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Purification and characterization of membrane-bound hydrogenase from Methanosarcina barkeri MS

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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 μmol H2 oxidized · min−1 · min−1 with methyl viologen as an electron acceptor, a maximal hydrogen production rate of 45±4 μmol H2 · min−1 · mg−1 with methyl viologen as electron donor, and an apparent K m for hydrogen oxidation of 5.6±1.7 μM. 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.

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Abbreviations

CoM-S-S-HTP:

the heterodisulfide of 7-mercaptoheptanoylthrconine phosphate and coenzyme M (mercaptoethanesulfonic acid)

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Author notes

  1. John M. Kemner

    Present address: Department of Microbiology SC-42, University of Washington, 98195, Seattle, WA, USA

  2. J. Gregory Zeikus

    Present address: Michigan Biotechnology Institute, 3900 Collins Road, 48910, Lansing, MI, USA

Authors and Affiliations

  1. Department of Microbiology and Public Health, Michigan State University, 48824, East Lansing, MI, USA

    John M. Kemner & J. Gregory Zeikus

  2. Department of Biochemistry, Michigan State University, 48824, East Lansing, MI, USA

    J. Gregory Zeikus

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Kemner, J.M., Zeikus, J.G. Purification and characterization of membrane-bound hydrogenase from Methanosarcina barkeri MS. Arch. Microbiol. 161, 47–54 (1994). https://doi.org/10.1007/BF00248892

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  • DOI: https://doi.org/10.1007/BF00248892

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