Abstract
BackgroundAcetate is the major source of methane in nature. The majority of investigations have focused on acetotrophic methanogens for which energy-conserving electron transport is dependent on the production and consumption of H2 as an intermediate, although the great majority of acetotrophs are unable to metabolize H2. The presence of cytochrome c and a complex (Ma-Rnf) homologous to the Rnf (Rhodobacter nitrogen fixation) complexes distributed in the domain Bacteria distinguishes non-H2-utilizing Methanosarcina acetivorans from H2-utilizing species suggesting fundamentally different electron transport pathways. Thus, the membrane-bound electron transport chain of acetate-grown M. acetivorans was investigated to advance a more complete understanding of acetotrophic methanogens.ResultsA component of the CO dehydrogenase/acetyl-CoA synthase (CdhAE) was partially purified and shown to reduce a ferredoxin purified using an assay coupling reduction of the ferredoxin to oxidation of CdhAE. Mass spectrometry analysis of the ferredoxin identified the encoding gene among annotations for nine ferredoxins encoded in the genome. Reduction of purified membranes from acetate-grown cells with ferredoxin lead to reduction of membrane-associated multi-heme cytochrome c that was re-oxidized by the addition of either the heterodisulfide of coenzyme M and coenzyme B (CoM-S-S-CoB) or 2-hydoxyphenazine, the soluble analog of methanophenazine (MP). Reduced 2-hydoxyphenazine was re-oxidized by membranes that was dependent on addition of CoM-S-S-CoB. A genomic analysis of Methanosarcina thermophila, a non-H2-utilizing acetotrophic methanogen, identified genes homologous to cytochrome c and the Ma-Rnf complex of M. acetivorans.ConclusionsThe results support roles for ferredoxin, cytochrome c and MP in the energy-conserving electron transport pathway of non-H2-utilizing acetotrophic methanogens. This is the first report of involvement of a cytochrome c in acetotrophic methanogenesis. The results suggest that diverse acetotrophic Methanosarcina species have evolved diverse membrane-bound electron transport pathways leading from ferredoxin and culminating with MP donating electrons to the heterodisulfide reductase (HdrDE) for reduction of CoM-S-S-CoB.
Highlights
Acetate is the major source of methane in nature
The analysis revealed that the product of MA0431 is closely related to the 2 × [4Fe-4S] ferredoxin purified from acetate-grown cells of M. thermophila [24,25,26,27] and the ferredoxin up-regulated in acetate- versus methanolgrown M. mazei [28]
The MP analog 2-hydroxyphenazine re-oxidized cytochrome c when added to membranes of acetate-grown cells previously reduced with ferredoxin (Figure 6). These results suggest that MP is either directly or indirectly linked to cytochrome c, a result further supporting the participation of MP and cytochrome c in the membrane-bound electron transport chain
Summary
Acetate is the major source of methane in nature. The majority of investigations have focused on acetotrophic methanogens for which energy-conserving electron transport is dependent on the production and consumption of H2 as an intermediate, the great majority of acetotrophs are unable to metabolize H2. Two genera of aceticlastic methanogens have been described, Methanosarcina and Methanosaeta [2] In both genera, the CO dehydrogenase/acetyl-CoA complex (Cdh) cleaves activated acetate into methyl and carbonyl groups. The proton gradient driving ATP synthesis is generated via a membrane-bound electron transport chain originating with oxidation of the carbonyl group of acetate by Cdh and terminating with reduction of CoM-S-S-CoB by Hdr. the pathway of carbon flow from the methyl group of acetate to methane is understood for both aceticlastic genera, the understanding of electron transport coupled to generation of the proton gradient is incomplete. The majority of investigations have focused on Methanosarcina barkeri and Methanosarcina mazei for which electron transport is dependent on the production and consumption of H2 as an intermediate, the great majority of Methanosarcina species [4] and all Methanosaeta species are unable to metabolize H2
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