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At least 19 recordsLinked to original sources
PubMed1979

Comparison of the membrane-bound and detergent-solubilised hydrogenase from paracoccus denitrificans. Isolation of the hydrogenase.

The hydrogenase from Paracoccus denitrificans is an integral membrane protein and has been solubilised by Triton X-100. The membrane-bound and detergent-solubilised forms of the enzyme have been compared. Both forms of the enzyme show a pH optimum for reduction of benzyl viologen at pH 8.5--9.0 and are both inhibited by concentrations of NaCl greater than 30 mM. An Arrhenius plot of the activity of hydrogenase in the membrane shows no 'break'. The form of the Arrhenius plot and the activation energy are not significantly changed on solubilisation of the enzyme. The Km and V values for benzyl viologen, methyl viologen and H2 are unaltered when the enzyme is extracted from the membrane. Therefore, solubilisation of hydrogenase from the membrane by Triton X-400 is unlikely to disrupt the native conformation of the enzyme. The detergent-solubilised hydrogenase has subsequently been purified using ammonium sulphate precipitation, sucrose density gradient centrifugation and chromatography on hydroxyapatite. The overall yield of activity is 23%, with a final purification of over 100-fold.

Cell Membrane↗
PubMed1976

The iron-sulfur centers and the function of hydrogenase from Clostridium pasteurianum.

Hydrogenase from C. pasteurianum is an iron-sulfur protein containing at least two tetrameric iron-sulfur centers. Information on the structure of the remaining iron atoms must await future investigation. Although the EPR spectra of dithionite-reduced hydrogenase and eight-iron Fd showed some similarity, the CD spectra clearly indicated a difference. The tetrameric iron-sulfur centers of hydrogenase were shown to undergo redox changes when hydrogenase was oxidized or reduced. However, no evidence is now available to support a role for the tetrameric Fe-S centers, responsible for the EPR spectrum A, as the primary site for H2 binding and activation. Because we have found that the [Fe4S4(SR)4]-containing ferredoxins do not have hydrogenase activity, it is conceivable that the additional iron atoms and/or certain amino acid residues of hydrogenase also contribute to the unique catalytic properties of this enzyme. Chemical synthesis of Fe-S clusters with different peptide environments and with hydrogenase function would lead to the identification of these functional groups. X-ray diffraction studies on hydrogenase will certainly complement the other approaches. Knowledge of the structure of the active site of hydrogenase will certainly accelerate research into: (1) the synthesis of a stable catalyst to replace hydrogenase in systems designed to produce H2 by coupling this catalyst to a photoreducing system; and (2) the elucidation of the active sites of more complicated iron-sulfur enzymes such as nitrogenase.

Binding Sites↗
PubMed1979

Regulation of hydrogenase in Rhizobium japonicum.

Factors that regulate the expression of an H2 uptake system in free-living cultures of Rhizobium japonicum have been investigated. Rapid rates of H2 uptake by R. japonicum were obtained by incubation of cell suspensions in a Mg-phosphate buffer under a gas phase of 86.7% N2, 8.3% H2, 4.2% CO2, and 0.8% O2. Cultures incubated under conditions comparable with those above, with the exception that Ar replaced H2, showed no hydrogenase activity. When H2 was removed after initiation of hydrogenase derepression, further increase in hydrogenase activity ceased. Nitrogenase activity was not essential for expression of hydrogenase activity. All usable carbon substrates tested repressed hydrogenase formation, but none of them inhibited hydrogenase activity. No effect on hydrogenase formation was observed from the addition of KNO3 or NH4Cl at 10 mM. Oxygen repressed hydrogenase formation, but did not inhibit activity of the enzyme in whole cells. The addition of rifampin or chloramphenicol to derepressed cultures resulted in inhibition of enzyme formation similar to that observed by O2 repression. The removal of CO2 during derepression caused a decrease in the rate of hydrogenase formation. No direct effect of CO2 on hydrogenase activity was observed.

Carbon Dioxide↗
PubMedDate not supplied

[Localization of hydrogenase in the cells of the thermophilic hydrogen bacterium, Pseudomonas thermophila].

