Coupling of ferredoxin and heterodisulfide reduction via electron bifurcation in hydrogenotrophic methanogenic archaea

Abstract

In methanogenic archaea growing on H(2) and CO(2) the first step in methanogenesis is the ferredoxin-dependent endergonic reduction of CO(2) with H(2) to formylmethanofuran and the last step is the exergonic reduction of the heterodisulfide CoM-S-S-CoB with H(2) to coenzyme M (CoM-SH) and coenzyme B (CoB-SH). We recently proposed that in hydrogenotrophic methanogens the two reactions are energetically coupled via the cytoplasmic MvhADG/HdrABC complex. It is reported here that the purified complex from Methanothermobacter marburgensis catalyzes the CoM-S-S-CoB-dependent reduction of ferredoxin with H(2). Per mole CoM-S-S-CoB added, 1 mol of ferredoxin (Fd) was reduced, indicating an electron bifurcation coupling mechanism: 2H(2) + Fd(OX) + CoM-S-S-CoB-->Fd(red)(2-) + CoM-SH + CoB-SH + 2H(+). This stoichiometry of coupling is consistent with an ATP gain per mole methane from 4 H(2) and CO(2) of near 0.5 deduced from an H(2)-threshold concentration of 8 Pa and a growth yield of up to 3 g/mol methane.

Fig. 1.

CoM-S-S-CoB dependence of (A) ferredoxin (Fd) reduction and of (B) metronidazole (MTZ) reduction with H₂ catalyzed by a cell extract of M. marburgensis. The assays were performed at 60 °C in 1.5-mL anaerobic cuvettetes containing 0.75 mL assay mixture and 100% H₂ as the gas phase. The 0.75-mL assay mixture contained 1.6 M potassium phosphate (pH 7), 1 mM CoM-S-S-CoB, ