Identification of an uptake hydrogenase required for hydrogen-dependent reduction of Fe(III) and other electron acceptors by Geobacter sulfurreducens

Geobacter sulfurreducens, a representative of the family Geobacteraceae that predominates in Fe(III)-reducing subsurface environments, can grow by coupling the oxidation of hydrogen to the reduction of a variety of electron acceptors, including Fe(III), fumarate, and quinones. An examination of the G. sulfurreducens genome revealed two operons, hya and hyb, which appeared to encode periplasmically oriented respiratory uptake hydrogenases. In order to assess the roles of these two enzymes in hydrogen-dependent growth, Hya- and Hyb-deficient mutants were generated by gene replacement. Hyb was found to be required for hydrogen-dependent reduction of Fe(III), anthraquinone-2,6-disulfonate, and fumarate by resting cell suspensions and to be essential for growth with hydrogen and these three electron acceptors. Hya, in contrast, was not. These findings suggest that Hyb is an essential respiratory hydrogenase in G. sulfurreducens.     

Identification of an uptake hydrogenase for hydrogen-dependent dissimilatory azoreduction by <Emphasis Type="Italic">Shewanella decolorationis</Emphasis> S12

Shewanella decolorationis S12, a representative dissimilatory azo-reducing bacterium of Shewanella genus, can grow by coupling the oxidation of hydrogen to the reduction of azo compounds as the sole electron acceptor, indicating that an uptake hydrogenase is an important component for electron transfer for azoreduction. For searching to the uptake hydrogenase in the genome of S. decolorationis, two operons, hyd and hya, were cloned and sequenced, which encode periplasmically oriented Fe-only hydrogenase and a Ni-Fe hydrogenase, respectively, according to the homologous comparison with other bacterial hydrogenases. In order to assess the roles of these two enzymes in hydrogen-dependent azoreduction and growth, hyd- and hya-deficient mutants were generated by gene replacement. Hya was found to be required for hydrogen-dependent reduction of azo compound by resting cell suspensions and to be essential for growth with hydrogen as electron donor and azo compound as electron acceptor. Hyd, in contrast, was not. These findings suggest that Hya is an essential respiratory hydrogenase of dissimilatory azoreduction in S. decolorationis.     

[NiFe] hydrogenases from the hyperthermophilic bacterium<Emphasis Type="Italic"> Aquifex aeolicus</Emphasis>: properties,function, and phylogenetics

Genes potentially coding for three distinct [NiFe] hydrogenases are present in the genome of Aquifex aeolicus. We have demonstrated that all three hydrogenases are expressed under standard growth conditions of the organism. Two hydrogenases were further purified to homogeneity. A periplasmically oriented hydrogenase was obtained in two forms, i.e., as a soluble enzyme containing only the two essential subunits and as a detergent-solubilized complex additionally containing a membrane-integral b-type cytochrome. The second hydrogenase purified was identified as a soluble cytoplasmic enzyme. The isolated enzymes were characterized with respect to biochemical/biophysical parameters, activity, thermostability, and substrate specificity. The phylogenetic positioning of all three hydrogenases was analyzed. A model for the metabolic roles of the three enzymes is proposed on the basis of the obtained results.     

Oxygen-tolerant H2 oxidation by membrane-bound [NiFe] hydrogenases of ralstonia species. Coping with low level H2 in air

Knallgas bacteria such as certain Ralstonia spp. are able to obtain metabolic energy by oxidizing trace levels of H2 using O2 as the terminal electron acceptor. The [NiFe] hydrogenases produced by these organisms are unusual in their ability to oxidize H2 in the presence of O2, which is a potent inactivator of most hydrogenases through attack at the active site. To probe the origin of this unusual O2 tolerance, we conducted a study on the membrane-bound hydrogenase from Ralstonia eutropha H16 and that of the closely related organism Ralstonia metallidurans CH34, which was purified using a new heterologous overproduction system. Direct electrochemical methods were used to determine apparent inhibition constants for O2 inhibition of H2 oxidation (K I(app)O2) for each enzyme. These values were at least 2 orders of magnitude higher than those of "standard" [NiFe] hydrogenases. Amino acids close to the active site were exchanged in the membrane-bound hydrogenase of R. eutropha H16 for those from standard hydrogenases to probe the role of individual residues in conferring O2 sensitivity. Michaelis constants for H2 (K M H2) were determined, and for some mutants these were increased more than 20-fold relative to the wild type. Mutations resulting in membrane-bound hydrogenase enzymes with increased K M H2 or decreased K I(app)O2 values were associated with impaired lithoautotrophic growth in the presence of high O2 concentrations.     

