Characterization of hydrogenase from the hyperthermophilic archaebacterium, Pyrococcus furiosus

The archaebacterium, Pyrococcus furiosus, grows optimally at 100 degrees C by a fermentative type metabolism in which H2 and CO2 are the only detectable products. The organism also reduces elemental sulfur (S0) to H2S. Cells grown in the absence of S0 contain a single hydrogenase, located in the cytoplasm, which has been purified 350-fold to apparent homogeneity. The yield of H2 evolution activity from reduced methyl viologen at 80 degrees C was 40%. The hydrogenase has a Mr value of 185,000 +/- 15,000 and is composed of three subunits of Mr 46,000 (alpha), 27,000 (beta), and 24,000 (gamma). The enzyme contains 31 +/- 3 g atoms of iron, 24 +/- 4 g atoms of acid-labile sulfide, and 0.98 +/- 0.05 g atoms of nickel/185,000 g of protein. The H2-reduced hydrogenase exhibits an electron paramagnetic resonance (EPR) signal at 70 K typical of a single [2Fe-2S] cluster, while below 15 K, EPR absorption is observed from extremely fast relaxing iron-sulfur clusters. The oxidized enzyme is EPR silent. The hydrogenase is reversibly inhibited by O2 and is remarkably thermostable. Most of its H2 evolution activity is retained after a 1-h incubation at 100 degrees C. Reduced ferredoxin from P. furiosus also acts as an electron donor to the enzyme, and a 350-fold increase in the rate of H2 evolution is observed between 45 and 90 degrees C. The hydrogenase also catalyzes H2 oxidation with methyl viologen or methylene blue as the electron acceptor. The temperature optimum for both H2 oxidation and H2 evolution is greater than 95 degrees C. Arrhenius plots show two transition points at approximately 60 and approximately 80 degrees C independent of the mode of assay. That occurring at 80 degrees C is associated with a dramatic increase in H2 production activity. The enzyme preferentially catalyzes H2 production at all temperatures examined and appears to represent a new type of "evolution" hydrogenase.     

EPR and electron nuclear double resonance investigation of oxidized hydrogenase II (uptake) from Clostridium pasteurianum W5. Effects of carbon monoxide binding

Two different hydrogenases have been isolated from Clostridium pasteurianum W5. Hydrogenase II (uptake) is active in H2 oxidation while hydrogenase I (bidirectional) is active both in H2 oxidation and evolution. Previous EPR and electron nuclear double resonance (ENDOR) studies of oxidized hydrogenase I have now been complemented by analogous studies on oxidized 57Fe-enriched hydrogenase II and its CO derivative (using 12CO and 13CO). Binding of CO greatly changes the EPR spectrum of oxidized hydrogenase II, and use of 13CO leads to resolved hyperfine splitting from interaction with a single 13CO molecule (AC approximately 34 MHz). This coupling is over 50% larger than that seen for hydrogenase I. 57Fe ENDOR disclosed two types of iron site in both oxidized hydrogenase II and its CO derivative. Combination of EPR, ENDOR, and M?ssbauer results shows that site 1 has AFe1 = 18 MHz shifting to approximately 30 MHz upon CO binding and consisting of two Fe atoms and site 2 has A2 approximately 7 MHz shifting to approximately 10 MHz and containing a single Fe. These results are very similar to those seen for hydrogenase I, which indicates that a structurally similar 3Fe cluster, believed to be the catalytically active site, is present in both. Proton ENDOR shows a solvent exchangeable resonance only in the CO derivative of hydrogenase II. This indicates a structural difference between hydrogenases I and II that is brought out by CO binding. No evidence of 14N coordination to the cluster is seen for either enzyme.     

Purification and characterization of an iron-nickel hydrogenase from Thermococcus celer.

