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.     

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.     

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 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.     

Characterization and in vitro interaction study of a [NiFe] hydrogenase large subunit from the hyperthermophilic archaeon Thermococcus kodakarensis KOD1

The large subunit of the [NiFe] hydrogenases harbors a NiFe(CN)(2)(CO) cluster. Maturation proteins HypA, B, C, D, E, and F are required for the NiFe cluster biosynthesis. While the maturation machinery has been hitherto studied intensively, little is known about interactions between the Hyp proteins and the large subunit of the [NiFe] hydrogenase. In this study, we have purified and characterized the cytosolic [NiFe] hydrogenase large subunit HyhL from Thermococcus kodakarensis (Tk-HyhL). Tk-HyhL exists in equilibrium between monomeric and dimeric forms. In vitro interaction analyses showed that Tk-HyhL monomer forms a tight complex with Tk-HypA and weakly interacts with Tk-HypC. The expected ternary complex formation was not detected. These observations reflect a diversity in the mechanism of Ni insertion in [NiFe] hydrogenase maturation depending on the organism.     

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)     

Connection between the membrane electron transport system and Hyn hydrogenase in the purple sulfur bacterium, Thiocapsa roseopersicina BBS

Thiocapsa. roseopersicina BBS has four active [NiFe] hydrogenases, providing an excellent opportunity to examine their metabolic linkages to the cellular redox processes. Hyn is a periplasmic membrane-associated hydrogenase harboring two additional electron transfer subunits: Isp1 is a transmembrane protein, while Isp2 is located on the cytoplasmic side of the membrane. In this work, the connection of HynSL to various electron transport pathways is studied. During photoautotrophic growth, electrons, generated from the oxidation of thiosulfate and sulfur, are donated to the photosynthetic electron transport chain via cytochromes. Electrons formed from thiosulfate and sulfur oxidation might also be also used for Hyn-dependent hydrogen evolution which was shown to be light and proton motive force driven. Hyn-linked hydrogen uptake can be promoted by both sulfur and nitrate. The electron flow from/to HynSL requires the presence of Isp2 in both directions. Hydrogenase-linked sulfur reduction could be inhibited by a Q_B site competitive inhibitor, terbutryne, suggesting a redox coupling between the Hyn hydrogenase and the photosynthetic electron transport chain. Based on these findings, redox linkages of Hyn hydrogenase are modeled.     

A central functional role for the 49-kDa subunit within the catalytic core of mitochondrial complex I

We have analyzed a series of eleven mutations in the 49-kDa protein of mitochondrial complex I (NADH:ubiquinone oxidoreductase) from Yarrowia lipolytica to identify functionally important domains in this central subunit. The mutations were selected based on sequence homology with the large subunit of [NiFe] hydrogenases. None of the mutations affected assembly of complex I, all decreased or abolished ubiquinone reductase activity. Several mutants exhibited decreased sensitivities toward ubiquinone-analogous inhibitors. Unexpectedly, seven mutations affected the properties of iron-sulfur cluster N2, a prosthetic group not located in the 49-kDa subunit. In three of these mutants cluster N2 was not detectable by electron-paramagnetic resonance spectroscopy. The fact that the small subunit of hydrogenase is homologous to the PSST subunit of complex I proposed to host cluster N2 offers a straightforward explanation for the observed, unforeseen effects on this iron-sulfur cluster. We propose that the fold around the hydrogen reactive site of [NiFe] hydrogenase is conserved in the 49-kDa subunit of complex I and has become part of the inhibitor and ubiquinone binding region. We discuss that the fourth ligand of iron-sulfur cluster N2 missing in the PSST subunit may be provided by the 49-kDa subunit.     

