enzyme showed the presence of [3Fe-xS] and [4Fe-4S] clusters. Spectra were also observed in the various states of activation of the enzyme. In these respects, the hydrogenase of D. desulfuricans resembles that from D. gigas, although the latter may have an additional iron-sulphur cluster.     

A new function of the Desulfovibrio vulgaris Hildenborough [Fe] hydrogenase in the protection against oxidative stress

Sulfate-reducing bacteria, like Desulfovibrio vulgaris Hildenborough, have developed a set of reactions allowing them to survive in oxic environments and even to reduce molecular oxygen to water. D. vulgaris contains a cytoplasmic superoxide reductase (SOR) and a periplasmic superoxide dismutase (SOD) involved in the elimination of superoxide anions. To assign the function of SOD, the periplasmic [Fe] hydrogenase activity was followed in both wild-type and sod deletant strains. This activity was lower in the strain lacking the SOD than in the wild-type when the cells were exposed to oxygen for a short time. The periplasmic SOD is thus involved in the protection of sensitive iron-sulfur-containing enzyme against superoxide-induced damages. Surprisingly, production of the periplasmic [Fe] hydrogenase was higher in the cells exposed to oxygen than in those kept in anaerobic conditions. A similar increase in the amount of [Fe] hydrogenase was observed when an increase in the redox potential was induced by addition of chromate. Viability of the strain lacking the gene encoding [Fe] hydrogenase after exposure to oxygen for 1 h was lower than that of the wild-type. These data reveal for the first time that production of the periplasmic [Fe] hydrogenase is up-regulated in response to an oxidative stress. A new function of the periplasmic [Fe] hydrogenase in the protective mechanisms of D. vulgaris Hildenborough toward an oxidative stress is proposed.     

Liberation of hydrogen sulfide during the catalytic action of Desulfovibrio hydrogenase under the atmosphere of hydrogen

The active site of [NiFe] hydrogenase from Desulfovibrio species is composed of a binuclear Ni-Fe complex bearing three diatomic nonprotein ligands to Fe and three bridges between the two metals, two of which are thiolate side chains of the protein moiety. The third bridging atom in the enzyme isolated from D. vulgaris Miyazaki F was suggested to be sulfur species, but was suggested to be oxygen species in D. gigas enzyme. When the hydrogenase from D. vulgaris Miyazaki F was incubated under the atmosphere of H2, H2S was liberated from the enzyme only in the presence of its electron carrier, cytochrome c3 or methylviologen. The amount of H2S liberation was little in the absence of electron carrier or essentially null when the enzyme was incubated under N2. The amount of H2S liberated was about 37% of the hydrogenase contained in the reaction vial in molar basis. These observations are in agreement with the recent observation that the third bridging site at the Ni-Fe active site is vacant in the reduced form of the enzyme revealed by X-ray crystallography.     

The presence of multiple intrinsic membrane nickel-containing hydrogenases in Desulfovibrio vulgaris (Hildenborough)

Three intrinsic membrane proteins exhibiting oxygen stable hydrogenase activity have been isolated from D. vulgaris. In contrast to the periplasmic exclusively non-heme iron hydrogenase, all three hydrogenases contain Ni in addition to non-heme iron, have low specific activities and are insensitive to inhibition by CO. None of the three hydrogenases cross react with IgA against the periplasmic hydrogenase of D. vulgaris but two of the new hydrogenases cross react with IgA against the periplasmic nickel containing hydrogenase of D. gigas and the other new hydrogenase cross reacts with IgA against the periplasmic nickel and selenium hydrogenase of D. desulfuricans (Norway -4).