Thermococcus celer cells contain a single hydrogenase located in the cytoplasm, which has been purified to apparent homogeneity using three chromatographic steps: Q-Sepharose, DEAE-Fast Flow, and Sephacryl S-200. In vitro assays demonstrated that this enzyme was able to catalyze the oxidation as well as the evolution of H2. T. celer hydrogenase had an apparent MW of 155,000+/-30,000 by gel filtration. When analyzed by SDS polyacrylamide gel electrophoresis a single band of 41,000+/-2,000 was detected. Hydrogenase activity was also detected in situ in a SDS polyacrylamide gel followed by an activity staining procedure revealing a single band corresponding to a protein of apparent Mr 84,000+/-3,000. Measurements of iron and acid-labile sulfide in different preparations of T. celer hydrogenase gave values ranging from 24 to 30 g-atoms Fe/mole of protein and 24 to 36 g-atoms of acid-labile sulfide per mole of protein. Nickel is present in 1.9-2.3 atoms per mole of protein. Copper, tungsten, and molybdenum were detected in amounts lower than 0.5 g-atoms per mole of protein. T. celer hydrogenase was inactive at ambient temperature, exhibited a dramatic increase in activity above 70 degrees C, and had an optimal activity above 90 degrees C. This enzyme showed no loss of activity after incubation at 80 degrees C for 28 h, but lost 50% of its initial activity after incubation at 96 degrees C for 20 h. Hydrogenase exhibited a half-life of approximately 25 min in air. However, after treating the air-exposed sample with sodium dithionite, more than 95% of the original activity was recovered. Copper sulfate, magnesium chloride and nitrite were also inactivators of this enzyme.     

The hyperthermophilic bacterium, Thermotoga maritima, contains an unusually complex iron-hydrogenase: amino acid sequence analyses versus biochemical characterization.

The hyperthermophilic bacterium, Thermotoga maritima, grows up to 90 degrees C by fermenting carbohydrates and it disposes of excess reductant by H(2) production. The H(2)-evolving cytoplasmic hydrogenase of this organism was shown to consist of three different subunits of masses 73 (alpha), 68 (beta) and 19 (gamma) kDa and to contain iron as the only metal. The genes encoding the subunits were clustered in a single operon in the order hydC (gamma), hydB (beta), and hydA (alpha). Sequence analyses indicated that: (a) the enzyme is an Fe-S-cluster-containing flavoprotein which uses NADH as an electron donor; and (b) the catalytic Fe-S cluster resides within the alpha-subunit, which is equivalent to the single subunit that constitutes most mesophilic Fe-hydrogenases. The alpha- and beta-subunits of the purified enzyme were separated by chromatography in the presence of 4 M urea. As predicted, the H(2)-dependent methyl viologen reduction activity of the holoenzyme (45-70 U mg(-1)) was retained in the alpha-subunit (130-160 U mg(-1)) after subunit separation. However, the holoenzyme did not contain flavin and neither it nor the alpha-subunit used NAD(P)(H) or T. maritima ferredoxin as an electron carrier. The holoenzyme, but not the alpha-subunit, reduced anthraquinone-2,6-disulfonate (apparent K(m), 690 microM) with H(2). The EPR properties of the reduced holoenzyme, when compared with those of the separated and reduced subunits, indicate the presence of a catalytic 'H-cluster' and three [4Fe-4S] and one [2Fe-2S] cluster in the alpha-subunit, together with one [4Fe-4S] and two [2Fe-2S] clusters in the beta-subunit. Sequence analyses predict that the alpha-subunit should contain an additional [2Fe-2S] cluster, while the beta-subunit should contain one [2Fe-2S] and three [4Fe-4S] clusters. The latter cluster contents are consistent with the measured Fe contents of about 32, 20 and 14 Fe mol(-1) for the holoenzyme and the alpha- and beta-subunits, respectively. The T. maritima enzyme is the first 'complex' Fe-hydrogenase to be purified and characterized, although the reason for its complexity remains unclear.     

A cytoplasmic nickel-iron hydrogenase with high specific activity from Desulfovibrio multispirans sp. N., a new species of sulfate reducing bacterium

A hydrogenase from a new species of sulfate reducing bacterium has been isolated and characterized. In contrast to other hydrogenases isolated from Desulfovibrio, this enzyme is found in the cytoplasmic fraction rather than in the periplasm. The specific activity of the enzyme, as measured in the hydrogen evolution assay, is twice as high as the specific activity of the hydrogenase from D. gigas. It also differentiates itself from the periplasmic Desulfovibrio hydrogenases by being more active in the hydrogen evolution rather than in the hydrogen uptake assay. The enzyme was shown to contain 0.9 atoms of nickel, 11 atoms of iron and 10 atoms of labile sulfide per mole of enzyme. It exhibits an unusually low intensity of the g = 2.31 nickel EPR signal in the isolated enzyme but shows a normal intensity for the g = 2.19 nickel EPR signal when reduced under hydrogen.     

On the novel H2-activating iron-sulfur center of the "Fe-only" hydrogenases

The two hydrogenases (I and II) of the anaerobic N2-fixing bacterium Clostridium pasteurianum (Cp) and the hydrogenases of the anaerobes Megasphaera elsdenii (Me) and Desulfovibrio vulgaris (strain Hildenborough, Dv), contain iron-sulfur clusters but not nickel. They are the most active hydrogenases known. All four enzymes in their reduced states give rise to EPR signals typical of [4Fe-4S]1+ clusters but exhibit novel EPR signals in their oxidized states. For example, Cp hydrogenase I exhibits a sharp rhombic EPR signal when oxidized under mild conditions but the enzyme is inactivated by over-oxidation and then exhibits an axial EPR signal. A similar axial signal is observed from mildly oxidized hydrogenase I after treatment with CO. EPR, M?ssbauer and ENDOR spectroscopy indicate that the EPR signals from the oxidized enzyme and its CO derivative arise from a novel spin-coupled Fe center. Low temperature magnetic circular dichroism (MCD) studies reveal that an EPR-silent Fe-S cluster with S greater than 1/2 is also present in oxidized hydrogenase I. From a study of all spectroscopic properties of Cp, Dv, and Me hydrogenases, it is concluded that the H2-activating site of all four is a novel Fe-S cluster with S greater than 0 and integer, which in the oxidized state is exchange-coupled to a S = 1/2 species. The data are most consistent with the S = 1/2 species being a low spin Fe(III) center. The H2-activating site is susceptible to oxidative rearrangements to yield both active and inactive states of the enzyme. We discuss the possible implications of these finding to methods of enzyme oxidation and purification procedures currently used for hydrogenases.     

The novel tungsten-iron-sulfur protein of the hyperthermophilic archaebacterium, Pyrococcus furiosus, is an aldehyde ferredoxin oxidoreductase. Evidence for its participation in a unique glycolytic pathway

The anaerobic archaebacterium, Pyrococcus furiosus, grows optimally at 100 degrees C by a fermentative-type metabolism in which H2, CO2, and organic acids are end products. The growth of this organism is stimulated by tungsten, and, from it, a novel, red-colored, tungsten-iron-sulfur protein, abbreviated RTP, has been purified (Mukund, S., and Adams, M. W. W. (1990) J. Biol. Chem. 265, 11508-11516). RTP (Mr approximately 85,000) contained approximately 1W, 7Fe, and 5 acid-labile sulfide atoms/molecule and exhibited unique EPR properties. The physiological function of the protein, however, was unknown. We show here that RTP is an inactive form of an aldehyde ferredoxin oxidoreductase (AOR). The active enzyme was obtained by rapid purification under anaerobic conditions using buffers containing dithiothreitol and glycerol. AOR catalyzed the oxidation of a range of aliphatic aldehydes with an optimum temperature for activity above 90 degrees C, but it did not oxidize glucose or glyceraldehyde 3-phosphate, nor reduce NAD(P), and its activity was independent of CoA. The active (AOR) and inactive (RTP) forms of the enzyme were indistinguishable in their contents of metals and acid-labile sulfide and in their EPR properties. The latter are though to originate from two nonidentical and spin-coupled iron-sulfur clusters, whereas the tungsten in this enzyme, which was not detectable by EPR, appears to be present as a novel pterin cofactor. Inhibition and activation studies indicated that AOR contains a catalytically essential W-SH group that is not present in RTP, the inactive form. AOR is a new type of aldehyde-oxidizing enzyme and is the first aldehyde oxidoreductase to be purified from an archaebacterium or a nonactogenic anaerobic bacterium. Its physiological role in P. furiosus is proposed as the oxidation of glyceraldehyde to glycerate in a unique, partially nonphosphorylated, glycolytic pathway that generates acetyl-CoA from glucose without the participation of nicotinamide nucleotides.     

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

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

Effects of acetylene on hydrogenases from the sulfate reducing and methanogenic bacteria

The effect of acetylene on the activity of the three types of hydrogenase from the anaerobic sulfate reducing bacteria has been investigated. The (Fe) hydrogenase is resistant to inhibition by acetylene while the nickel-containing hydrogenases are inhibited by acetylene with the (NiFe) hydrogenase being 10-50 fold more sensitive than the (NiFeSe) hydrogenase. In addition the Ni(III) EPR signal (g approximately 2.3) of the "as isolated" (NiFe) hydrogenase was significantly decreased in intensity upon exposure to acetylene.     

