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At least 19 recordsLinked to original sources
PubMed1987

An investigation of hydrogenase I and hydrogenase II from Clostridium pasteurianum by resonance Raman spectroscopy. Evidence for a [2Fe-2S] cluster in hydrogenase I.

Resonance Raman spectra are reported for hydrogenase I and II from Clostridium pasteurianum. These spectra show overlapping bands with contributions from [4Fe-4S] clusters, known to be present in these enzymes, and from novel FeS centers of hitherto undefined structure. For hydrogenase I there are strong bands at 288 and 394 cm-1, which are seen in [2Fe-2S] proteins and in no other FeS species so far examined. In contrast these bands do not appear for hydrogenase II, whose resonance Raman spectrum is dominated by [4Fe-4S] cluster modes. These results provide the first structural information on the hydrogenase I FeS center involved in H2 activation and demonstrate structural differences between hydrogenase I and hydrogenase II.

Clostridium↗
PubMed1984

The physical and catalytic properties of hydrogenase II of Clostridium pasteurianum. A comparison with hydrogenase I.

Hydrogenase II of Clostridium pasteurianum is a monomeric protein of Mr = 53,000 containing 8 iron and 8 acid-labile sulfide atoms/mol. It is distinct from hydrogenase I from the same organism (Mr = 60,000 12 Fe and 12 S2-/mol). Metal analyses showed that neither hydrogenase contains nickel or any other metals in significant amounts. The iron atoms of hydrogenase II resisted chelation by 2,2'-bipyridyl but all were susceptible when the enzyme was treated with ferricyanide. Core extrusion indicated the presence of two [4Fe-4S] clusters in hydrogenase II and EPR spectroscopy showed two distinct paramagnetic species which could be interpreted as one [4Fe-4S]2+(2+,1+) and one [4Fe-4S]2+(2+,3+) per molecule. The absorption coefficient of H2-reduced hydrogenase II at 420 nm was 23,000 M-1 cm-1 with a A420 / A275 ratio of 0.27. There were large differences between hydrogenase I and hydrogenase II in the absorption spectra of the air-oxidized, H2-reduced, and dithionite-reduced forms of the enzymes. Hydrogenase II catalyzed H2 evolution with methyl viologen or ferredoxin as the electron carrier, and H2 oxidation with methylene blue or methyl viologen as the electron acceptor. Apparent Km values were determined for all these reactions with both hydrogenases. Hydrogenase II is a relatively inactive enzyme, except in the reduction of methylene blue by H2. The pH dependencies of H2 oxidation were similar for both hydrogenases but were very different in H2 evolution. The activation energy values were much higher for H2 catalysis by hydrogenase II than for hydrogenase I. The two hydrogenases have the same sensitivity to inactivation by O2 but differ in their sensitivity to metal-chelating reagents and to CO. Hydrogenase I is more readily inhibited by CO but hydrogenase II binds CO irreversibly. From the above data, a mechanism is proposed to account for the observed differences in the catalytic activities of hydrogenase I and hydrogenase II.

Binding Sites↗
PubMed2002

Effect of redox potential on activity of hydrogenase 1 and hydrogenase 2 in Escherichia coli.

This report elucidates the distinctions of redox properties between two uptake hydrogenases in Escherichia coli. Hydrogen uptake in the presence of mediators with different redox potential was studied in cell-free extracts of E. coli mutants HDK103 and HDK203 synthesizing hydrogenase 2 or hydrogenase 1, respectively. Both hydrogenases mediated H(2) uptake in the presence of high-potential acceptors (ferricyanide and phenazine methosulfate). H(2) uptake in the presence of low-potential acceptors (methyl and benzyl viologen) was mediated mainly by hydrogenase 2. To explore the dependence of hydrogen consumption on redox potential of media in cell-free extracts, a chamber with hydrogen and redox ( E(h)) electrodes was used. The mutants HDK103 and HDK203 exhibited significant distinctions in their redox behavior. During the redox titration, maximal hydrogenase 2 activity was observed at the E(h) below -80 mV. Hydrogenase 1 had maximum activity in the E(h) range from +30 mV to +110 mV. Unlike hydrogenase 2, the activated hydrogenase 1 retained activity after a fast shift of redox potential up to +500 mV by ferricyanide titration and was more tolerant to O(2). Thus, two hydrogenases in E. coli are complementary in their redox properties, hydrogenase 1 functioning at higher redox potentials and/or at higher O(2) concentrations than hydrogenase 2.

