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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 H(2) 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/H(2)/CO(2), H(2)/CO(2), 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/H(2)/CO(2) but not on H(2)/CO(2) 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 H(2) formation from reduced ferredoxin, generated by the oxidation of the carbonyl group of acetate to CO(2). (ii) Under autotrophic growth conditions, the enzyme catalyzes the energetically unfavorable reduction of ferredoxin by H(2), 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 H(2)/CO(2), the reduction of CO(2) to formylmethanofuran. Thus, in vivo genetic analysis has led to the identification of the electron donor of this key initial step of methanogenesis.

SUBMITTER: Meuer J

PROVIDER: S-EPMC122822 | biostudies-literature | 2002 Apr

REPOSITORIES: biostudies-literature

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

Meuer Jörn J Kuettner H Craig HC Zhang Jun Kai JK Hedderich Reiner R Metcalf William W WW

Proceedings of the National Academy of Sciences of the United States of America 20020402 8

Ech hydrogenase (Ech) from the methanogenic archaeon Methanosarcina barkeri catalyzes the reversible reduction of ferredoxin by H(2) 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/H(2)/CO(2), H(2)/CO(2), or acetate as carbon and energy sources but show ...[more]

PMID: 11929975

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Project description:The methanogenic archaeon Methanosarcina barkeri encodes three distinct types of hydrogenase, whose functions vary depending on the growth substrate. These include the F420-dependent (Frh), methanophenazine-dependent (Vht), and ferredoxin-dependent (Ech) hydrogenases. To investigate their physiological roles, we characterized a series of mutants lacking each hydrogenase in various combinations. Mutants lacking Frh, Vht, or Ech in any combination failed to grow on H2-CO2, whereas only Vht and Ech were essential for growth on acetate. In contrast, a mutant lacking all three grew on methanol with a final growth yield similar to that of the wild type and produced methane and CO2 in the expected 3:1 ratio but had a ca. 33% lower growth rate. Thus, hydrogenases play a significant, but nonessential, role during growth on this substrate. As previously observed, mutants lacking Ech failed to grow on methanol-H2 unless they were supplemented with biosynthetic precursors. Interestingly, this phenotype was abolished in the ?ech ?frh and ?ech ?frh ?vht mutants, consistent with the idea that hydrogenases inhibit methanol oxidation in the presence of H2, which prevents production of the reducing equivalents needed for biosynthesis. Quantification of the methane and CO2 produced from methanol by resting cell suspensions of various mutants supported this conclusion. On the basis of the global transcriptional profiles, none of the hydrogenases were upregulated to compensate for the loss of the others. However, the transcript levels of the F420 dehydrogenase operon were significantly higher in all strains lacking frh, suggesting a mechanism to sense the redox state of F420 The roles of the hydrogenases in energy conservation during growth with each methanogenic pathway are discussed.IMPORTANCE Methanogenic archaea are key players in the global carbon cycle due to their ability to facilitate the remineralization of organic substrates in many anaerobic environments. The consequences of biological methanogenesis are far-reaching, with impacts on atmospheric methane and CO2 concentrations, agriculture, energy production, waste treatment, and human health. The data presented here clarify the in vivo function of hydrogenases during methanogenesis, which in turn deepens our understanding of this unique form of metabolism. This knowledge is critical for a variety of important issues ranging from atmospheric composition to human health.

Dataset Information

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

Publications

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

Similar Datasets

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