Purification, characterization and redox properties of hydrogenase from Methanosarcina barkeri (DSM 800)

A soluble hydrogenase from the methanogenic bacterium, Methanosarcina barkeri (DSM 800) has been purified to apparent electrophoretic homogeneity, with an overall 550-fold purification, a 45% yield and a final specific activity of 270 mumol H2 evolved min-1 (mg protein)-1. The hydrogenase has a high molecular mass of approximately equal to 800 kDa and subunits with molecular masses of approximately equal to 60 kDa. The enzyme is stable to heating at 65 degrees C and to exposure to air at 4 degrees C in the oxidized state for periods up to a week. The overall stability of this enzyme is compared with other hydrogenase isolated from strict anaerobic sulfate-reducing bacteria. Ms. barkeri hydrogenase shows an absorption spectrum typical of a non-heme iron protein with maxima at 275 nm, 380 nm and 405 nm. A flavin component, identified as FMN or riboflavin was extracted under acidic conditions and quantified to approximately one flavin molecule per subunit. In addition to this component, 8-10 iron atoms and 0.6-0.8 nickel atom were also detected per subunit. The electron paramagnetic resonance (EPR) spectrum of the native enzyme shows a rhombic signal with g values at 2.24, 2.20 and approximately equal to 2.0. probably due to nickel which is optimally measured at 40 K but still detectable at 77 K. In the reduced state, using dithionite or molecular hydrogen as reductants, at least two types of g = 1.94 EPR signals, due to iron-sulfur centers, could be detected and differentiated on the basis of power and temperature dependence. Center I has g values at 2.04, 1.90 and 1.86, while center II has g values at 2.08, 1.93 and 1.85. When the hydrogenase is reduced by hydrogen or dithionite the rhombic EPR species disappears and is replaced by other EPR-active species with g values at 2.33, 2.23, 2.12, 2.09, 2.04 and 2.00. These complex signals may represent different nickel species and are only observable at temperatures higher than 20 K. In the native preparation, at high temperatures (T greater than 35 K) or in partially reduced samples, a free radical due to the flavin moiety is observed. The EPR spectrum of reduced hydrogenase in 80% Me2SO presents an axial type of spectrum only detectable below 30 K.     

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.     

Characterization of a [2Fe-2S] protein encoded in the iron-hydrogenase operon of<Emphasis Type="Italic"> Thermotoga maritima</Emphasis>

Thermotoga maritima grows optimally at 80 degrees C by fermenting carbohydrates to organic acids, CO(2), and H(2). The production of H(2) is catalyzed by a cytoplasmic, heterotrimeric (alphabetagamma) Fe-hydrogenase. This is encoded by three genes, hydC (gamma), hydB (beta) and hydA (alpha), organized within a single operon that contains five additional open reading frames (ORFs). The recombinant form of the first ORF of the operon, TM1420, was produced in Escherichia coli. It has a molecular mass of 8537+/-3 Da as determined by mass spectrometry, in agreement with the predicted amino acid sequence. Purified TM1420 is red in color, has a basic p I (8.8), and contains 1.9 Fe atoms/mol that are present as a single [2Fe-2S] cluster, as determined by UV-visible absorption and EPR spectroscopy. The protein contains five cysteine residues, but their arrangement is characteristic of a subunit or domain rather than of a ferredoxin-type protein. The reduction potential of the [2Fe-2S] cluster (-233 mV at pH 6.5 and 25 degrees C) is pH independent but decreases linearly with temperature to -296 mV (-1.15 mV/ degrees C) at 80 degrees C. TM1420 is not reduced, in vitro, by the Fe-hydrogenase nor by a pyruvate ferredoxin oxidoreductase. The protein was unstable at 70 degrees C under anaerobic conditions with a half-life of approximately 30 min. The basic nature of TM1420, its instability at the growth temperature of T. maritima, and the unusual spacing of its cysteine residues suggest that this protein does not function as a ferredoxin-type electron carrier for the Fe-hydrogenase. Instead, TM1420 is more likely part of a thermostable multi-protein complex that is involved in metal cluster assembly of the hydrogenase holoenzyme.     