Spheroplasts that were osmotically stable in 0.2M Tris-HCl--0.02M EDTA were prepared from the autotrophically grown cells of Pseudomonas thermophila K-2. The spheroplasts possessed 90--95% of the hydrogenase activity of the whole cells. The half-life time of hydrogenase in the spheroplasts at 80 degrees C was 8.5 min. A spectrophotometric technique was developed for determining the membrane-bound hydrogenase in the presence of sulfhydryl compounds with methylene blue as electron acceptor. The maximal specific activity of hydrogenase in extracts prepared in the anaerobic conditions in the presence of dithiothreitol and Mg2+ and Mn2+ ions was 10 +/- 3 units per 1 mg of protein, which closely corresponded with the activity of hydrogenase in the whole cells. Almost all activity of hydrogenase assayed with methylene blue was localized in the membrane fraction. The activity of soluble NAD-specific hydrogenase was not detected. Large particles located in 60-70% sucrose had the highest hydrogenase activity upon fractionation in a continuous sucrose concentration gradient. The second, lower peak of the hydrogenase activity was detected in fractions of 40--50% sucrose. As was found by electron microscopy, the size of membrane vesicles with the hydrogenase activity varied within the range of 68--156 nm. The membrane preparations possessed the activity of NADH-dehydrogenase, NADH-oxidase and succinate dehydrogenase as well.

Centrifugation, Density Gradient↗
PubMed1976

Purification and properties of soluble hydrogenase from Alcaligenes eutrophus H 16.

The soluble hydrogenase (hydrogen: NAD+ oxidoreductase, EC 1.12.1.2) from Alcaligenes eutrophus H 16 was purified 68-fold with a yield of 20% and a final specific activity (NAD reduction) of about 54 mumol H2 oxidized/min per mg protein. The enzyme was shown to be homogenous by polyacrylamide gel electrophoresis. Its molecular weight and isoelectric point were determined to be 205 000 and 4.85 respectively. The oxidized hydrogenase, as purified under aerobic conditions, was of high stability but not reactive. Reductive activation of the enzyme by H2, in the presence of catalytic amounts of NADH, or by reducing agents caused the hydrogenase to become unstable. The purified enzyme, in its active state, was able to reduce NAD, FMN, FAD, menaquinone, ubiquinone, cytochrome c, methylene blue, methyl viologen, benzyl viologen, phenazine methosulfate, janus green, 2,6-dichlorophenoloindophenol, ferricyanide and even oxygen. In addition to hydrogenase activitiy, the enzyme exhibited also diaphorase and NAD(P)H oxidase activity. The reversibility of hydrogenase function (i.e. H2 evolution from NADH, methyl viologen and benzyl viologen) was demonstrated. With respect to H2 as substrate, hydrogenase showed negative cooperativity; the Hill coefficient was n = 0.4. The apparent Km value for H2 was found to be 0.037 mM. The absorption spectrum of hydrogenase was typical for non-heme iron proteins, showing maxima (shoulders) at 380 and 420 nm. A flavin component could be extracted from native hydrogenase characterized by its absorption bands at 375 and 447 nm and a strong fluorescense at 526 nm.

Alcaligenes↗
PubMed1979

The iron-sulphur centres of soluble hydrogenase from Alcaligenes eutrophus.

