Partial purification and characterization of the first hydrogenase isolated from a thermophilic sulfate-reducing bacterium.

A soluble [NiFe] hydrogenase has been partially purified from the obligate thermophilic sulfate-reducing bacterium Thermodesulfobacterium mobile. A 17% purification yield was obtained after four chromatographic steps and the hydrogenase presents a purity index (A398 nm/A277 nm) equal to 0.21. This protein appears to be 75% pure on SDS-gel electrophoresis showing two major bands of molecular mass around 55 and 15 kDa. This hydrogenase contains 0.6-0.7 nickel atom and 7-8 iron atoms per mole of enzyme and has a specific activity of 783 in the hydrogen uptake reaction, of 231 in the hydrogen production assay and of 84 in the deuterium-proton exchange reaction. The H2/HD ratio is lower than one in the D2-H+ exchange reaction. The enzyme is very sensitive to NO, relatively little inhibited by CO but unaffected by NO2-. The EPR spectrum of the native hydrogenase shows the presence of a [3Fe-4S] oxidized cluster and of a Ni(III) species.

A gas chromatographic-mass spectrometric technique for studying simultaneous hydrogen-deuteron exchange and para-orthohydrogen conversion in hydrogenases of Desulfovibrio vulgaris Hildenborough.

An original gas chromatographic-mass spectrometric technique is described for studying simultaneous dihydrogen-deuteron exchange and para-ortho H2 conversion catalyzed by different Desulfovibrio hydrogenases. Para and orthohydrogens are separated on an alumina column at the temperature of liquid nitrogen, but if both HD and ortho H2 are present, their retention times are too close to each other for total separation and only one peak is observed with a thermal conductivity detector. In order to resolve the peaks from one another, a fraction of the gas released from the gas chromatograph column is admitted to the ion source of a mass spectrometer, where the gases are separated according to their respective masses. Because of a peak-jumping system, the different components involved in the exchange and in the conversion reactions can be scanned so that the spectra corresponding to mass m/e 2 (para and ortho H2), m/e 3 (HD), and m/e 4 (D2) can be obtained simultaneously. This technique has been employed to resolve a controversial problem concerning the occurrence or lack of any para-orthohydrogen conversion in heavy water. Actually both exchange and conversion were demonstrated to occur with a (NiFe) hydrogenase, whereas with a (NiFeSe) hydrogenase, which had an exchange activity equivalent to that of the former, practically no para-ortho conversion could be observed in D2O. These findings are related to the constitutional and catalytic properties of the hydrogenases belonging to the different classes.

Mass-spectrometric studies of the interrelations among hydrogenase, carbon monoxide dehydrogenase, and methane-forming activities in pure and mixed cultures of Desulfovibrio vulgaris, Desulfovibrio desulfuricans, and Methanosarcina barkeri.

The activities of pure and mixed cultures of Desulfovibrio vulgaris and Methanosarcina barkeri in the exponential growth phase were monitored by measuring changes in dissolved-gas concentration by membrane-inlet mass spectrometry. M. barkeri grown under H2-CO2 or methanol produced limited amounts of methane and practically no hydrogen from either substrate. The addition of CO resulted in a transient H2 production concomitant with CO consumption. Hydrogen was then taken up, and CH4 production increased. All these events were suppressed by KCN, which inhibited carbon monoxide dehydrogenase activity. Therefore, with both substrates, H2 appeared to be an intermediate in CO reduction to CH4. The cells grown on H2-CO2 consumed 4 mol of CO and produced 1 mol of CH4. Methanol-grown cells reduced CH3OH with H2 resulting from carbon monoxide dehydrogenase activity, and the ratio was then 1 mol of CH4 to 1 mol of CO. Only 12CH4 and no 13CH4 was obtained from 13CO, indicating that CO could not be the direct precursor of CH4. In mixed cultures of D. vulgaris and M. barkeri on lactate, an initial burst of H2 was observed, followed by a lower level of production, whereas methane synthesis was linear with time. Addition of CO to the mixed culture also resulted in transient extra H2 production but had no inhibitory effect upon CH4 formation, even when the sulfate reducer was D. vulgaris Hildenborough, whose periplasmic iron hydrogenase is very sensitive to CO. The hydrogen transfer is therefore probably mediated by a less CO-sensitive nickel-iron hydrogenase from either of both species.