CO-dependent H2 evolution by Rhodospirillum rubrum: role of CODH:CooF complex

Upon exposure to CO during anaerobic growth, the purple phototrophic bacterium Rhodospirillum rubrum expresses a CO-oxidizing H(2) evolving enzymatic system. The CO-oxidizing enzyme, carbon monoxide dehydrogenase (CODH), has been purified and extensively characterized. However the electron transfer pathway from CODH to the CO-induced hydrogenase that evolves H(2) is not well understood. CooF is an Fe-S protein that is the proposed mediator of electron transfer between CODH and the CO-induced hydrogenase. Here we present the spectroscopic and biochemical properties of the CODH:CooF complex. The characteristic EPR signals observed for CODH are largely insensitive to CooF complexation. Metal analysis and EPR spectroscopy show that CooF contains 2 Fe(4)S(4) clusters. The observation of 2 Fe(4)S(4) clusters for CooF contradicts the prediction of 4 Fe(4)S(4) clusters based on analysis of the amino acid sequence of CooF and structural studies of CooF homologs. Comparison of in vivo and in vitro CO-dependent H(2) evolution indicates that approximately 90% of the activity is lost upon cell lysis. We propose that the loss of two labile Fe-S clusters from CooF during cell lysis may be responsible for the low in vitro CO-dependent H(2) evolution activity. During the course of these studies, a new assay for CODH:CooF was developed using membranes from an R. rubrum mutant that did not express CODH:CooF, but expressed high levels of the CO-induced hydrogenase. The assay revealed that the CO-induced hydrogenase requires the presence of CODH:CooF for optimal H(2) evolution activity.     

The structure and mechanism of iron-hydrogenases

Hydrogenases devoid of nickel and containing only Fe-S clusters have been found so far only in some strictly anaerobic bacteria. Four Fe-hydrogenases have been characterized: from Megasphaera elsdenii, Desulfovibrio vulgaris (strain Hildenborough), and two from Clostridium pasteurianum. All contain two or more [4Fe-4S]1+,2+ or F clusters and a unique type of Fe-S center termed the H cluster. The H cluster appears to be remarkably similar in all the hydrogenases, and is proposed as the site of H2 oxidation and H2 production. The F clusters serve to transfer electrons between the H cluster and the external electron carrier. In all of the hydrogenases the H cluster is comprised of at least three Fe atoms, and possibly six. In the oxidized state it contains two types of magnetically distinct Fe atoms, has an S = 1/2 spin state, and exhibits a novel rhombic EPR signal. The reduced cluster is diamagnetic (S = 0). The oxidized H cluster appears to undergo a conformation change upon reduction with H2 with an increase in Fe-Fe distances of about 0.5 A. Studies using resonance Raman, magnetic circular dichroism and electron spin echo spectroscopies suggest that the H cluster has significant non-sulfur coordination. The H cluster has two binding sites for CO, at least one of which can also bind O2. Binding to one site changes the EPR properties of the cluster and gives a photosensitive adduct, but does not affect catalytic activity. Binding to the other site, which only becomes exposed during the catalytic cycle, leads to loss of catalytic activity. Mechanisms of H2 activation and electron transfer are proposed to explain the effects of CO binding and the ability of one of the hydrogenases to preferentially catalyze H2 oxidation.     

The metabolism of hydrogen by extremely thermophilic, sulfur-dependent bacteria

Abstract In just the last few years, a group of bacteria have been discovered that have the remarkable property of growing near and above 100°C. These extremely thermophilic organisms, defined here as having the ability to grow at 90°C with optimum growth at 80°C and above, have been isolated mainly from sulfur-rich, marine geothermal environments, both shallow and deep sea. They comprise over a dozen different genera, and except for one novel eubacterium, all may be classified as archaebacteria. The majority of the extremely thermophilic genera metabolize elemental sulfur (S°) and a survey of the various organisms reveals that most of them also depend upon the oxidation of hydrogen gas (H₂) as an energy source. In addition, two extremely thermophilic genera are known that actively produce H₂ as end-products of novel fermentative metabolisms. The enzyme hydrogenase, which is responsible for catalysing H₂ activation and H₂ production, appears to play several roles in electron and energy transfer during the growth of these organisms. Hydrogenase has so far been purified from only one extremely thermophilic species, from Pyrococcus furiosus ( T _opt = 100°C), and hydrogenase activity has been exmained in cell-free extracts of only a few others. However, a comparison of their properties with those of hydrogenases from mesophilic bacteria suggests that (a) the hydrogenase responsible for catalysing H₂ oxidation in extremely thermophilic organisms may be an extremely thermostable version of the mesophilic enzyme, and (b) a new type of 'evolution' hydrogenase, lacking the Ni-S or Fe-S catalytic sites of the mesophilic enzymes, is required for catalysing H₂ evolution at temperatures near and above 100°C.     

