Purification and characterization of membrane-bound hydrogenase from Methanosarcina barkeri MS |
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Authors: | John M Kemner J Gregory Zeikus |
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Institution: | (1) Department of Microbiology and Public Health, Michigan State University, 48824 East Lansing, MI, USA;(2) Department of Biochemistry, Michigan State University, 48824 East Lansing, MI, USA;(3) Present address: Department of Microbiology SC-42, University of Washington, 98195 Seattle, WA, USA;(4) Present address: Michigan Biotechnology Institute, 3900 Collins Road, 48910 Lansing, MI, USA |
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Abstract: | Hydrogenase was solubilized from the membrane of acetate-grown Methanosarcina barkeri MS and purification was carried out under aerobic conditions. The enzyme was reactivated under reducing conditions in the presence of H2. The enzyme showed a maximal activity of 120±40 mol H2 oxidized · min–1 · min–1 with methyl viologen as an electron acceptor, a maximal hydrogen production rate of 45±4 mol H2 · min–1 · mg–1 with methyl viologen as electron donor, and an apparent K
m for hydrogen oxidation of 5.6±1.7 M. The molecular weight estimated by gel filtration was 98,000. SDS-PAGE showed the enzyme to consist of two polypeptides of 57,000 and 35,000 present in a 1:1 ratio. The native protein contained 8±2 mol Fe, 8±2 mol S2–, and 0.5 mol Ni/mol enzyme. Cytochrome b was reduced by hydrogen in a solubilized membrane preparation. The hydrogenase did not couple with autologous F420 or ferredoxin, nor with FAD, FMN, or NAD(P)+. The physiological function of the membrane-bound hydrogenase in hydrogen consumption is discussed.Abbreviation CoM-S-S-HTP
the heterodisulfide of 7-mercaptoheptanoylthrconine phosphate and coenzyme M (mercaptoethanesulfonic acid) |
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Keywords: | Methanosarcina barkeri Hydrogenase — Membrane bound F420 nonreactive Cytochrome b Hydrogen uptake |
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