Electron Transport↗
PubMed2006

Occurrence of hydrogenases in cyanobacteria and anoxygenic photosynthetic bacteria: implications for the phylogenetic origin of cyanobacterial and algal hydrogenases.

Hydrogenases are important enzymes in the energy metabolism of microorganisms. Therefore, they are widespread in prokaryotes. We analyzed the occurrence of hydrogenases in cyanobacteria and deduced a FeFe-hydrogenase in three different heliobacterial strains. This allowed the first phylogenetic analysis of the hydrogenases of all five major groups of photosynthetic bacteria (heliobacteria, green nonsulfur bacteria, green sulfur bacteria, photosynthetic proteobacteria, and cyanobacteria). In the case of both hydrogenases found in cyanobacteria (uptake and bidirectional), the green nonsulfur bacterium Chloroflexus aurantiacus was found to be the closest ancestor. Apart from a close relation between the archaebacterial and the green sulfur bacterial sulfhydrogenase, we could not find any evidence for horizontal gene transfer. Therefore, it would be most parsimonious if a Chloroflexus-like bacterium was the ancestor of Chloroflexus aurantiacus and cyanobacteria. After having transmitted both hydrogenase genes vertically to the different cyanobacterial species, either no, one, or both enzymes were lost, thus producing the current distribution. Our data and the available data from the literature on the occurrence of cyanobacterial hydrogenases show that the cyanobacterial uptake hydrogenase is strictly linked to the occurrence of the nitrogenase. Nevertheless, we did identify a nitrogen-fixing Synechococcus strain without an uptake hydrogenase. Since we could not find genes of a FeFe-hydrogenase in any of the tested cyanobacteria, although strains performing anoxygenic photosynthesis were also included in the analysis, a cyanobacterial origin of the contemporary FeFe-hydrogenase of algal plastids seems unlikely.

Bacterial Proteins↗
PubMed1987

The mechanisms of H2 activation and CO binding by hydrogenase I and hydrogenase II of Clostridium pasteurianum.

Hydrogenase I (bidirectional) and hydrogenase II (uptake) of Clostridium pasteurianum have been investigated by electron paramagnetic resonance (EPR) spectroscopy, in the presence and absence of the inhibitor, CO. These hydrogenases contain both a novel type of iron-sulfur cluster (H), which is the proposed site of H2 catalysis, and ferredoxin-type [4Fe-4S] clusters (F). The results show that the H clusters of these two hydrogenases have very different properties. The H cluster of oxidized hydrogenase II (Hox-II) exhibits three distinct EPR signals, two of which are pH-dependent. Hox-II binds CO reversibly to give a single, pH-independent species with a novel, rhombic EPR spectrum. The H cluster of reduced hydrogenase II (Hred-II) does not react with CO. In contrast, the EPR spectrum of Hox-I appears homogeneous and independent of pH. Hox-I has a much lower affinity for CO than Hox-II, and binds CO irreversibly to give an axial EPR signal. Hred-I also binds CO irreversibly. The EPR spectra of Fred-I and Fred-II show little or no change after CO treatment. Prior exposure to CO does not affect the catalytic activity of the reduced or oxidized hydrogenases when assayed in the absence of CO, but both enzymes are irreversibly inactivated if CO is present during catalysis. Mechanisms for H2 activation by hydrogenase I and hydrogenase II are proposed from the determined midpoint potentials (Em, pH 8.0) of H-I and H-II (Em approximately -400 mV, -CO; approximately -360 mV, +CO), F-I (Em = -420 mV, +/- CO), and F-II (Em = -180 mV, +/- CO). These allow one to rationalize the different modes of CO binding to the two hydrogenases and suggest why hydrogenase II preferentially catalyzes H2 oxidation. The results are discussed in light of recent spectroscopic data on the structures of the two H clusters.

Carbon Monoxide↗
PubMed1989

Iron-sulfur clusters of hydrogenase I and hydrogenase II of Clostridium pasteurianum.

