Solubilization and Properties of a Particulate Hydrogenase from
            <i>Methanobacterium</i>
            Strain G2R

McKellar, Robert; Sprott, G. Dennis

doi:10.1128/jb.139.1.231-238.1979

Cited by 43 publications

(11 citation statements)

References 46 publications

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“…The present paper is the first report of the production of superoxide radicals by a bacterial hydrogenase, although all hydrogenases so far studied contain iron-sulphur centres (see , and some of them, the membranebound hydrogenases of Chromatium (Kakuno et al, 1977), A. eutrophus (Schink & Schlegel, 1979) and Methanobacterium strain G2R (McKellar & Sprott, 1979), were found to be unstable under reducing conditions or inhibited by reductants respectively.…”

Section: Discussionmentioning

confidence: 81%

Production of superoxide radicals by soluble hydrogenase from Alcaligenes eutrophus H16

Schneider¹,

Schlegel²

1981

Biochemical Journal

120

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The soluble hydrogenase (hydrogen-NAD+ oxidoreductase, EC 1.12.1.2) of Alcaligenes eutrophus H16 was shown to be stabilized by oxidation with oxygen and ferricyanide as long as electron donors and reducing compounds were absent. The simultaneous presence of H2, NADH and O2 in the enzyme solution, however, caused an irreversible inactivation of hydrogenase that was dependent on the O2 concentration. The half-life periods of 4 degrees C under partial pressures of 0.1, 5, 20 and 50% O2 were 11, 5, 2.5 and 1.5 h respectively. Evidence has been obtained that hydrogenase produces superoxide free radical anions (O2-.), which were detected by their ability to oxidize hydroxylamine to nitrite. The correlation between O2 concentration, nitrite formation and inactivation rates and the stabilization of hydrogenase by addition of superoxide dismutase indicated that superoxide radicals are responsible for enzyme inactivation. During short-term activity measurements (NAD+ reduction, H2 evolution from NADH), hydrogenase activity was inhibited by O2 only very slightly. In the presence of 0.7 mM-O2 an inhibition of about 20% was observed.

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Section: Discussionmentioning

confidence: 81%

Production of superoxide radicals by soluble hydrogenase from Alcaligenes eutrophus H16

Schneider¹,

Schlegel²

1981

Biochemical Journal

120

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“…The reason for the specific enhancement of methanogenesis from acetate by pronase is not clear, but selective effects of proteases have been reported for membrane-bound proteins (5,12,25). In Methanobacterium ruminantium and M. thermoaiatotrophiciam CH3-S-CoM methylreductase (22,23), hydrogenase (19), and the hydrogenase-ATP synthetase complex (8) are closely associated with the particulate fraction. In this study the aceticlastic components were also associated with the particulate fraction.…”

Section: Discussionmentioning

confidence: 99%

Methanogenic cleavage of acetate by lysates of Methanosarcina barkeri

Baresi¹

1984

J Bacteriol

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Cell lysates of acetate-grown Methanosarcina barkeri 227 were found to cleave acetate to CH4 and CO2. The aceticlastic reaction was identified by using radioactive methyl-labeled acetate. Cell lysates decarboxylated acetate in a nitrogen atmosphere, conserving the methyl group in methane. The rate of methanogenesis from acetate in the cell lysates was comparable to that observed with whole cells. Aceticlastic activity was found in the particulate fraction seperate from methylcoenzyme M methylreductase activity, which occurs in the soluble fraction. Pronase treatment eliminated methylcoenzyme M methylreductase activity in lysates and stimulated aceticlastic activity, indicating the aceticlastic activity was not derived from unbroken cells, which are unaffected by proteolytic treatment.for the gift of CH3-S-CoM. I also thank M. Smith, D. Boone, and J. G. Ferry for constructive comments on the manuscript.

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“…One unit of activity was defined as 2 ,umol of methyl viologen reduced (equivalent to 1 ,umol of H2 oxidized) per min. The same definition was utilized with benzyl viologen (8578 = 8.65 mM-1 cm-1) (26). The concentrations of H2 and CO in the liquid phase of reaction mixtures were calculated from the standard solubility tables of Linke (20).…”

Section: Methodsmentioning

confidence: 99%

Demonstration of hydrogenase in extracts of the homoacetate-fermenting bacterium Clostridium thermoaceticum

Drake¹

1982

J Bacteriol

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Cell-free extracts of the homoacetate-fermenting bacterium Clostridium thermoaceticum were shown to catalyze the hydrogen-dependent reduction of various artificial electron acceptors. The activity of the hydrogenase was optimal at pH 8.5 to 9 and was extremely sensitive to,aeration. EDTA did not significantly reduce the lability of the enzymic activity to oxidation (aeration). At 50°C, when both methyl viologen and hydrogen were at saturating concentrations with respect to hydrogenase, the specific activity of cell-free extracts approximated 4 p,mol of H2 oxidized per min per mg of protein; fourfold higher specific activities were obtained when benzyl viologen was utilized as an electron acceptor. Activity stains of polyacrylamide gels demonstrated the presence of a single hydrogenase band, suggesting that the catalytic activity in cell extracts was due to a single enzyme. The activity was stable for at least 32 min at 55°C but was slowly inactivated at 700C. NAD, NADP, flavin adenine dinucleotide, flavin mononucleotide, and ferredoxin were not significantly reduced, but possible reduction of the particulate b-type cyctochrome of C. thermoaceticum was observed. NaCl, sodium dodecyl sulfate, iodoacetamide, and CO were shown to inhibit catalysis. A kinetic study is presented, and the possible physiological roles for hydrogenase in C. thermoaceticum are discussed.

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Solubilization and Properties of a Particulate Hydrogenase from Methanobacterium Strain G2R

Cited by 43 publications

References 46 publications

Production of superoxide radicals by soluble hydrogenase from Alcaligenes eutrophus H16

Production of superoxide radicals by soluble hydrogenase from Alcaligenes eutrophus H16

Methanogenic cleavage of acetate by lysates of Methanosarcina barkeri

Demonstration of hydrogenase in extracts of the homoacetate-fermenting bacterium Clostridium thermoaceticum

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