Genetic analysis of the archaeon Methanosarcina barkeri Fusaro reveals a central role for Ech hydrogenase and ferredoxin in methanogenesis and carbon fixation

Abstract

Ech hydrogenase (Ech) from the methanogenic archaeon Methanosarcina barkeri catalyzes the reversible reduction of ferredoxin by H₂ and is a member of a distinct group of membrane-bound [NiFe] hydrogenases with sequence similarity to energy-conserving NADH:quinone oxidoreductase (complex I). To elucidate the physiological role(s) of Ech a mutant lacking this enzyme was constructed. The mutant was unable to grow on methanol/H₂/CO₂, H₂/CO₂, or acetate as carbon and energy sources but showed wild-type growth rates with methanol as sole substrate. Addition of pyruvate to the growth medium restored growth on methanol/H₂/CO₂ but not on H₂/CO₂ or acetate. Results obtained from growth experiments, cell suspension experiments, and enzyme activity measurements in cell extracts provide compelling evidence for essential functions of Ech and a 2[4Fe-4S] ferredoxin in the metabolism of M. barkeri. The following conclusions were made. (i) In acetoclastic methanogenesis, Ech catalyzes H₂ formation from reduced ferredoxin, generated by the oxidation of the carbonyl group of acetate to CO₂. (ii) Under autotrophic growth conditions, the enzyme catalyzes the energetically unfavorable reduction of ferredoxin by H₂, most probably driven by reversed electron transport, and the reduced ferredoxin thus generated functions as low potential electron donor for the synthesis of pyruvate in an anabolic pathway. (iii) Reduced ferredoxin in addition provides the reducing equivalents for the first step of methanogenesis from H₂/CO₂, the reduction of CO₂ to formylmethanofuran. Thus, in vivo genetic analysis has led to the identification of the electron donor of this key initial step of methanogenesis.