The soluble hydrogenase (hydrogen:NAD+ oxidoreductase (EC 1.12.1.2) from Alcaligenes eutrophus has been purified to homogeneity by an improved procedure, which includes preparative electrophoresis as final step. The specific activity of 57 mumol H2 oxidized/min per mg protein was achieved and the yield of pure enzyme from 200 g cells (wet weight) was about 16 mg/purification. After removal of non-functional iron, analysis of iron and acid-labile sulphur yielded average values of 11.5 and 12.9 atoms/molecule of enzyme, respectively. p-Chloromercuribenzoate was a strong inhibitor of hydrogenase and apparently competed with NAD not with H2. Chelating agents, CO and O2 failed to inhibit enzyme activity. The oxidized hydrogenase showed an EPR spectrum with a small signal at g = 2.02. On reduction the appearance of a high temperature (50--77 K) signal at g = 2.04, 1.95 and a more complex low temperature (less than 30 K) spectrum at g = 2.04, 2.0, 1.95, 1.93, 1.86 was observed. The pronounced temperature dependence and characteristic lineshape of the signals obtained with hydrogenase in 80--85% dimethylsulphoxide demonstrated that iron-sulphur centres of both the [2Fe-2S] and [4Fe-4S] types are present in the enzyme. Quantitation of the EPR signals indicated the existence of two identical centres each of the [4Fe-4S] and of the [2Fe-2S] type. The midpoint redox potentials of the [4Fe-4S] and the [2Fe-2S] centres were determined to be -445 mV and -325 mV, respectively. Spin coupling between two centres, indicated by the split feature of the low temperature spectrum of the native hydrogenase around g = 1.95, 1.93, has been established by power saturation studies. On reduction of the [Fe-4S] centres, the electron spin relaxation rate of the [2Fe-2S] centres was considerably increased. Treatment of hydrogenase with CO caused no change in EPR spectra.

Alcaligenes↗
PubMed1979

Purification of the membrane-bound hydrogenase of Escherichia coli.

The membrane-bound hydrogenase (EC class 1.12) of aerobically grown Escherichia coli cells was solubilized by treatment with deoxycholate and pancreatin. The enzyme was further purified to electrophoretic homogeneity by chromoatographic methods, including hydrophobic-interaction chromatography, with a yield of 10% as judged by activity and an overall purification of 2140-fold. The hydrogenase was a dimer of identical subunits with a mol.wt. of 113,000 and contained 12 iron and 12 acid-labile sulphur atoms per molecule. The epsilon 400 was 49,000M-1 . cm-1. The hydrogenase catalysed both H2 evolution and H2 uptake with a variety of artificial electron carriers, but would not interact with flavodoxin, ferredoxin or nicotinamide and flavin nucleotides. We were unable to identify any physiological electron carrier for the hydrogenase. With Methyl Viologen as the electron carrier, the pH optimum for H2 evolution and H2 uptake was 6.5 and 8.5 respectively. The enzyme was stable for long periods at neutral pH, low temperatures and under anaerobic conditions. The half-life of the hydrogenase under air at room temperature was about 12 h, but it could be stabilized by Methyl Viologen and Benzyl Viologen, both of which are electron carriers for the enzyme, and by bovine serum albumin. The hydrogenase was strongly inhibited by carbon monoxide (Ki = 1870Pa), heavy-metal salts and high concentrations of buffers, but was resistant to inhibition by thiol-blocking and metal-complexing reagents. These aerobically grown E. coli cells lacked formate hydrogenlyase activity and cytochrome c552.

Carbon Monoxide↗
PubMed1979

Hydrodynamic parameters of the detergent-solubilised hydrogenase from Paracoccus denitrificans.

The hydrogenase from Paracoccus denitrificans, which is an intrinsic membrane protein, has been solubilised from membranes by Triton X-100. The partial specific volume of the solubilised protein has been determined using sucrose density gradient centrifugation in H2O and 2H2O. The values of the specific volumes of hydrogenase, measured in the presence or absence of Triton X-100, are 0.73 and 0.74 ml . g-1, respectively, indicating that hydrogenase binds much less than one micelle of Triton X-100. The sedimentation coefficient of hydrogenase is increased from 10.4 S to 15.9 S on removal of detergent. The Stokes' radius of hydrogenase, determined by gel filtration on Sepharose 6B, is 5.5 nm in the presence of Triton X-100 compared to 6.7 nm in the absence of detergent. The apparent molecular weight therefore increases from 242,500 to 466,000 on removal of detergent. In the presence of urea and sodium dodecylsulphate, the hydrogenase has an apparent molecular weight of 63,000. The enzyme therefore behaves as a non-covalently linked tetramer in the presence of Triton X-100. Removal of Triton X-100 results in association of tetramers to form octamers.

