Electron transfer between hydrogenases and mono- and multiheme cytochromes in Desulfovibrio ssp

 A comparative study of electron transfer between the 16 heme high molecular mass cytochrome (Hmc) from Desulfovibrio vulgaris Hildenborough and the [Fe] and [NiFe] hydrogenases from the same organism was carried out, both in the presence and in the absence of catalytic amounts of cytochrome c ₃. For comparison, this study was repeated with the [NiFe] hydrogenase from D. gigas. Hmc is very slowly reduced by the [Fe] hydrogenase, but faster by either of the two [NiFe] hydrogenases. In the presence of cytochrome c ₃, in equimolar amounts to the hydrogenases, the rates of electron transfer are significantly increased and are similar for the three hydrogenases. The results obtained indicate that the reduction of Hmc by the [Fe] or [NiFe] hydrogenases is most likely mediated by cytochrome c ₃. A similar study with D. vulgaris Hildenborough cytochrome c ₅₅₃ shows that, in contrast, this cytochrome is reduced faster by the [Fe] hydrogenase than by the [NiFe] hydrogenases. However, although catalytic amounts of cytochrome c ₃ have no effect in the reduction by the [Fe] hydrogenase, it significantly increases the rate of reduction by the [NiFe] hydrogenases. Received: 14 April 1998 / Accepted: 25 June 1998     

Characterization of a HoxEFUYH type of [NiFe] hydrogenase from <Emphasis Type="Italic">Allochromatium vinosum</Emphasis> and some EPR and IR properties of the hydrogenase module

A soluble hydrogenase from Allochromatium vinosum was purified. It consisted of a large (M _r = 52 kDa) and a small (M _r = 23 kDa) subunit. The genes encoding for both subunits were identified. They belong to an open reading frame where they are preceded by three more genes. A DNA fragment containing all five genes was cloned and sequenced. The deduced amino acid sequences of the products characterized the complex as a member of the HoxEFUYH type of [NiFe] hydrogenases. Detailed sequence analyses revealed binding sites for eight Fe–S clusters, three [2Fe–2S] clusters and five [4Fe–4S] clusters, six of which are also present in homologous subunits of [FeFe] hydrogenases and NADH:ubiquione oxidoreductases (complex I). This makes the HoxEFUYH type of hydrogenases the one that is evolutionary closest to complex I. The relative positions of six of the potential Fe–S clusters are predicted on the basis of the X-ray structures of the Clostridium pasteurianum [FeFe] hydrogenase I and the hydrophilic domain of complex I from Thermus thermophilus. Although the HoxF subunit contains binding sites for flavin mononucleotide and NAD(H), cell-free extracts of A. vinosum did not catalyse a H₂-dependent reduction of NAD⁺. Only the hydrogenase module (HoxYH) could be purified. Its electron paramagnetic resonance (EPR) and IR spectral properties showed the presence of a Ni–Fe active site and a [4Fe–4S] cluster. Its activity was sensitive to carbon monoxide. No EPR signals from a light-sensitive Ni_a–C* state could be observed. This study presents the first IR spectroscopic data on the HoxYH module of a HoxEFUYH type of [NiFe] hydrogenase.     

Membrane-bound F420H2-dependent heterodisulfide reduction in Methanococcus voltae

Washed membranes prepared from H₂+CO₂- or formate-grown cells of Methanococcus voltae catalyzed the oxidation of coenzyme F₄₂₀H₂ and the reduction of the heterodisulfide (CoB–S–S–CoM) of 2-mercaptoethanesulfonate and 7-mercaptoheptanoylthreonine phosphate, which is the terminal electron acceptor of the methanogenic pathway. The reaction followed a 1:1 stoichiometry according to the equation: F₄₂₀H₂ + COB–S–S–CoM → F₄₂₀ + CoM–SH + CoB–SH. These findings indicate that the reaction depends on a membrane-bound F₄₂₀H₂-oxidizing enzyme and on the heterodisulfide reductase, which remains partly membrane-bound after cell lysis. To elucidate the nature of the F₄₂₀H₂-oxidizing protein, washed membranes were solubilized with detergent, and the enzyme was purified by sucrose density centrifugation, anion-exchange chromatography, and gel filtration. Several lines of evidence indicate that F₄₂₀H₂ oxidation is catalyzed by a membrane-associated F₄₂₀-reducing hydrogenase. The purified protein catalyzed the H₂-dependent reduction of methyl viologen and F₄₂₀. The apparent molecular mass and the subunit composition (43, 37, and 27 kDa) are almost identical to those of the F₄₂₀-reducing hydrogenase that has already been purified from Mc. voltae. Moreover, the N-terminus of the 37-kDa subunit is identical to the amino acid sequence deduced from the fruG gene of the operon encoding the selenium-containing F₄₂₀-reducing hydrogenase from Mc. voltae. A distinct F₄₂₀H₂ dehydrogenase, which is present in methylotrophic methanogens, was not found in this organism. Received: 18 September 1998 / Accepted: 2 November 1998     