Hydrogen production and deuterium-proton exchange reactions catalyzed by Desulfovibrio nickel(II)-substituted rubredoxins.

The nickel tetrahedral sulfur-coordinated core formed upon metal replacement of the native iron in Desulfovibrio sp. rubredoxins is shown to mimic the reactivity pattern of nickel-containing hydrogenases with respect to hydrogen production, deuterium-proton exchange, and inhibition by carbon monoxide.

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)

Nickel-[iron-sulfur]-selenium-containing hydrogenases from Desulfovibrio baculatus (DSM 1743). Redox centers and catalytic properties.

The hydrogenase from Desulfovibrio baculatus (DSM 1743) was purified from each of three different fractions: soluble periplasmic (wash), soluble cytoplasmic (cell disruption) and membrane-bound (detergent solubilization). Plasma-emission metal analysis detected in all three fractions the presence of iron plus nickel and selenium in equimolecular amounts. These hydrogenases were shown to be composed of two non-identical subunits and were distinct with respect to their spectroscopic properties. The EPR spectra of the native (as isolated) enzymes showed very weak isotropic signals centered around g approximately 2.0 when observed at low temperature (below 20 K). The periplasmic and membrane-bound enzymes also presented additional EPR signals, observable up to 77 K, with g greater than 2.0 and assigned to nickel(III). The periplasmic hydrogenase exhibited EPR features at 2.20, 2.06 and 2.0. The signals observed in the membrane-bound preparations could be decomposed into two sets with g at 2.34, 2.16 and approximately 2.0 (component I) and at 2.33, 2.24, and approximately 2.0 (component II). In the reduced state, after exposure to an H2 atmosphere, all the hydrogenase fractions gave identical EPR spectra. EPR studies, performed at different temperatures and microwave powers, and in samples partially and fully reduced (under hydrogen or dithionite), allowed the identification of two different iron-sulfur centers: center I (2.03, 1.89 and 1.86) detectable below 10 K, and center II (2.06, 1.95 and 1.88) which was easily saturated at low temperatures. Additional EPR signals due to transient nickel species were detected with g greater than 2.0, and a rhombic EPR signal at 77 K developed at g 2.20, 2.16 and 2.0. This EPR signal is reminiscent of the Ni-signal C (g at 2.19, 2.14 and 2.02) observed in intermediate redox states of the well characterized Desulfovibrio gigas hydrogenase (Teixeira et al. (1985) J. Biol. Chem. 260, 8942]. During the course of a redox titration at pH 7.6 using H2 gas as reductant, this signal attained a maximal intensity around -320 mV. Low-temperature studies of samples at redox states where this rhombic signal develops (10 K or lower) revealed the presence of a fast-relaxing complex EPR signal with g at 2.25, 2.22, 2.15, 2.12, 2.10 and broad components at higher field. The soluble hydrogenase fractions did not show a time-dependent activation but the membrane-bound form required such a step in order to express full activity.(ABSTRACT TRUNCATED AT 400 WORDS)

Inhibition studies of three classes of Desulfovibrio hydrogenase: application to the further characterization of the multiple hydrogenases found in Desulfovibrio vulgaris Hildenborough.

The three types of hydrogenase hitherto characterized in genus Desulfovibrio exhibit distinctive inhibition patterns of their proton-deuterium exchange activity by CO, NO and NO2-. The (Fe) and (NiFeSe) hydrogenases are the most sensitive to all three inhibitors while the (NiFe) enzymes, relatively little inhibited by CO, are still very sensitive to NO but unaffected by NO2-. These differences together with some specific catalytic properties, in particular the pH profile and the H2 to HD ratio in the exchange reaction, constitute a simple means of characterizing multiple hydrogenases present in one or different species.

Hydrogenase activity in aged, nonviable Desulfovibrio vulgaris cultures and its significance in anaerobic biocorrosion.

Batch cultures of Desulfovibrio vulgaris stored at 32 degrees C for 10 months have been found to retain 50% of the hydrogenase activity of a 1-day culture. The hydrogenase found in old cultures needs reducing conditions for its activation. Viable cell counts are negative after 6 months, showing that the hydrogenase activity does not depend on the presence of viable cells. These observations are of importance in the understanding of anaerobic biocorrosion of metals caused by depolarization phenomena.

