Membrane-bound F420H2-dependent heterodisulfide reduction in Methanococcus voltae

Abstract

Washed membranes prepared from H₂+CO₂- or formate-grown cells of Methanococcus voltae catalyzed the oxidation of coenzyme F₄₂₀H₂ and the reduction of the heterodisulfide (CoB–S–S–CoM) of 2-mercaptoethanesulfonate and 7-mercaptoheptanoylthreonine phosphate, which is the terminal electron acceptor of the methanogenic pathway. The reaction followed a 1:1 stoichiometry according to the equation: F₄₂₀H₂ + COB–S–S–CoM → F₄₂₀ + CoM–SH + CoB–SH. These findings indicate that the reaction depends on a membrane-bound F₄₂₀H₂-oxidizing enzyme and on the heterodisulfide reductase, which remains partly membrane-bound after cell lysis. To elucidate the nature of the F₄₂₀H₂-oxidizing protein, washed membranes were solubilized with detergent, and the enzyme was purified by sucrose density centrifugation, anion-exchange chromatography, and gel filtration. Several lines of evidence indicate that F₄₂₀H₂ oxidation is catalyzed by a membrane-associated F₄₂₀-reducing hydrogenase. The purified protein catalyzed the H₂-dependent reduction of methyl viologen and F₄₂₀. The apparent molecular mass and the subunit composition (43, 37, and 27 kDa) are almost identical to those of the F₄₂₀-reducing hydrogenase that has already been purified from Mc. voltae. Moreover, the N-terminus of the 37-kDa subunit is identical to the amino acid sequence deduced from the fruG gene of the operon encoding the selenium-containing F₄₂₀-reducing hydrogenase from Mc. voltae. A distinct F₄₂₀H₂ dehydrogenase, which is present in methylotrophic methanogens, was not found in this organism.