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Re‐evaluation of the function of the F 420 dehydrogenase in electron transport of Methanosarcina mazei
Author(s) -
Welte Cornelia,
Deppenmeier Uwe
Publication year - 2011
Publication title -
the febs journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 204
eISSN - 1742-4658
pISSN - 1742-464X
DOI - 10.1111/j.1742-4658.2011.08048.x
Subject(s) - oxidoreductase , hydrogenase , ferredoxin , chemistry , methanosarcina barkeri , electron transport chain , biochemistry , dehydrogenase , mutant , enzyme , redox , methanogenesis , inorganic chemistry , gene , organic chemistry , methane
Methanosarcina mazei is a methanogenic archaeon that is able to thrive on various substrates and therefore contains a variety of redox‐active proteins involved in both cytoplasmic and membrane‐bound electron transport. The organism possesses a complex branched respiratory chain that has the ability to utilize different electron donors. In this study, two knockout mutants of the membrane‐bound F 420 dehydrogenase (ΔfpoF and ΔfpoA‐O) were constructed and analyzed. They exhibited severe growth deficiencies with trimethylamine, but not with acetate, as substrates. In cell lysates of the fpo mutants, the F 420 :heterodisulfide oxidoreductase activity was strongly reduced, although soluble F 420 hydrogenase was still present. This led to the conclusion that the predominant part of cellular oxidation of the reduced form of F 420 (F 420 H 2 ) in Ms. mazei is performed by F 420 dehydrogenase. Enzyme assays of cytoplasmic fractions revealed that ferredoxin (Fd):F 420 oxidoreductase activity was essentially absent in the ΔfpoF mutant. Subsequently, FpoF was produced in Escherichia coli and purified for further characterization. The purified FpoF protein catalyzed the Fd:F 420 oxidoreductase reaction with high specificity (the K M for reduced Fd was 0.5 μ m ) but with low velocity ( V max = 225 mU·mg −1 ) and was present in the Ms. mazei cytoplasm in considerable amounts. Consequently, soluble FpoF might participate in electron carrier equilibrium and facilitate survival of the Ms. mazei Δech mutant that lacks the membrane‐bound Fd‐oxidizing Ech hydrogenase.