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.     

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)     

Accessory proteins functioning selectively and pleiotropically in the biosynthesis of [NiFe] hydrogenases in Thiocapsa roseopersicina.

There are at least two membrane-bound (HynSL and HupSL) and one soluble (HoxEFUYH) [NiFe] hydrogenases in Thiocapsa roseopersicina BBS, a purple sulfur photosynthetic bacterium. Genes coding for accessory proteins that participate in the biosynthesis and maturation of hydrogenases seem to be scattered along the chromosome. Transposon-based mutagenesis was used to locate the hydrogenase accessory genes. Molecular analysis of strains showing mutant phenotypes led to the identification of hupK (hoxV ), hypC1, hypC2, hypD, hypE, and hynD genes. The roles of hynD, hupK and the two hypC genes were investigated in detail. The putative HynD was found to be a hydrogenase-specific endoprotease type protein, participating in the maturation of the HynSL enzyme. HupK plays an important role in the formation of the functionally active membrane-bound [NiFe] hydrogenases, but not in the biosynthesis of the soluble enzyme. In-frame deletion mutagenesis showed that HypC proteins were not specific for the maturation of either hydrogenase enzyme. The lack of either HypC protein drastically reduced the activity of every hydrogenase. Hence both HypCs might participate in the maturation of [NiFe] hydrogenases. Homologous complementation with the appropriate genes substantiated the physiological roles of the corresponding gene products in the H2 metabolism of T. roseopersicina.     

The role of Hox hydrogenase in the H2 metabolism of Thiocapsa roseopersicina

The purple sulfur phototrophic bacterium Thiocapsa roseopersicina BBS synthesizes at least three NiFe hydrogenases (Hox, Hup, Hyn). We characterized the physiological H(2) consumption/evolution reactions in mutants having deletions of the structural genes of two hydrogenases in various combinations. This made possible the separation of the functionally distinct roles of the three hydrogenases. Data showed that Hox hydrogenase (unlike the Hup and Hyn hydrogenases) catalyzed the dark fermentative H(2) evolution and the light-dependent H(2) production in the presence of thiosulfate. Both Hox(+) and Hup(+) mutants demonstrated light-dependent H(2) uptake stimulated by CO(2) but only the Hup(+) mutant was able to mediate O(2)-dependent H(2) consumption in the dark. The ability of the Hox(+) mutant to evolve or consume hydrogen was found to depend on a number of interplaying factors including both growth and reaction conditions (availability of glucose, sulfur compounds, CO(2), H(2), light). The study of the redox properties of Hox hydrogenase supported the reversibility of its action. Based on the results a scheme is suggested to describe the role of Hox hydrogenase in light-dependent and dark hydrogen metabolism in T. roseopersicina BBS.     

Activation and active sites of nickel-containing hydrogenases

Hydrogenases that contain nickel and iron-sulphur clusters also have a regulatory mechanism, by which exposure to oxidants such as oxygen prevents their reaction with hydrogen. Treatment with reducing agents then causes reactivation. In some hydrogenases from Desulfovibrio species, there is evidence that there are at least two different deactivated states, which differ in their rates of reductive reactivation. The membrane-bound hydrogenase of D. desulfuricans, Norway strain, the periplasmic hydrogenase of D. gigas and the membrane-bound hydrogenase of Alcaligenes eutrophus can be isolated in a state (termed "Unready") which requires up to several hours for full activation by hydrogen. By contrast the soluble hydrogenases of D. desulfuricans and A. eutrophus can be reactivated relatively rapidly. In all of these enzymes, with the exception of the latter one, the existence of the activated and deactivated states can be correlated with different ESR-detectable forms of nickel. The possible functions of nickel and [Fe-4S] clusters in catalysis are discussed.     

