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

摘要

Ech hydrogenase (Ech) from the methanogenic archaeonMethanosarcina barkeri catalyzes the reversiblereduction of ferredoxin by H2 and is a member of a distinctgroup of membrane-bound [NiFe] hydrogenases with sequence similarityto energy-conserving NADH:quinone oxidoreductase (complex I). Toelucidate the physiological role(s) of Ech a mutant lacking thisenzyme was constructed. The mutant was unable to grow onmethanol/H2/CO2,H2/CO2, or acetate as carbon and energysources but showed wild-type growth rates with methanol as solesubstrate. Addition of pyruvate to the growth medium restored growth onmethanol/H2/CO2 but not onH2/CO2 or acetate. Results obtained fromgrowth experiments, cell suspension experiments, and enzyme activitymeasurements in cell extracts provide compelling evidence for essentialfunctions of Ech and a 2[4Fe-4S] ferredoxin in the metabolism ofM. barkeri. The following conclusions were made.(i) In acetoclastic methanogenesis, Ech catalyzesH2 formation from reduced ferredoxin, generated by theoxidation of the carbonyl group of acetate to CO2.(ii) Under autotrophic growth conditions, the enzymecatalyzes the energetically unfavorable reduction of ferredoxin byH2, most probably driven by reversed electron transport,and the reduced ferredoxin thus generated functions as low potentialelectron donor for the synthesis of pyruvate in an anabolic pathway.(iii) Reduced ferredoxin in addition provides thereducing equivalents for the first step of methanogenesis fromH2/CO2, the reduction of CO2 toformylmethanofuran. Thus, in vivo genetic analysis hasled to the identification of the electron donor of this keyinitial step of methanogenesis.