Cell Membrane↗
PubMed1979

Solubilization and properties of a particulate hydrogenase from Methanobacterium strain G2R.

Mechanical disruption of cells of Methanobacterium strain G2R resulted in a 78-fold increase in the specific activity of the hydrogenase as measured by the benzyl viologen reduction assay. Approximately 50% of the activity in disrupted cells was associated with the particulate fraction. Between 69 and 85% of the particulate hydrogenase was released by treatment with the detergents Triton X-100, deoxycholate, and octyl-beta-d-glucopyranoside. The relative electrophoretic mobilities of the soluble hydrogenases were identical, indicating that G2R possessed a single electrophoretically distinct hydrogenase. The particulate enzyme was inactivated by oxygen and could be reactivated with dithionite or glucose plus glucose oxidase. The enzyme had a pH optimum of 8.5 and resisted heating at 52 but not 77 degrees C. A number of nonspecific dyes, flavin adenine dinucleotide, and riboflavin 5'-phosphate were effective electron acceptors; oxidized nicotinamide adenine dinucleotide, nicotinamide adenine dinucleotide phosphate, and factor 420 were apparently not reduced. Hydrogenase activity was inhibited by p-hydroxymercuribenzoate, cyanide, chloroform, and chloramphenicol. The molecular weight of the solubilized enzyme was 900,000, with subunits of molecular weights 38,500, 50,700, and approximately 80,000. It is suggested that, in intact cells of G2R, the large hydrogenase complex is loosely bound to the cell wall or membrane.

Cell Fractionation↗
PubMed1979

Hydrogenase activity in the dry state: isotope exchange and reversible oxidoreduction of cytochrome c3.

Hydrogenase (hydrogen:ferricytochrome c3 oxidoreductase, EC 1.12.2.1) catalyzes three types of reactions, i.e., (1) conversion between hydrogen modifications, para-H2 and ortho-H2, (2) exchange reaction between hydrogen isotopes, and (3) reversible oxidoreduction of an electron carrier with H2 and protons. We observed that purified desulfovibrio hydrogenase in the dry state could catalyze not only the conversion and exchange reactions (Yagi, T., Tsuda, M., Mori, Y. and Inokuchi, H. (1969) J. Am. Chem. Soc. 91, 2801) but also the reversible oxidoreduction of the electron carrier, cytochrome c3 with H2. The rate of the conversion was in the range from 0.1 to 0.65 mol H2 converted per mol hydrogenase per s, and the ratio of the conversion rate to the exchange rate was near 5. The rate of oxidoreduction of cytochrome c3 in the dry state was 0.015 mol H2 taken up in the forward reaction and 0.003 mol H2 released in the reverse reaction per mol hydrogenase per s. The process of these reactions could be explained by the observations that the hydrogenase molecule in the dry state has protons which are directly exchangeable with H2 during catalytic process. The reversible oxidoreduction of cytochrome c3 is also explained by inter- and intramolecular electron transfer among cytochrome c3 molecules.

Cytochrome c Group↗
PubMed1975

Structural and catalytic properties of hydrogenase from Chromatium.

The enzyme hydrogenase, from the photosynthetic bacterium Chromatium, was purified to homogeneity after solubilization of the particulate enzyme with deoxycholate. The purification procedure included ammonium sulfate fractionation, treatment with manganous phosphate gel, heating at 63 degrees, DEAE-cellulose chromatography, and isoelectric focusing. The last step gave two active enzyme fractions with isoelectric points of 4.2 and 4.4. It was shown that the two fractions were different forms of the same protein. The enzyme was obtained in 23% yield and was purified 1700-fold. The enzyme had a molecular weight of 98,000, a sedimentation coefficient of 5.16 S and gave a single protein and activity band on disc gel electrophoresis. Sodium dodecyl sulfate gel electrophoresis gave a single band of mol wt 50,000, suggesting that the active enzyme was composed of two subunits of the same molecular weight. The pure hydrogenase contained four atoms of iron and four atoms of acid-labile sulfide, and had a visible absorption peak at 410 nm. Electron paramagnetic resonance (EPR) spectroscopy at 10--15 K showed a free-radical signal at g' = 2.003 in the oxidized enzyme and signals at g' = 2.2 and 2.06 in the reduced enzyme. These findings suggest that Chromatium hydrogenase is an iron-sulfur protein. The pure hydrogenase catalyzed the exchange reaction between H2 and HDO or HTO, the reduction of Benzyl Viologen and Methylene Blue, and the evolution of hydrogen from reduced Methyl Viologen. The mechanism of hydrogen activation was shown to be heterolytic cleavage to an enzyme hydride and a proton. Hydrogenase could not catalyze reduction of pyridine nucleotides or ferredoxin with H2. The effect of pH and various inhibitors on the enzymatic activity has been studied.