Detection and localization of two hydrogenases in Methylococcus capsulatus (Bath) and their potential role in methane metabolism

Methylococcus capsulatus (Bath) was shown to contain two distinct hydrogenases, a soluble hydrogenase and a membrane-bound hydrogenase. This is the first report of a membrane-bound hydrogenase in methanotrophs. Both enzymes were expressed apparently constitutively under normal growth conditions. The soluble hydrogenase was capable of reducing NAD(+) with molecular hydrogen. The activities of both soluble and particulate methane monooxygenases could be driven by molecular hydrogen. This confirmed that molecular hydrogen could be used as a source of reducing power for methane oxidation. Hydrogen-driven methane monooxygenase activities tolerated elevated temperatures and moderate oxygen concentrations. The significance of these findings for biotechnological applications of methanotrophs is discussed.     

Energy-converting [NiFe] hydrogenases: more than just H2 activation

The well-characterized [NiFe] hydrogenases have a key function in the H2 metabolism of various microorganisms. A subfamily of the [NiFe] hydrogenases with unique properties has recently been identified. The six conserved subunits that build the core of these membrane-bound hydrogenases share sequence similarity with subunits that form the catalytic core of energy-conserving NADH:quinone oxidoreductases (complex I). The physiological role of some of these hydrogenases is to catalyze the reduction of H+ with electrons derived from reduced ferredoxins or polyferredoxins. This exergonic reaction is coupled to energy conservation by means of electron-transport phosphorylation. Other members of this hydrogenase subfamily mainly function in providing the cell with reduced ferredoxin using H2 as electron donor in a reaction driven by reverse electron transport. These hydrogenases have therefore been designated as energy-converting [NiFe] hydrogenases.     

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.     

Heterologous expression of Alteromonas macleodii and Thiocapsa roseopersicina [NiFe] hydrogenases in Synechococcus elongatus

Oxygen-tolerant [NiFe] hydrogenases may be used in future photobiological hydrogen production systems once the enzymes can be heterologously expressed in host organisms of interest. To achieve heterologous expression of [NiFe] hydrogenases in cyanobacteria, the two hydrogenase structural genes from Alteromonas macleodii Deep ecotype (AltDE), hynS and hynL, along with the surrounding genes in the gene operon of HynSL were cloned in a vector with an IPTG-inducible promoter and introduced into Synechococcus elongatus PCC7942. The hydrogenase protein was expressed at the correct size upon induction with IPTG. The heterologously-expressed HynSL hydrogenase was active when tested by in vitro H(2) evolution assay, indicating the correct assembly of the catalytic center in the cyanobacterial host. Using a similar expression system, the hydrogenase structural genes from Thiocapsa roseopersicina (hynSL) and the entire set of known accessory genes were transferred to S. elongatus. A protein of the correct size was expressed but had no activity. However, when the 11 accessory genes from AltDE were co-expressed with hynSL, the T. roseopersicina hydrogenase was found to be active by in vitro assay. This is the first report of active, heterologously-expressed [NiFe] hydrogenases in cyanobacteria.     

The H(2) sensor of Ralstonia eutropha is a member of the subclass of regulatory [NiFe] hydrogenases

Two energy-generating hydrogenases enable the aerobic hydrogen bacterium Ralstonia eutropha (formerly Alcaligenes eutrophus) to use molecular hydrogen as the sole energy source. The complex synthesis of the nickel-iron-containing enzymes has to be efficiently regulated in response to H(2), which is available in low amounts in aerobic environments. H(2) sensing in R. eutropha is achieved by a hydrogenase-like protein which controls the hydrogenase gene expression in concert with a two-component regulatory system. In this study we show that the H(2) sensor of R. eutropha is a cytoplasmic protein. Although capable of H(2) oxidation with redox dyes as electron acceptors, the protein did not support lithoautotrophic growth in the absence of the energy-generating hydrogenases. A specifically designed overexpression system for R. eutropha provided the basis for identifying the H(2) sensor as a nickel-containing regulatory protein. The data support previous results which showed that the sensor has an active site similar to that of prototypic [NiFe] hydrogenases (A. J. Pierik, M. Schmelz, O. Lenz, B. Friedrich, and S. P. J. Albracht, FEBS Lett. 438:231-235, 1998). It is demonstrated that in addition to the enzymatic activity the regulatory function of the H(2) sensor is nickel dependent. The results suggest that H(2) sensing requires an active [NiFe] hydrogenase, leaving the question open whether only H(2) binding or subsequent H(2) oxidation and electron transfer processes are necessary for signaling. The regulatory role of the H(2)-sensing hydrogenase of R. eutropha, which has also been investigated in other hydrogen-oxidizing bacteria, is intimately correlated with a set of typical structural features. Thus, the family of H(2) sensors represents a novel subclass of [NiFe] hydrogenases denoted as the "regulatory hydrogenases."     

