PURIFICATION AND CHARACTERIZATION OF F420H2-DEHYDROGENASE FROM METHANOLOBUS-TINDARIUS

The oxidation of F420H2 (reduced coenzyme F420) is a key reaction in the final step of methanogenesis. This step is catalyzed in Methanolobus tindarius by the membrane-bound F420H2-dehydrogenase which was purified 31-fold to apparent homogeneity. The apparent molecular mass of the native enzyme was 120 kDa. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis revealed the presence of five different subunits of apparent molecular masses of 45 kDa, 40 kDa, 22 kDa, 18 kDa and 17 kDa. The purified F420H2-dehydrogenase, which was yellowish, contained 16 +/- 2 mol iron and 16 +/- 3 mol acid-labile sulfur/mol enzyme. No flavin could be detected. The oxygen-stable enzyme catalyzed the oxidation of F420H2 (apparent K(m) = 5.4-mu-M) with methylviologen and metronidazole as electron acceptors at a specific rate of 13-mu-mol . min-1 . mg-1 (k(cat) = 25.5 s-1). The isoelectric point was at pH 5.0. The temperature optimum was at 37-degrees-C and the pH optimum at 6.8.