Amino Acids↗
PubMed1979

Measurement in vivo of hydrogenase-catalysed hydrogen evolution in the presence of nitrogenase enzyme in cyanobacteria.

A method was devised that allows measurement in vivo of hydrogenase-catalysed H2 evolution from the cyanobacterium Anabaena cylindrica, independent of nitrogenase activity, which is also present. Addition of low concentrations of reduced Methyl Viologen (1-10mM) to intact heterocystous filaments of the organism resulted in H2 evolution, but produced conditions giving total inhibition of nitrogenase (acetylene-reducing and H2-evolving) activity. That the H2 formed under these conditions was not contributed to by nitrogenase was also supported by the observation that its rate of formation was similar in the dark or with Ar replaced by N2 in the gas phase, and also in view of the pattern of H2 evolution at very low Methyl Viologen concentrations. Conclusive evidence that the H2 formed in the presence of Methyl Viologen was solely hydrogenase-mediated was its evolution even from nitrogenase-free (non-heterocystous) cultures; by contrast 'uptake' hydrogenase activity in such cultures was greatly decreased. The hydrogenase activity was inhibited by CO and little affected by acetylene. Finally the hydrogenase activity was shown to be relatively constant at different stages during the batch growth of the organism, as opposed to nitrogenase activity, which varied.

Acetylene↗
PubMed1978

Hydrogen-dependent growth of Escherichia coli in anaerobic respiration and the presence of hydrogenases with different functions.

E. coli K10 was found to grow anaerobically on molecular hydrogen by reducing nitrate, fumarate, and trimethylamine N-oxide when peptone was added to the culture medium. Molar growth yields based on consumed hydrogen estimated from the amounts of reduction products were all 7.8 g cells/mol, suggesting that 1 mol of ATP was produced in the oxidation of 1 mol of hydrogen. Hydrogenase activity measured in terms of hydrogen evolution was several times higher in cells grown on glucose than in cells grown on hydrogen in the presence of fumarate and trimethylamine N-oxide, while hydrogenase activity measured in terms of hydrogen uptake was unchanged in both cases. The ratio of hydrogenase activities measured in terms of hydrogen uptake and evolution was also high in the extract and centrifugal fractions from cells grown in hydrogen. The soluble fraction and trypsin digest of the precipitate at 100,000 X g were subjected to polyacrylamide disc gel electrophoresis and hydrogenase bands were stained by reduction of benzyl viologen with hydrogen and by oxidation of reduced methyl viologen. The resulting patterns suggest that multiple forms of hydrogenase are present and that the amounts of forms functioning in hydrogen evolution were greatly decresed in cells grown on hydrogen in the presence of acceptors.

Anaerobiosis↗
PubMed1979

The interaction of polymeric viologens with hydrogenases from Desulfovibrio desulfuricans and Clostridium pasteurianum.

