Purification and properties of an F420H2 dehydrogenase from Methanosarcina mazei Gö1

Abstract

Abstract The F420H2 dehydrogenase is part of the energy-conserving F420H2:heterodisulfide oxidoreductase system in Methanosarcina mazei Go1. The enzyme was purified 75-fold to apparent homogeneity from washed membranes. The molecular mass as determined by both native gel electrophoresis and gel filtration was 115 000. The purified enzyme was composed of five different polypeptides with molecular masses of 40, 37, 22, 20, and 16 kDa and contained 7 mol S2− and 7 mol Fe. The specific activity was 17 U mg protein−1 (apparent Vmax) using F420H2 as electron donor (Km=7 μM) and methylviologen and metronidazole as electron acceptors at pH 8.5 at a temperature of 39°C. The enzyme also catalyzed the reduction of 2,3-dimethyl-1,4-naphthoquinone, 2-methyl-1,4-naphthoquinone and tetramethyl-p-benzoquinone. The protein did not exhibit hydrogenase activity since F420 was not reduced by hydrogen and H2 production from F420H2 was not observed. In contrast to the F420H2 dehydrogenase from Archaeoglobus fulgidus, the enzyme from Methanosarcina mazei Go1 was similar to the corresponding protein of Methanolobus tindarius with respect to molecular mass and subunit composition. However, the proteins of the methanogenic organisms are different in cofactor content, since evidence is presented that the enzyme from Methanosarcina mazei Go1 contains FAD.