The iron and acid-labile sulfide contents and the electron paramagnetic resonance (EPR) properties of hydrogenase I (bidirectional) and hydrogenase II (uptake) of Clostridium pasteurianum (strain W5) have been determined on the basis of quantitative amino acid analyses. The iron and acid-labile sulfide values are approximately 20 and 18 atoms per molecule of hydrogenase I and 14 and 11 atoms per molecule of hydrogenase II, respectively. These amounts are substantially greater than previously reported values, which relied on protein concentration determined by colorimetric assay. The oxidized hydrogenases exhibit unusual EPR signals that originate from a novel type of iron-sulfur center, termed the hydrogenase or H cluster, which covalently binds the inhibitor CO. This EPR signal represents approximately one unpaired electron per molecule in each enzyme with and without bound CO, which is consistent with the presence of one oxidized H cluster (S = 1/2) per enzyme molecule. The two enzymes also contain ferredoxin-type four-iron centers or F clusters. The EPR signals from the F clusters observed in the reduced forms of hydrogenase I and hydrogenase II account for approximately four and one unpaired electron per molecule, respectively. We conclude from the iron determinations and the EPR results, together with a reevaluation of previous spectroscopic data, that in both hydrogenases the H cluster probably comprises six iron atoms. Mechanistic models of the two hydrogenases are presented that account for their cluster compositions and the dramatic differences in their catalytic activities.

Amino Acids↗
PubMed2002

Engineering the Rhizobium leguminosarum bv. viciae hydrogenase system for expression in free-living microaerobic cells and increased symbiotic hydrogenase activity.

Rhizobium leguminosarum bv. viciae UPM791 induces hydrogenase activity in pea (Pisum sativum L.) bacteroids but not in free-living cells. The symbiotic induction of hydrogenase structural genes (hupSL) is mediated by NifA, the general regulator of the nitrogen fixation process. So far, no culture conditions have been found to induce NifA-dependent promoters in vegetative cells of this bacterium. This hampers the study of the R. leguminosarum hydrogenase system. We have replaced the native NifA-dependent hupSL promoter with the FnrN-dependent fixN promoter, generating strain SPF25, which expresses the hup system in microaerobic free-living cells. SPF25 reaches levels of hydrogenase activity in microaerobiosis similar to those induced in UPM791 bacteroids. A sixfold increase in hydrogenase activity was detected in merodiploid strain SPF25(pALPF1). A time course induction of hydrogenase activity in microaerobic free-living cells of SPF25(pALPF1) shows that hydrogenase activity is detected after 3 h of microaerobic incubation. Maximal hydrogen uptake activity was observed after 10 h of microaerobiosis. Immunoblot analysis of microaerobically induced SPF25(pALPF1) cell fractions indicated that the HupL active form is located in the membrane, whereas the unprocessed protein remains in the soluble fraction. Symbiotic hydrogenase activity of strain SPF25 was not impaired by the promoter replacement. Moreover, bacteroids from pea plants grown in low-nickel concentrations induced higher levels of hydrogenase activity than the wild-type strain and were able to recycle all hydrogen evolved by nodules. This constitutes a new strategy to improve hydrogenase activity in symbiosis.

Bacterial Proteins↗
PubMed1998

An Escherichia coli hydrogenase-3-type hydrogenase in methanogenic archaea.

Methanogenic archaea are known to contain two types of [NiFe] hydrogenases designated F420-reducing hydrogenase and F420-non-reducing hydrogenase. We report here that they additionally contain Escherichia coli hydrogenase-3-type [NiFe] hydrogenases. The evidence is based on biochemical studies and analysis of the subunit primary structure of this hydrogenase (designated Ech) purified from membranes of acetate-grown cells of Methanosarcina barkeri. The subunits EchE and EchC of the EchABCDEF complex showed 34% and 45% sequence identity to the nickel-containing large subunit HycE and to the iron-sulfur cluster containing small subunit HycG, respectively, of the hydrogenase in the formate hydrogen lyase complex from E. coli. Analysis of the totally sequenced genomes of Methanococcus jannaschii and Methanobacterium thermoautotrophicum strain deltaH revealed that these organisms contain similar open reading frames, indicating the presence of an E. coli hydrogenase-3-type hydrogenase also in these methanogenic archaea.