The Three-Dimensional Structure of [NiFeSe] Hydrogenase from Desulfovibrio vulgaris Hildenborough: A Hydrogenase without a Bridging Ligand in the Active Site in Its Oxidised, “as-Isolated” State

Hydrogen is a good energy vector, and its production from renewable sources is a requirement for its widespread use. [NiFeSe] hydrogenases (Hases) are attractive candidates for the biological production of hydrogen because they are capable of high production rates even in the presence of moderate amounts of O₂, lessening the requirements for anaerobic conditions. The three-dimensional structure of the [NiFeSe] Hase from Desulfovibrio vulgaris Hildenborough has been determined in its oxidised “as-isolated” form at 2.04-Å resolution. Remarkably, this is the first structure of an oxidised Hase of the [NiFe] family that does not contain an oxide bridging ligand at the active site. Instead, an extra sulfur atom is observed binding Ni and Se, leading to a SeCys conformation that shields the NiFe site from contact with oxygen. This structure provides several insights that may explain the fast activation and O₂ tolerance of these enzymes.     

A pseudo-SECIS element in <Emphasis Type="Italic">Methanococcus voltae</Emphasis> documents evolution of a selenoprotein into a sulphur-containing homologue

Methanococcus maripaludis possesses two sets of F₄₂₀-non-reducing hydrogenases which are differentially expressed in response to the selenium content of the medium. One of the subunits of the selenium-containing hydrogenase, VhuD, contains two selenocysteine residues, whereas the homologue of M. voltae possesses cysteine residues in the equivalent positions. Analysis of the 3 non-translated region of the M. voltae vhuD mRNA revealed the existence of a structure resembling the consensus of archaeal SECIS elements but with deviations rendering it non-functional in determining selenocysteine insertion. The presence of a pseudo-SECIS element in the 3 non-translated region of the vhuD mRNA from M. voltae suggests that VhuD from this organism has developed from a selenocysteine-containing ancestor. The 3 non-translated region from the VhcD homologues neither contained a SECIS nor a pseudo SECIS element.     

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)     

Function of coenzyme F420-dependent NADP reductase in methanogenic archaea containing an NADP-dependent alcohol dehydrogenase

Methanogenic archaea growing on ethanol or isopropanol as the electron donor for CO₂ reduction to CH₄ contain either an NADP-dependent or a coenzyme F₄₂₀-dependent alcohol dehydrogenase. We report here that in both groups of methanogens, the N ⁵, N ¹⁰-methylenetetrahydromethanopterin dehydrogenase and the N ⁵, N ¹⁰-methylenetetrahydromethanopterin reductase, two enzymes involved in CO₂ reduction to CH₄, are specific for F₄₂₀. This raised the question how F₄₂₀H₂ is regenerated in the methanogens with an NADP-dependent alcohol dehydrogenase. We found that these organisms contain catabolic activities of an enzyme catalyzing the reduction of F₄₂₀ with NADPH. The F₄₂₀-dependent NADP reductase from Methanogenium organophilum was purified and characterized. The N-terminal amino acid sequence showed 42% sequence identity to a putative gene product in Methanococcus jannaschii, the total genome of which has recently been sequenced. Received: 12 May 1997 / Accepted: 1 July 1997     

Partial Purification and Characterization of Two Hydrogenases from the Extreme Thermophile Methanococcus jannaschii

