Open AccessPurification and characterization of F 420 H 2 ‐dehydrogenase from Methanolobus tindarius
Author(s) -
HAASE Peter,
DEPPENMEIER Uwe,
BLAUT Michael,
GOTTSCHALK Gerhard
Publication year - 1992
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1992.tb16579.x
Subject(s) - isoelectric point , chemistry , molecular mass , flavin group , enzyme , enzyme kinetics , sodium dodecyl sulfate , gel electrophoresis , polyacrylamide gel electrophoresis , dehydrogenase , isoelectric focusing , biochemistry , chromatography , active site
The oxidation of F 420 H 2 (reduced coenzyme F 420 ) is a key reaction in the final step of methanogenesis. This step is catalyzed in Methanolobus tindarius by the membrane‐bound F 420 H 2 ‐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 F 420 H 2 ‐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 F 420 H 2 (apparent K m = 5.4 μM) with methylviologen and metronidazole as electron acceptors at a specific rate of 13 μmol · min −1 ± min −1 ± mg −1 ( K cat = 25.5 s −1 ). The isoelectric point was at pH 5.0. The temperature optimum was at 37°C and the pH optimum at 6.8.