Amino Acid Sequence↗
PubMed2004

A multisubunit membrane-bound [NiFe] hydrogenase and an NADH-dependent Fe-only hydrogenase in the fermenting bacterium Thermoanaerobacter tengcongensis.

Thermoanaerobacter tengcongensis is a thermophilic Gram-positive bacterium able to dispose of the reducing equivalents generated during the fermentation of glucose to acetate and CO(2) by reducing H(+) to H(2). A unique combination of hydrogenases, a ferredoxin-dependent [NiFe] hydrogenase and an NADH-dependent Fe-only hydrogenase, were found to be responsible for H(2) formation in this organism. Both enzymes were purified and characterized. The tightly membrane-bound [NiFe] hydrogenase belongs to a small group of complex-I-related [NiFe] hydrogenases and has highest sequence similarity to energy-converting [NiFe] hydrogenase (Ech) from Methanosarcina barkeri. A ferredoxin isolated from Ta. tengcongensis was identified as the physiological substrate of this enzyme. The heterotetrameric Fe-only hydrogenase was isolated from the soluble fraction. It contained FMN and multiple iron-sulfur clusters, and exhibited a typical H-cluster EPR signal after autooxidation. Sequence analysis predicted and kinetic studies confirmed that the enzyme is an NAD(H)-dependent Fe-only hydrogenase. When H(2) was allowed to accumulate in the culture, the fermentation was partially shifted to ethanol production. In cells grown at high hydrogen partial pressure [p(H(2))] the NADH-dependent hydrogenase activity was fourfold lower than in cells grown at low p(H(2)), whereas aldehyde dehydrogenase and alcohol dehydrogenase activities were higher in cells grown at elevated p(H(2)). These results indicate a regulation in response to the p(H(2)).

Amino Acid Sequence↗
PubMed1996

Similarities in the architecture of the active sites of Ni-hydrogenases and Fe-hydrogenases detected by means of infrared spectroscopy.

Three groups that absorb in the 2100-1800-cm-1 infrared spectral region have recently been detected in Ni-hydrogenase from Chromatium vinosum [Bagley, K.A., Duin, E.C., Roseboom, W., Albracht, S. P.J. & Woodruff, W.H. (1995) Biochemistry 34, 5527-5535]. To assess the significance and generality of this observation, we have carried out an infrared-spectroscopic study of eight hydrogenases of three different types (nickel, iron and metal-free) and of 11 other iron-sulfur and/or nickel proteins. Infrared bands in the 2100-1800-cm-1 spectral region were found in spectra of all Ni-hydrogenases and Fe-hydrogenases and were absent from spectra of any of the other proteins, including a metal-free hydrogenase. The positions of these bands are dependent on the redox state of the hydrogenase. The three groups in Ni-hydrogenases that are detected by infrared spectroscopy are assigned to the three unidentified small non-protein ligands that coordinate iron in the dinuclear Ni/Fe active site as observed in the X-ray structure of the enzyme from Desulfovibrio gigas [Volbeda, A., Charon, M.-H., Piras, C., Hatchikian, E.C., Frey, M. & Fontecilla-Camps, J.C. (1995) Nature 373, 580-587]. It is concluded that these groups occur exclusively in metal-containing H2-activating enzymes. It is proposed that the active sites of Ni-hydrogenases and of Fe-hydrogenases have a similar architecture, that is required for the activation of molecular hydrogen.

Binding Sites↗
PubMed2001

H2 consumption by Escherichia coli coupled via hydrogenase 1 or hydrogenase 2 to different terminal electron acceptors.

Hydrogen uptake in the presence of various terminal electron acceptors was examined in Escherichia coli mutants synthesizing either hydrogenase 1 or hydrogenase 2. Both hydrogenases mediated nitrate-dependent H2 consumption but neither of them was coupled with nitrite. Unlike hydrogenase 2, hydrogenase 1 demonstrated poor activity with electron acceptors of low midpoint redox potential. Oxygen-linked H2 uptake via hydrogenase 1 was observed over a wide range of air concentrations. Hydrogenase 2 catalyzed this reaction only at low air concentrations. Thus, hydrogenase 1 works in cells at higher redox potential, being more tolerant to oxygen than hydrogenase 2.