F₄₂₀-nonreactive and F₄₂₀-reactive hydrogenases have been partially purified from Methanococcus jannaschii, an extremely thermophilic methanogen isolated from a submarine hydrothermal vent. The molecular weights of both hydrogenases were determined by native gradient electrophoresis in 5 to 27% polyacrylamide gels. The F₄₂₀-nonreactive hydrogenase produced one major band (475 kilodaltons), whereas the F₄₂₀-reactive hydrogenase produced two major bands (990 and 115 kilodaltons). The F₄₂₀-nonreactive hydrogenase consisted of two subunits (43 and 31 kilodaltons), and the F₄₂₀-reactive hydrogenase contained three subunits (48, 32, and 25 kilodaltons). Each hydrogenase was active at very high temperatures. Methyl viologen-reducing activity of the F₄₂₀-nonreactive hydrogenase was maximal at 80°C but was still detectable at 103°C. The maximum activities of F₄₂₀-reactive hydrogenase for F₄₂₀ and methyl viologen were measured at 80 and 90°C, respectively. Low but measureable activity toward methyl viologen was repeatedly observed at 103°C. Moreover, the half-life of the F₄₂₀-nonreactive hydrogenase at 70°C was over 9 h, and that of the F₄₂₀-reactive enzyme was over 3 h.     

Structure-function relationships among the nickel-containing hydrogenases.

The enzymology of the heterodimeric (NiFe) and (NiFeSe) hydrogenases, the monomeric nickel-containing hydrogenases plus the multimeric F420-(NiFe) and NAD(+)-(NiFe) hydrogenases are summarized and discussed in terms of subunit localization of the redox-active nickel and non-heme iron clusters. It is proposed that nickel is ligated solely by amino acid residues of the large subunit and that the non-heme iron clusters are ligated by other cysteine-rich polypeptides encoded in the hydrogenase operons which are not necessarily homologous in either structure or function. Comparison of the hydrogenase operons or putative operons and their hydrogenase genes indicate that the arrangement, number and types of genes in these operons are not conserved among the various types of hydrogenases except for the gene encoding the large subunit. Thus, the presence of the gene for the large subunit is the sole feature common to all known nickel-containing hydrogenases and unites these hydrogenases into a large but diverse gene family. Although the different genes for the large subunits may possess only nominal general derived amino acid homology, all large subunit genes sequenced to date have the sequence R-X-C-X-X-C fully conserved in the amino terminal region of the polypeptide chain and the sequence of D-P-C-X-X-C fully conserved in the carboxyl terminal region. It is proposed that these conserved motifs of amino acids provide the ligands required for the binding of the redox-active nickel. The existing EXAFS (Extended X-ray Absorption Fine Structure) information is summarized and discussed in terms of the numbers and types of ligands to the nickel and the various redox species of nickel defined by EPR spectroscopy. New information concerning the ligands to nickel is presented based on site-directed mutagenesis of the gene encoding the large subunit of the (NiFe) hydrogenase-1 of Escherichia coli. Based on considerations of the biochemical, molecular and biophysical information, ligand environments of the nickel in different redox states of the (NiFe) hydrogenase are proposed.     

The F420-Reducing [NiFe]-Hydrogenase Complex from Methanothermobacter marburgensis, the First X-ray Structure of a Group 3 Family Member

The reversible redox reaction between coenzyme F₄₂₀ and H₂ to F₄₂₀H₂ is catalyzed by an F₄₂₀-reducing [NiFe]-hydrogenase (FrhABG), which is an enzyme of the energy metabolism of methanogenic archaea. FrhABG is a group 3 [NiFe]-hydrogenase with a dodecameric quaternary structure of 1.25 MDa as recently revealed by high-resolution cryo-electron microscopy. We report on the crystal structure of FrhABG from Methanothermobacter marburgensis at 1.7 Å resolution and compare it with the structures of group 1 [NiFe]-hydrogenases, the only group structurally characterized yet. FrhA is similar to the large subunit of group 1 [NiFe]-hydrogenases regarding its core structure and the embedded [NiFe]-center but is different because of the truncation of ca 160 residues that results in similar but modified H₂ and proton transport pathways and in suitable interfaces for oligomerization. The small subunit FrhG is composed of an N-terminal domain related to group 1 enzymes and a new C-terminal ferredoxin-like domain carrying the distal and medial [4Fe-4S] clusters. FrhB adopts a novel fold, binds one [4Fe-4S] cluster as well as one FAD in a U-shaped conformation and provides the F₄₂₀-binding site at the Si-face of the isoalloxazine ring. Similar electrochemical potentials of both catalytic reactions and the electron-transferring [4Fe-4S] clusters, determined to be E°′ ≈ − 400 mV, are in full agreement with the equalized forward and backward rates of the FrhABG reaction. The protein might contribute to balanced redox potentials by the aspartate coordination of the proximal [4Fe-4S] cluster, the new ferredoxin module and a rather negatively charged FAD surrounding.     

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.     