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     

Mutants of Alcaligenes eutrophus defective in autotrophic metabolism

Forty-four mutants of Alcaligenes eutrophus H 16 were isolated which grew poorly or not at all under autotrophic conditions. Four types were characterized with respect to their defects and their physiological properties. One mutant lacked both enzymes specific for autotrophic CO₂ fixation, another one lacked both hydrogenases, and two mutants lacked either the membrane-bound or the soluble hydrogenase. Comparing the results of studies on these mutant types, the following conclusions were drawn: the lack of each hydrogenase enzyme could be partially compensated by the other one; the lack of membrane-bound hydrogenase did not affect autotrophic growth, whereas the lack of the soluble hydrogenase resulted in a decreased autotrophic growth rate. When pyruvate as well as hydrogen were supplied to the wild-type, the cell yield was higher than in the presence of pyruvate alone. Mutant experiments under these conditions indicated that either of both hydrogenases was able to add to the energy supply of the cell. Only the soluble hydrogenase was involved in the control of the rate of hydrogen oxidation by carbon dioxide; the mutant lacking this enzyme did not respond to the presence or absence of CO₂. The suppression of growth on fructose by hydrogen could be mediated by either of both hydrogenases alone.     

Analyses of the large subunit histidine-rich motif expose an alternative proton transfer pathway in [NiFe] hydrogenases

A highly conserved histidine-rich region with unknown function was recognized in the large subunit of [NiFe] hydrogenases. The HxHxxHxxHxH sequence occurs in most membrane-bound hydrogenases, but only two of these histidines are present in the cytoplasmic ones. Site-directed mutagenesis of the His-rich region of the T. roseopersicina membrane-attached Hyn hydrogenase disclosed that the enzyme activity was significantly affected only by the replacement of the His104 residue. Computational analysis of the hydrogen bond network in the large subunits indicated that the second histidine of this motif might be a component of a proton transfer pathway including Arg487, Asp103, His104 and Glu436. Substitutions of the conserved amino acids of the presumed transfer route impaired the activity of the Hyn hydrogenase. Western hybridization was applied to demonstrate that the cellular level of the mutant hydrogenases was similar to that of the wild type. Mostly based on theoretical modeling, few proton transfer pathways have already been suggested for [NiFe] hydrogenases. Our results propose an alternative route for proton transfer between the [NiFe] active center and the surface of the protein. A novel feature of this model is that this proton pathway is located on the opposite side of the large subunit relative to the position of the small subunit. This is the first study presenting a systematic analysis of an in silico predicted proton translocation pathway in [NiFe] hydrogenases by site-directed mutagenesis.     

Differential expression of hydrogenase isoenzymes in Escherichia coli K-12: evidence for a third isoenzyme.

The cellular contents of the nickel-containing, membrane-bound hydrogenase isoenzymes 1 and 2 (hydrogenases 1 and 2) were analyzed by crossed immunoelectrophoresis. Their expression was differentially influenced by nutritional and genetic factors. Hydrogenase 2 content was enhanced after growth with either hydrogen and fumarate or glycerol and fumarate and correlated reasonably with cellular hydrogen uptake capacity. Hydrogenase 1 content was negligible under the above conditions but was enhanced by exogenous formate. Its expression was greatly reduced in a pfl mutant, which is unable to synthesise formate, but was restored to normal levels when the growth medium included formate. A mutation in the anaerobic regulatory gene, fnr, led to low overall hydrogenase activity and greatly reduced levels of both isoenzymes and abolished the formate enhancement of hydrogenase 1 content. Formate hydrogenlyase activity was similarly reduced in the fnr strain but, in contrast, was restored, as was overall hydrogenase activity, to normal levels by growth in the presence of formate. Low H2 uptake activity was found for the fnr strain under all growth conditions examined. Hydrogenase 1 content, therefore, does not correlate with formate hydrogenlyase activity and its role is unclear. A third hydrogenase isoenzyme, immunologically distinct from hydrogenases 1 and 2, whose expression is enhanced by formate, is present and forms part of the formate hydrogenlyase. We suggest that the effect of the fnr gene product on formate hydrogenlyase expression is mediated via internal formate.