Electron Transport↗
PubMed2006

Mutagenesis of hydrogenase accessory genes of Synechocystis sp. PCC 6803. Additional homologues of hypA and hypB are not active in hydrogenase maturation.

Genes homologous to hydrogenase accessory genes are scattered over the whole genome in the cyanobacterium Synechocystis sp. PCC 6803. Deletion and insertion mutants of hypA1 (slr1675), hypB1 (sll1432), hypC, hypD, hypE and hypF were constructed and showed no hydrogenase activity. Involvement of the respective genes in maturation of the enzyme was confirmed by complementation. Deletion of the additional homologues hypA2 (sll1078) and hypB2 (sll1079) had no effect on hydrogenase activity. Thus, hypA1 and hypB1 are specific for hydrogenase maturation. We suggest that hypA2 and hypB2 are involved in a different metal insertion process. The hydrogenase activity of DeltahypA1 and DeltahypB1 could be increased by the addition of nickel, suggesting that HypA1 and HypB1 are involved in the insertion of nickel into the active site of the enzyme. The urease activity of all the hypA and hypB single- and double-mutants was the same as in wild-type cells. Therefore, there seems to be no common function for these two hyp genes in hydrogenase and urease maturation in Synechocystis. Similarity searches in the whole genome yielded Slr1876 as the best candidate for the hydrogenase-specific protease. The respective deletion mutant had no hydrogenase activity. Deletion of hupE had no effect on hydrogenase activity but resulted in a mutant unable to grow in a medium containing the metal chelator nitrilotriacetate. Growth was resumed upon the addition of cobalt or methionine. Because the latter is synthesized by a cobalt-requiring enzyme in Synechocystis, HupE is a good candidate for a cobalt transporter in cyanobacteria.

Base Sequence↗
PubMed1994

Nickel availability to pea (Pisum sativum L.) plants limits hydrogenase activity of Rhizobium leguminosarum bv. viciae bacteroids by affecting the processing of the hydrogenase structural subunits.

Rhizobium leguminosarum bv. viciae UPM791 induces the synthesis of an [NiFe] hydrogenase in pea (Pisum sativum L.) bacteroids which oxidizes the H2 generated by the nitrogenase complex inside the root nodules. The synthesis of this hydrogenase requires the genes for the small and large hydrogenase subunits (hupS and hupL, respectively) and 15 accessory genes clustered in a complex locus in the symbiotic plasmid. We show here that the bacteroid hydrogenase activity is limited by the availability of nickel to pea plants. Addition of Ni2+ to plant nutrient solutions (up to 10 mg/liter) resulted in sharp increases (up to 15-fold) in hydrogenase activity. This effect was not detected when other divalent cations (Zn2+, Co2+, Fe2+, and Mn2+) were added at the same concentrations. Determinations of the steady-state levels of hupSL-specific mRNA indicated that this increase in hydrogenase activity was not due to stimulation of transcription of structural genes. Immunoblot analysis with antibodies raised against the large and small subunits of the hydrogenase enzyme demonstrated that in the low-nickel situation, both subunits are mainly present in slow-migrating, unprocessed forms. Supplementation of the plant nutrient solution with increasing nickel concentrations caused the conversion of the slow-migrating forms of both subunits into fast-moving, mature forms. This nickel-dependent maturation process of the hydrogenase subunits is mediated by accessory gene products, since bacteroids from H2 uptake-deficient mutants carrying Tn5 insertions in hupG and hupK and in hypB and hypE accumulated the immature forms of both hydrogenase subunits even in the presence of high nickel levels.

Cobalt↗
PubMed1963

TEMPERATURE-SENSITIVE HYDROGENASE AND HYDROGENASE SYNTHESIS IN A PSYCHROPHILIC BACTERIUM.

