Electron transport in acetate-grown Methanosarcina acetivorans

Abstract

Background: 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, 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.

Results: A 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.

Conclusions: The 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.

Figure 1

Reduction of ferredoxin by CdhAE. The 70-μl reaction mixture consisted of 2.2 μg of CdhAE and 28 μM (final concentration) of ferredoxin contained in 50 mM MOPS buffer (pH 6.8) under 1 atm CO. The reaction was initiated with CdhAE. A, complete reaction mixture initial absorbance 0.61. B, reaction mixture minus CdhAE, initial absorbance 0.72. C, reaction mixture minus ferredoxin, initial absorbance 0.72. The reduction of ferredoxin was followed by the decrease in absorbance at 402 nm.

Figure 2

Ferredoxin:heterodisulfide oxidoreductase activity of membranes. The 100-μl reaction mixture consisted of 20 mM NADPH, 2 μg FNR (Sigma), the membrane fraction of acetate-grown cells (60 μg protein), 1.1 mM CoM-S-S-CoB and the indicated concentrations of ferredoxin contained in 50 mM MOPS buffer (pH 6.8). Total thiols were determined by the DTNB assay. Symbols: (filled triangles)1.2 μM ferredoxin, (filled circles) 0.6 μM ferredoxin, (filled squares) 0.3 μM ferredoxin, (open circles) minus ferredoxin.

Figure 3

Ferredoxin-dependent reduction of membrane-bound cytochrome c. The 100-μl reaction mixture consisted of purified membranes (300 μg protein), the indicated amount of ferredoxin, 1 μg FNR and 1 mM NADPH in 50 mM MOPS (pH 6.8). The reaction was initiated by addition of FNR (Sigma). The reduction of cytochrome c was followed at 554 nm. Panel A, time-course for the reduction of cytochrome c. Symbols: (filled squares) 4 μM ferredoxin; (open circles) 0.2 μM ferredoxin; (open squares) minus ferredoxin; (open triangles) minus FNR; (filled circles) minus NADPH. Panel B, reduced minus oxidized spectra recorded at the indicated times after initiation of the reaction containing 4 μM ferredoxin.

Figure 4

Oxidation of membrane-bound cytochrome c by CoM-S-S-CoB. The reduction of cytochrome c was performed as described in the caption to Figure 3. The 100-μl reaction mixture consisted of membranes (400 μg protein), 2 μM ferredoxin, 1 μg FNR and 1 mM NADPH. FNR was added at time zero and 0.32 mM (final concentration) CoM-S-S-CoB was added (arrow). Reduction and oxidation of cytochrome c was monitored by the absorbance at 554 nm.

Figure 5

Reduction of 2-hydroxyphenazine and re-oxidation dependent on membranes and CoM-S-S-CoB. The 100-μl reaction mixture consisted of membranes (107 μg protein), 4 μM ferredoxin, 100 μM 2-hydroxyphenazine and CdhAE (40 μg) in 50 mM MOPS (pH 6.8) under 1 atm CO. The reduction and oxidation of 2-hydroxyphenazine was followed by the absorbance at 475 nm (ε₄₇₅ = 2.5 mM^-1 cm^-1). CdhAE was added to initiate the reduction at time zero. At point A the cuvette was flushed with 100% N₂ and 2 μl of MOPS buffer (pH 6.8) was added. At points B and C, 2 μl of MOPS buffer (pH 6.8) containing CoM-S-S-CoB was added to the reaction reaching final concentrations of 240 and 480 μM.

Figure 6

Oxidation of membrane-bound cytochrome c by 2-hydroxyphenazine. The 100-μl reaction mixture consisted of membranes (750 μg protein), 4 μM ferredoxin 1 mM NADPH and1 μg FNR contained in 50 mM MOPS buffer (pH 6.8). The reduction of cytochrome c was initiated by addition of FNR. The reduction and re-oxidation was monitored at 554 nm. When fully reduced, 200 μM 2-hydroxyphenazine (2 μl) was added (arrow). Panel A, time course for the reduction and re-oxidation by 2-hydroxyphenazine added at the arrow. Panel B, reduced minus oxidized UV-visible spectra of membranes before (lower trace) and after (upper trace) addition of 2-hydroxyphenazine.

Figure 7

Comparison of electron transport pathways for Methanosarcina mazei and Methanosarcina barkeri versus Methanosarcina acetivorans. Panel A, M. mazei and M. barkeri. Panel B, M. acetivorans. Ech, Ech hydrogenase; Fd_r, ferredoxin reduced; Fd_o, ferredoxin oxidized; Vho, Vho hydrogenase; MP, methanophenazine; HdrDE, heterodisulfide reductase; CoM-SH, coenzyme M; CoB-SH, coenzyme B; Atp, ATP synthase; Cyt c, cytochrome c; Ma-Rnf, Rnf complex from M. acetivorans; Mrp, putative sodium/proton antiporter.