The pH dependence of proton-deuterium exchange, hydrogen production and uptake catalyzed by hydrogenases from sulfate-reducing bacteria.

Different patterns have been found in the pH dependence of hydrogenase activity with enzymes purified from different species of Desulfovibrio. With the cytoplasmic hydrogenase from Desulfovibrio baculatus strain 9974, the pH optima in H2 production and uptake were respectively 4.0 and 7.5 with a higher activity in production than in uptake. The highest D2-H+ exchange activity was found also at pH 4.0 but the optima differed for the HD and the H2 components. Both similarly rose when the pH decreased from 9.0 to 4.5, but the rate of H2 evolution slowed whereas the HD evolution continued rising till pH values around 3.0 were reached. The H2 to HD ratio at pH above 4.5 was higher than one. With the periplasmic hydrogenase from Desulfovibrio vulgaris Hildenborough, the highest exchange activity was near pH 5.5, the same value as in hydrogen production. The periplasmic hydrogenase from Desulfovibrio gigas had in contrast the same pH optimum in the exchange (7.5-8.0) as in the H2 uptake. The ratio of H2 to HD was below one for both enzymes. These different patterns may be related to functional and structural differences in the three hydrogenases so far studied, particularly in the composition of their catalytic centers.

Redox properties and activity studies on a nickel-containing hydrogenase isolated from a halophilic sulfate reducer Desulfovibrio salexigens.

A soluble hydrogenase from the halophilic sulfate reducing bacterium Desulfovibrio salexigens, strain British Guiana (NCIB 8403) has been purified to apparent homogeneity with a final specific activity of 760 mumoles H2 evolved/min/mg (an overall 180-fold purification with 20% recovery yield). The enzyme is composed of two non-identical subunits of molecular masses 62 and 36 kDa, respectively, and contains approximately 1 Ni, 12-15 Fe and 1 Se atoms/mole. The hydrogenase shows a visible absorption spectrum typical of an iron-sulfur containing protein (A400/A280 = 0.275) and a molar absorbance of 54 mM-1cm-1 at 400 nm. In the native state (as isolated, under aerobic conditions), the enzyme is almost EPR silent at 100 K and below. However, upon reduction under H2 atmosphere a rhombic EPR signal develops at g-values 2.22, 2.16 and around 2.0, which is optimally detected at 40 K. This EPR signal is reminiscent of the nickel signal C (g-values 2.19, 2.16 and 2.02) observed in intermediate redox states of the well characterized D. gigas nickel containing hydrogenase and assigned to nickel by 61 Ni isotopic substitution (J.J.G. Moura, M. Teixeira, I. Moura, A.V. Xavier and J. Le Gall (1984), J. Mol. Cat., 23, 305-314). Upon longer incubation with H2 the "2.22" EPR signal decreases. During the course of a redox titration under H2, this EPR signal attains a maximal intensity around--380 mV. At redox states where this "2.22" signal develops (or at lower redox potentials), low temperature studies (below 10 K) reveals the presence of other EPR species with g-values at 2.23, 2.21, 2.14 with broad components at higher fields. This new signal (fast relaxing) exhibits a different microwave power dependence from that of the "2.22" signal, which readily saturates with microwave power (slow relaxing). Also at low temperature (8 K) typical reduced iron-sulfur EPR signals are concomitantly observed with gmed approximately 1.94. The catalytic properties of the enzyme were also followed by substrate isotopic exchange D2/H+ and H2 production measurements.

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.

Continuous monitoring, by mass spectrometry, of H2 production and recycling in Rhodopseudomonas capsulata.

Hydrogen evolution and consumption by cell and chromatophore suspensions of the photosynthetic bacterium Rhodopseudomonas capsulata was measured with a sensitive and specific mass spectrometric technique which directly monitors dissolved gases. H2 production by nitrogenase was inhibited by acetylene and restored by carbon monoxide. An H2 evolution activity coupled with HD formation and D2 uptake (H-D exchange) was unaffected by C2H2 and CO. Cultures lacking nitrogenase activity also exhibited H-D exchange activity, which was catalyzed by a membrane-bound hydrogenase present in the chromatophores of R. capsulata. A net hydrogen uptake, mediated by hydrogenase, was observed when electron acceptors such as CO2, O2, or ferricyanide were present in the medium.