Upadhyay, J. (Washington State University, Pullman) and J. L. Stokes. Temperature-sensitive hydrogenase and hydrogenase synthesis in a psychrophilic bacterium. J. Bacteriol. 86:992-998. 1963.-Hydrogenase and its synthesis were more heat-sensitive in psychrophilic strain 82 than in mesophilic Escherichia coli. The enzyme was not formed above 20 C by the psychrophile, whereas it was formed by E. coli and other mesophiles at 45 C. Aerobically grown cells of strain 82 do not contain hydrogenase but could be induced to form the enzyme by incubation with glucose and amino acids. Hydrogenase adaptation proceeded best at pH 8.0. The psychrophile hydrogenase was destroyed 50% by exposure to 60 C for 2 hr compared with 25% destruction of mesophile hydrogenase under the same conditions. The psychrophile hydrogenase was most active at pH 9.0, and the mesophile hydrogenase was most active at pH 10.0 or higher.

Bacteria↗
PubMed1980

The membrane-bound hydrogenase of Alcaligenes eutrophus: II. Localization and immunological comparison with other hydrogenase systems.

Immunological comparison of the soluble and the membrane-bound hydrogenase of Alcaligenes eutrophus revealed no common antigenic determinants shared by the native proteins, however, a small amount of cross-reacting material was detected after freezing and thawing. Immune precipitation assays supported previous observations indicating the membrane-bound hydrogenase to be localized in the outer surface of the cytoplasmic membrane. The membrane-bound hydrogenases of A. eutrophus and Pseudomonas pseudoflava showed close immunological relationship, and material cross-reacting to both antisera was found in membrane extracts of all hydrogen-oxidizing strains of Pseudomonas. Alcaligenes and Aquaspirillum. Material cross-reacting to the membrane-bound hydrogenase of Xanthobacter autotraphicus GZ 29 was found only in a few hydrogen-oxidizing bacteria. Material cross-reacting to the soluble hydrogenase of A. eutrophus was detected in strains of A. eutrophus and A. ruhlandii only. Comparison of the membrane-bound hydrogenase of A. eutrophus, P. pseudoflava and X. autotrophicus with hydrogenases of other physiological bacterial groups revealed serological relationship to the membrane-bound hydrogenases of the hydrogen bacteria and of Chromatium vinosum only. The results are discussed in terms of physiological, taxonomical, and evolutionary aspects.

Alcaligenes↗
PubMed1990

Restoration of hydrogenase activity in hydrogenase-negative strains of Escherichia coli by cloned DNA fragments from Chromatium vinosum and Proteus vulgaris.

DNA fragments from Proteus vulgaris and Chromatium vinosum were isolated which restored hydrogenase activities in both hydA and hydB mutant strains of Escherichia coli. The hydA and hydB genes, which map near minute 59 of the genome map, 17 kb distant from each other, are not structural hydrogenase genes, but mutation in either of these genes leads to failure to synthesize any of the hydrogenase isoenzymes. The smallest DNA fragments which restored hydrogenase activity to both E. coli mutant strains were 4.7 kb from C. vinosum and 2.3 kb from P. vulgaris. These fragments were cleaved into smaller fragments which did not complement either of the E. coli mutations. The cloned heterologous genes also restored formate hydrogenlyase activity but they did not restore activity in hydE, hupA or hupB mutant strains of E. coli. The cloned genes, on plasmids, did not lead to the synthesis of proteins of sufficient size to be the hydrogenase catalytic subunit. The hydrogenase proteins synthesized by hydA and hydB mutant strains of E. coli transformed by cloned genes from P. vulgaris and C. vinosum were shown by isoelectric and immunological methods to be E. coli hydrogenase. Thus, these genes are not hydrogenase structural genes.

Blotting, Southern↗
PubMed1993

A genetic region downstream of the hydrogenase structural genes of Bradyrhizobium japonicum that is required for hydrogenase processing.

Deletion of a 2.9-kb chromosomal EcoRI fragment of DNA located 2.2 kb downstream from the end of the hydrogenase structural genes resulted in the complete loss of hydrogenase activity. The normal 65- and 35-kDa hydrogenase subunits were absent in the deletion mutants. Instead, two peptides of 66.5 and 41 kDa were identified in the mutants by use of anti-hydrogenase subunit-specific antibody. A hydrogenase structural gene mutant did not synthesize either the normal hydrogenase subunits or the larger peptides. Hydrogenase activity in the deletion mutants was complemented to near wild-type levels by plasmid pCF1, containing a 6.5-kb BglII fragment, and the 65- and 35-kDa hydrogenase subunits were also recovered in the mutants containing pCF1.

Cosmids↗