Characterization of a cyanobacterial-like uptake [NiFe] hydrogenase: EPR and FTIR spectroscopic studies of the enzyme from Acidithiobacillus ferrooxidans

Electron paramagnetic resonance (EPR) and Fourier transform IR studies on the soluble hydrogenase from Acidithiobacillus ferrooxidans are presented. In addition, detailed sequence analyses of the two subunits of the enzyme have been performed. They show that the enzyme belongs to a group of uptake [NiFe] hydrogenases typical for Cyanobacteria. The sequences have also a close relationship to those of the H₂-sensor proteins, but clearly differ from those of standard [NiFe] hydrogenases. It is concluded that the structure of the catalytic centre is similar, but not identical, to that of known [NiFe] hydrogenases. The active site in the majority of oxidized enzyme molecules, 97% in cells and more than 50% in the purified enzyme, is EPR-silent. Upon contact with H₂ these sites remain EPR-silent and show only a limited IR response. Oxidized enzyme molecules with an EPR-detectable active site show a Ni_r*-like EPR signal which is light-sensitive at cryogenic temperatures. This is a novelty in the field of [NiFe] hydrogenases. Reaction with H₂ converts these active sites to the well-known Ni_a-C* state. Illumination below 160 K transforms this state into the Ni_a-L* state. The reversal, in the dark at 200 K, proceeds via an intermediate Ni EPR signal only observed with the H₂-sensor protein from Ralstonia eutropha. The EPR-silent active sites in as-isolated and H₂-treated enzyme are also light-sensitive as observed by IR spectra at cryogenic temperatures. The possible origin of the light sensitivity is discussed. This study represents the first spectral characterization of an enzyme of the group of cyanobacterial uptake hydrogenases. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.     

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.     

Identification of a Novel Class of Membrane-Bound [NiFe]-Hydrogenases in Thermococcus onnurineus NA1 by In Silico Analysis

In silico analysis of group 4 [NiFe]-hydrogenases from a hyperthermophilic archaeon, Thermococcus onnurineus NA1, revealed a novel tripartite gene cluster consisting of dehydrogenase-hydrogenase-cation/proton antiporter subunits, which may be classified as the new subgroup 4b of [NiFe]-hydrogenases-based on sequence motifs.Hydrogenases are the key enzymes involved in the metabolism of H₂, catalyzing the following chemical reaction: 2H⁺ + 2e⁻ ↔ H₂. Hydrogenases can be classified into [NiFe]-hydrogenases, [FeFe]-hydrogenases, and [Fe]-hydrogenases, based on their distinctive functional core containing the catalytic metal center (11, 17).The genomic analysis of Thermococcus onnurineus NA1, a hyperthermophilic archaeon isolated from a deep-sea hydrothermal vent area, revealed the presence of several distinct gene clusters encoding seven [NiFe]-hydrogenases and one homolog similar to Mbx (membrane-bound oxidoreductase) from Pyrococcus furiosus (1, 6, 8, 12). According to the classification system of hydrogenases by Vignais et al. (17), three hydrogenases (one F₄₂₀-reducing and two NADP-reducing hydrogenases) belong to group 3 [NiFe]-hydrogenases, and four hydrogenases belong to group 4 [NiFe]-hydrogenases. The group 4 hydrogenases are widely distributed among bacteria and archaea (17), with Hyc and Hyf (hydrogenase 3 and 4, respectively) from Escherichia coli (19), Coo (CO-induced hydrogenase) from Rhodospirillum rubrum (4), Ech (energy-converting hydrogenase) from Methanosarcina barkeri (7), and Mbh (membrane-bound hydrogenase) from P. furiosus (6, 10, 12) being relatively well-characterized hydrogenases in this group. One of the four group 4 hydrogenases from T. onnurineus NA1 was found to be similar in sequence to that of P. furiosus Mbh (10).     

Purification and properties of a F420-nonreactive,membrane-bound hydrogenase from Methanosarcina strain Gö1