The interaction between hydrogenases from either Desulfovibrio desulfuricans or Clostridium pasteurianum and electron donors methyl viologen or polymeric viologens was examined. Extracts from each organism contained a single gel electophoretic band of active hydrogenase. The hydrogenase of D. desulfuricans was much more stable than that of Cl. pasteurianum. With methyl viologen apparent Km and Vm values were 0.5 mM and 0.62 mumole H2/min per milligram protein for the Cl. pasteurianum and 0.7 and 6.2 mumole H2/min per milligram protein, respectively, for the D. desulfuricans enzyme. The hydrogenases bound the polymeric viologens more tightly than methyl viologen, more so for the enzyme of D. desulfuricans than for Cl. pasteurianum. Maximal rate of hydrogen production was less with the polymeric than with methyl viologen. The results suggest that the D. desulfuricans enzyme in conjunction wiion than that from Cl. pasteurianum.

Clostridium↗
PubMed1979

Purification and properties of the membrane-bound hydrogenase from Proteus mirabilis.

The cytoplasmic membrane-bound hydrogenase of the facultative anaerobe, Proteus mirabilis, has been solubilized and purified to homogeneity. The purified enzyme exhibited a maximal specific activity of about 780 mumol H2 oxidized/min per mg protein (benzyl viologen reduction). The hydrogenase has a molecular weight of 205 000 and is composed of two subunits with a molecular weight of 63 000 and two of 33 000. The absorption spectrum of the enzyme was characteristic of non-heme iron proteins. The millimolar extinction coefficients at 400 and 280 nm are 106 and 390, respectively. The hydrogenase has about 24 iron atoms and 24 acid-labile sulfide atoms/molecule. Amino acid analyses revealed the presence of 39 half-cystine residues/molecule and a preponderance of acidic amino acids. The hydrogenase in its oxidized form exhibits an EPR signal of the HiPIP-type with g values at 2.025 and 2.018. Upon reduction with either dithionite or H2 the signal disappears; no other signals were detectable.

Amino Acids↗
PubMed1978

Relationships in hydrogen metabolism between hydrogenase and nitrogenase in phototrophic bacteria.

Purple bacteria Rhodospirillum rubrum and Thiocapsa roseopersicina form two enzymes, hydrogenase and nitrogenase, which participate in hydrogen metabolism. H2 photoproduction in these bacteria is associated mainly or completely with the action of nitrogenase. The soluble and membrane-bound hydrogenases of T. roseopersicina have similar physicochemical properties (mol. weight, subunit composition, N-terminal amino acids, Fe2+ and S2- content, pl. Eo'). In comparison with other hydrogenases the enzyme from R. rubrum and T. roseopersicina evolve H2 with high rate from reduced cytochrome c3, but not from ferredoxins. H2 production and N2 fixation take place in the presence of NAD(P)H. NADP-reductase, ferredoxin and cytochrome c3 participate in this reaction. Possible relationships between hydrogenase-nitrogenase in the metabolism of molecular hydrogen are discussed.

Bacteria↗
PubMedDate not supplied

[Kinetic patterns of bacterial hydrogenase inactivation].

Kinetics of inactivation of hydrogenases is exemplified with the enzyme from Chloropseudomonas ethylica. The effect of gaseous phase, temperature, pH on the inactivation kinetics has been studied. Inactivation of the hydrogenase during incubation of an enzyme solution in air has been studied. Analysis of the kinetic data allowed the conclusion to be made that inactivation involves two forms of the enzyme which differ in their activity and resistance to inactivating actions. Stabilities of the mean time of operation of hydrogenases from Chloropseudomonas ethylica and Thiocapsa roseopersicina are compared. The mechanism of inactivation of hydrogenases from the two sources were found to be similar in many ways.

Air↗
PubMed1975

A new assay method for hydrogenase based on an enzymic electrode reaction. The enzymic electric cell method.

A new assay method for hydrogenase [EC 1.12.2.1] based on the enzymic electrode reaction of H2-H+ equilibrium has been established. The method is based on the experimental fact that the short-circuit current of the electric cell composed of an electrode with hydrogenase and methylviologen as the mediator of H2-H+ equilibrium and a saturated calomel electrode as the counter electrode, is practically proportional to the amount of hydrogenase in the cell. The new method is referred to as the "enzymic electric cell method." This technique has applications not only to routine activity assay but also to the direct determination of the time course of enzyme denaturation, which has not previously been possible.

Cytochrome c Group↗