Delivery of iron-sulfur clusters to the hydrogen-oxidizing [NiFe]-hydrogenases in Escherichia coli requires the A-type carrier proteins ErpA and IscA

During anaerobic growth Escherichia coli synthesizes two membrane-associated hydrogen-oxidizing [NiFe]-hydrogenases, termed hydrogenase 1 and hydrogenase 2. Each enzyme comprises a catalytic subunit containing the [NiFe] cofactor, an electron-transferring small subunit with a particular complement of [Fe-S] (iron-sulfur) clusters and a membrane-anchor subunit. How the [Fe-S] clusters are delivered to the small subunit of these enzymes is unclear. A-type carrier (ATC) proteins of the Isc (iron-sulfur-cluster) and Suf (sulfur mobilization) [Fe-S] cluster biogenesis pathways are proposed to traffic pre-formed [Fe-S] clusters to apoprotein targets. Mutants that could not synthesize SufA had active hydrogenase 1 and hydrogenase 2 enzymes, thus demonstrating that the Suf machinery is not required for hydrogenase maturation. In contrast, mutants devoid of the IscA, ErpA or IscU proteins of the Isc machinery had no detectable hydrogenase 1 or 2 activities. Lack of activity of both enzymes correlated with the absence of the respective [Fe-S]-cluster-containing small subunit, which was apparently rapidly degraded. During biosynthesis the hydrogenase large subunits receive their [NiFe] cofactor from the Hyp maturation machinery. Subsequent to cofactor insertion a specific C-terminal processing step occurs before association of the large subunit with the small subunit. This processing step is independent of small subunit maturation. Using western blotting experiments it could be shown that although the amount of each hydrogenase large subunit was strongly reduced in the iscA and erpA mutants, some maturation of the large subunit still occurred. Moreover, in contrast to the situation in Isc-proficient strains, these processed large subunits were not membrane-associated. Taken together, our findings demonstrate that both IscA and ErpA are required for [Fe-S] cluster delivery to the small subunits of the hydrogen-oxidizing hydrogenases; however, delivery of the Fe atom to the active site might have different requirements.     

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     

Conservation of hydrogenase and polyferredoxin structures in the hyperthermophilic archaebacterium Methanothermus fervidus.

A 3.3-kilobase-pair region of the Methanothermus fervidus genome encoding part of the small subunit and all of the large subunit of the methyl viologen-reducing hydrogenase and a polyferredoxin was cloned and sequenced. The sequence of this hyperthermophilic hydrogenase conforms to the consensus sequence established for procaryotic [NiFe] hydrogenases. Although the M. fervidus polyferredoxin is the same size as the Methanobacterium thermoautotrophicum ferredoxin, containing six tandemly arranged bacterial ferredoxinlike domains, these two proteins are predicted to be only 64% identical in their primary sequences.     

Oxygen tolerance of the H2-sensing [NiFe] hydrogenase from Ralstonia eutropha H16 is based on limited access of oxygen to the active site

Hydrogenases, abundant proteins in the microbial world, catalyze cleavage of H2 into protons and electrons or the evolution of H2 by proton reduction. Hydrogen metabolism predominantly occurs in anoxic environments mediated by hydrogenases, which are sensitive to inhibition by oxygen. Those microorganisms, which thrive in oxic habitats, contain hydrogenases that operate in the presence of oxygen. We have selected the H2-sensing regulatory [NiFe] hydrogenase of Ralstonia eutropha H16 to investigate the molecular background of its oxygen tolerance. Evidence is presented that the shape and size of the intramolecular hydrophobic cavities leading to the [NiFe] active site of the regulatory hydrogenase are crucial for oxygen insensitivity. Expansion of the putative gas channel by site-directed mutagenesis yielded mutant derivatives that are sensitive to inhibition by oxygen, presumably because the active site has become accessible for oxygen. The mutant proteins revealed characteristics typical of standard [NiFe] hydrogenases as described for Desulfovibrio gigas and Allochromatium vinosum. The data offer a new strategy how to engineer oxygen-tolerant hydrogenases for biotechnological application.     

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.     