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)     

Electron paramagnetic resonance studies on the mechanism of activation and the catalytic cycle of the nickel-containing hydrogenase from Desulfovibrio gigas

Desulfovibrio gigas hydrogenase (EC 1.12.2.1) is a complex enzyme containing one nickel, one 3Fe, and two [Fe4S4] clusters (Teixeira, M., Moura, I., Xavier, A. V., Der Vartanian, D. V., LeGall, J., Peck, H. D., Jr., Huynh, B. H., and Moura, J. J. G. (1983) Eur. J. Biochem. 130, 481-484). This hydrogenase belongs to a class of enzymes that are inactive "as isolated" (the so-called "oxygen-stable hydrogenases") and must go through an activation process in order to express full activity. The state of characterization of the active centers of the enzyme as isolated prompted us to do a detailed analysis of the redox patterns, activation profile, and catalytic redox cycle of the enzyme in the presence of either the natural substrate (H2) or chemical reductants. The effect of natural cofactors, as cytochrome C3, was also studied. Special focus was given to the intermediate redox species generated during the catalytic cycle of the enzyme and to the midpoint redox potentials associated. The available information is discussed in terms of a "working hypothesis" for the mechanism of the [NiFe] hydrogenases from sulfate reducing organisms in the context of activation process and catalytic cycle.     

Hydrodynamic, structural and magnetic properties of Megasphaera elsdenii Fe hydrogenase reinvestigated

Megasphaera elsdenii hydrogenase has been purified to homogeneity using an FPLC procedure as the final step. The protein gives a single band in SDS/PAGE with an apparent molecular mass of 57-59 kDa. There is no second hydrogenase activity in the soluble fraction of M. elsdenii. The hydrodynamics of the enzyme have been compared to those of the two-subunit Fe hydrogenase from Desulfovibrio vulgaris (Hildenborough) in the analytical ultracentrifuge using the absorption of the intrinsic iron-sulfur clusters as the monitor. Sedimentation-velocity experiments indicate the M. elsdenii enzyme (s20,w = 4.95 S) to be essentially globular, while the D. vulgaris enzyme (s20,w = 4.1 S) has a less symmetric shape. From the sedimentation equilibrium measurements under a variety of conditions an average molecular mass is calculated of 58 kDa (M. elsdenii) and 54 kDa (D. vulgaris), respectively. Pure, maximally active M. elsdenii hydrogenase has A405/A280 = 0.36 and has a specific H2-production activity of 400 mumol H2.min-1.(mg protein)-1 at 30 degrees C and pH 8.0. The enzyme contains some 13-18 iron and acid-labile sulfur ions/58-kDa monomer. Eight of these Fe-S are present as two electron-transferring ferredoxin-like cubanes with Em approximately greater than -0.3 V, as indicated by pH-dependent EPR spectroscopy on the H2-reduced enzyme. In the (re)oxidized state the remainder iron gives rise to a single S = 1/2 rhombic EPR signal. Hydrogen-production activity, content of remainder iron and rhombic EPR signal intensity are mutually correlated. Purified hydrogenase appears to exist as a mixture of fully active holoenzyme and inactive protein still carrying the two cubanes but deficient in active-site iron.     

Purification and characterization of putative alkaline [Ni-Fe] hydrogenase from unicellular marine green alga, Tetraselmis kochinensis NCIM 1605

Hydrogenase enzyme from the unicellular marine green alga Tetraselmis kochinensis NCIM 1605 was purified 467 fold to homogeneity. The molecular weight was estimated to be approximately 89kDa by SDS-PAGE. This enzyme consists of two subunits with molecular masses of approximately 70 and approximately 19kDa. The hydrogenase was found to contain 10g atoms of Fe and 1g of atom of Ni per mole of protein. The specific activity of hydrogen evolution was 50micromol H(2)/mg/h of enzyme using reduced methyl viologen as an electron donor. This hydrogenase enzyme has pI value approximately 9.6 representing its alkaline nature. The absorption spectrum of the hydrogenase enzyme showed an absorption peak at 425nm indicating that the enzyme had iron-sulfur clusters. The total of 16 cysteine residues were found per mole of enzyme under the denaturing condition and 20 cysteine residues in reduced denatured enzyme indicating that it has two disulfide bridges.