title: Re-evaluation of the function of the F420 dehydrogenase in electron transport of Methanosarcina mazei.
abstract: 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 μM) 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.
authors.affiliation: Institute of Microbiology and Biotechnology, University of Bonn, Bonn, Germany.
authors.forename: Cornelia
authors.initials: C
authors.lastname: Welte
authors.forename: Uwe
authors.initials: U
authors.lastname: Deppenmeier
mesh_terms: D004579:Electron Transport / Q000502:physiology*; D017020:Methanosarcina / Q000201:enzymology* / Q000235:genetics; D010088:Oxidoreductases / Q000378:metabolism*
chemical_list: C072742:F420H2 dehydrogenase; D010088:Oxidoreductases; C400203:Ech hydrogenase