Maturation of Rhizobium leguminosarum Hydrogenase in the Presence of Oxygen Requires the Interaction of the Chaperone HypC and the Scaffolding Protein HupK

[NiFe] hydrogenases are key enzymes for the energy and redox metabolisms of different microorganisms. Synthesis of these metalloenzymes involves a complex series of biochemical reactions catalyzed by a plethora of accessory proteins, many of them required to synthesize and insert the unique NiFe(CN)₂CO cofactor. HypC is an accessory protein conserved in all [NiFe] hydrogenase systems and involved in the synthesis and transfer of the Fe(CN)₂CO cofactor precursor. Hydrogenase accessory proteins from bacteria-synthesizing hydrogenase in the presence of oxygen include HupK, a scaffolding protein with a moderate sequence similarity to the hydrogenase large subunit and proposed to participate as an intermediate chaperone in the synthesis of the NiFe cofactor. The endosymbiotic bacterium Rhizobium leguminosarum contains a single hydrogenase system that can be expressed under two different physiological conditions: free-living microaerobic cells (∼12 μm O₂) and bacteroids from legume nodules (∼10–100 nm O₂). We have used bioinformatic tools to model HupK structure and interaction of this protein with HypC. Site-directed mutagenesis at positions predicted as critical by the structural analysis have allowed the identification of HupK and HypC residues relevant for the maturation of hydrogenase. Mutant proteins altered in some of these residues show a different phenotype depending on the physiological condition tested. Modeling of HypC also predicts the existence of a stable HypC dimer whose presence was also demonstrated by immunoblot analysis. This study widens our understanding on the mechanisms for metalloenzyme biosynthesis in the presence of oxygen.     

HupW protease specifically required for processing of the catalytic subunit of the uptake hydrogenase in the cyanobacterium Nostoc sp. strain PCC 7120

The maturation process of [NiFe] hydrogenases includes a proteolytic cleavage of the large subunit. We constructed a mutant of Nostoc strain PCC 7120 in which hupW, encoding a putative hydrogenase-specific protease, is inactivated. Our results indicate that the protein product of hupW selectively cleaves the uptake hydrogenase in this cyanobacterium.     

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.     

The H2 sensor of Ralstonia eutropha: biochemical and spectroscopic analysis of mutant proteins modified at a conserved glutamine residue close to the [NiFe] active site

[NiFe] hydrogenases contain a highly conserved histidine residue close to the [NiFe] active site which is altered by a glutamine residue in the H(2)-sensing [NiFe] hydrogenases. In this study, we exchanged the respective glutamine residue of the H(2) sensor (RH) of Ralstonia eutropha, Q67 of the RH large subunit HoxC, by histidine, asparagine and glutamate. The replacement by histidine and asparagine resulted in slightly unstable RH proteins which were hardly affected in their regulatory and enzymatic properties. The exchange to glutamate led to a completely unstable RH protein. The purified wild-type RH and the mutant protein with the Gln/His exchange were analysed by continuous-wave and pulsed electron paramagnetic resonance (EPR) techniques. We observed a coupling of a nitrogen nucleus with the [NiFe] active site for the mutant protein which was absent in the spectrum of the wild-type RH. A combination of theoretical calculations with the experimental data provided an explanation for the observed coupling. It is shown that the coupling is due to the formation of a weak hydrogen bond between the protonated N(epsilon) nucleus of the histidine with the sulfur of a conserved cysteine residue which coordinates the metal atoms of the [NiFe] active site as a bridging ligand. The effect of this hydrogen bond on the local structure of the [NiFe] active site is discussed.     

Engineering a non-native hydrogen production pathway into Escherichia coli via a cyanobacterial [NiFe] hydrogenase

Biotechnology is a promising approach for the generation of hydrogen, but is not yet commercially viable. Metabolic engineering is a potential solution, but has largely been limited to native pathway optimisation. To widen opportunities for use of non-native [NiFe] hydrogenases for improved hydrogen production, we introduced a cyanobacterial hydrogen production pathway and associated maturation factors into Escherichia coli. Hydrogen production is observed in vivo in a hydrogenase null host, demonstrating coupling to host electron transfer systems. Hydrogenase activity is also detected in vitro. Hydrogen output is increased when formate production is abolished, showing that the new pathway is distinct from the native formate dependent pathway and supporting the conclusion that it couples cellular NADH and NADPH pools to molecular hydrogen. This work demonstrates non-native hydrogen production in E. coli, showing the wide portability of [NiFe] hydrogenase pathways and the potential for metabolic engineering to improve hydrogen yields.