The distribution of the F₄₂₀-reactive and F₄₂₀-nonreactive hydrogenases from the methylotrophic Methanosarcina strain Gö1 indicated a membrane association of the F₄₂₀-nonreactive enzyme. The membrane-bound F₄₂₀-nonreactive hydrogenase was purified 42-fold to electrophoretic homogeneity with a yield of 26.7%. The enzyme had a specific activity of 359 mol H₂ oxidized · min^-1 · mg protein^-1. The purification procedure involved dispersion of the membrane fraction with the detergent Chaps followed by anion exchange, hydrophobic and hydroxylapatite chromatography. The aerobically prepared enzyme had to be reactivated anaerobically. Maximal activity was observed at 80°C. The molecular mass as determined by native gel electrophoresis and gel filtration was 77000 and 79000, respectively. SDS gel electrophoresis revealed two polypeptides with molecular masses of 60000 and 40000 indicating a 1:1 stoichiometry. The purified enzyme contained 13.3 mol S^2-, 15.1 mol Fe and 0.8 mol Ni/mol enzyme. Flavins were not detected. The amino acid sequence of the N-termini of the subunits showed a higher degree of homology to cubacterial uptake-hydrogenases than to F₄₂₀-dependent hydrogenases from other methanogenic bacteria. The physiological function of the F₄₂₀-nonreactive hydrogenase from Methanosarcina strain Gö1 is discussed.Abbreviations transmembrane electrochemical gradient of H^- - CoM-SH 2-mercaptoethanesulfonate - F₄₂₀ (N-l-lactyl--l-glutamyl)-l-glutamic acid phospodiester of 7,8-didemethyl-8-hydroxy-5-deazariboflavin-5-phosphate - F₄₂₀H₂ reduced F₄₂₀ - HTP-SH 7-mercaptoheptanoylthreonine phosphate - Mb. Methanobacterium - PMSF phenylmethyl-sulfonylfluoride - Cl₃AcOH trichloroacetic acid     

Pathways of autotrophic CO2 fixation and of dissimilatory nitrate reduction to N2O in Ferroglobus placidus

The strictly anaerobic Archaeon Ferroglobus placidus was grown chemolithoautotrophically on H₂ and nitrate and analyzed for enzymes and coenzymes possibly involved in autotrophic CO₂ fixation. The following enzymes were found [values in parentheses = μmol min^–1 (mg protein)^–1]: formylmethanofuran dehydrogenase (0.2), formylmethanofuran:tetrahydromethanopterin formyltransferase (0.6), methenyltetrahydromethanopterin cyclohydrolase (10), F₄₂₀-dependent methylenetetrahydromethanopterin dehydrogenase (1.5), F₄₂₀-dependent methylenetetrahydromethanopterin reductase (0.4), and carbon monoxide dehydrogenase (0.1). The cells contained coenzyme F₄₂₀ (0.4 nmol/mg protein), tetrahydromethanopterin (0.9 nmol/ mg protein), and cytochrome b (4 nmol/mg membrane protein). From the enzyme and coenzyme composition of the cells, we deduced that autotrophic CO₂ fixation in F. placidus proceeds via the carbon monoxide dehydrogenase pathway as in autotrophically growing Archaeoglobus and Methanoarchaea species. Evidence is also presented that cell extracts of F. placidus catalyze the reduction of two molecules of nitrite to 1 N₂O with NO as intermediate (0.1 μmol N₂O formed per min and mg protein), showing that – at least in principle –F. placidus has a denitrifying capacity. Received: 23 August 1996 / Accepted: 6 November 1996     

Physiological characteristics and growth behavior of single and double hydrogenase mutants of Desulfovibrio fructosovorans

The presence of one periplasmic [NiFe] hydrogenase, one periplasmic [Fe] hydrogenase, and one cytoplasmic NADP-reducing hydrogenase has been previously established in Desulfovibrio fructosovorans. In the present work, marker-exchange mutagenesis was performed to determine the function of the tetrameric NADP-reducing hydrogenase encoded by the hndA, B, C, and D genes. The mutations performed were not lethal to the cells, although the H₂-dependent NADP reduction was completely abolished. The double-mutated DM4 (ΔhynABC, ΔhndD) strain was still able to grow on hydrogen plus sulfate as the sole energy source. The growth may have occurred under these culture conditions because of the presence of the remaining [Fe] hydrogenase. The cells grew differently on various substrates depending on whether fructose, lactate, or pyruvate was used in the presence of sulfate. The (hnd mutant growth rates were 25–70% lower than those of the wild-type strain, although the molar growth yield remained unchanged. By contrast, mutants devoid of both [NiFe] hydrogenase and NADP-reducing hydrogenase had 24-38% lower growth yields and showed a corresponding drop in the growth rates. We concluded that each of the three hydrogenases may contribute to the energy supply in D. fructosovorans and that the loss of one enzyme might be compensated for by another. However, the loss of two hydrogenases affected the phosphorylation accompanying the metabolism of fructose, lactate, and pyruvate. Received: 17 September 1996 / Accepted: 5 November 1996     

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.