Genetic analysis of the archaeon Methanosarcina barkeri Fusaro reveals a central role for Ech hydrogenase and ferredoxin in methanogenesis and carbon fixation - PubMed (original) (raw)

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

Jörn Meuer et al. Proc Natl Acad Sci U S A. 2002.

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.

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Figures

Figure 1

Construction and verification of an M. barkeri_Δ_echABCDEF mutant. (A) Ech1 was constructed by transformation of M. barkeri to PurR with linearized plasmid carrying the Δ_ech1_∷pac-ori-aph mutation. Recombination (dotted lines) between homologous sequences on the plasmid and chromosome resulted in replacement of the_ech_ operon with the mutation. A small fragment of_echF_ remains in the mutant. (B) Mutant (Ech1) and wild-type (WT) chromosome structure was verified by DNA hybridization as described. The predicted sizes of hybridizing bands (base pairs) are shown in parentheses: lanes 1 and 8, DNA markers; lane 2, Ech1-_Hin_dIII (1938, 2018); lane 3, WT-_Hin_dIII (2011, 4540); lane 4, Ech1-_Eco_RV (3076, 3984); lane 5, WT-_Eco_RV (2132, 3551); lane 6, Ech1-_Eco_RI (3912, >4000); lane 7, WT-_Eco_RI (2283, >6526). (C) EchE and HdrD were detected by Western blot as described (8). Lane 1, 5 μg of Ech1 cell extract; lane 2, 5 μg of Ech1 membrane protein; lane 3, 0.1 μg of purified Hdr (control); lane 4, 0.1 μg of purified Ech (control); lane 5, 5 μg of WT cell extract; lane 6, 5 μg of WT membrane protein. The molecular mass of subunit EchE is about 39 kDa, the molecular mass of HdrD is 43 kDa. The migration of molecular mass standards is shown on the left.

Figure 2

Ferredoxin-dependent formation of H2 and CO2 from CHO-MFR. Freshly prepared membrane fractions of methanol-grown M. barkeri were assayed for H2 and CO2 production as described. Ferredoxin was added at the time point indicated. The rate of H2 and CO2 formation was 200 nmol min−1 (mg membrane protein)−1.

Figure 3

Proposed functional roles for Ech and ferredoxin during growth of_M. barkeri_ on various substrates. Models for growth on methanol plus H2/CO2 (A), acetate (B), H2/CO2(C), and methanol (D) are shown. The methanogenic pathway is shown in blue, the energy-conserving electron transport chain in red, and biosynthetic pathway in purple. The steps catalyzed by Ech and ferredoxin-dependent enzymes are labeled in green. See text for detailed discussion.

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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 - PubMed (original) (raw)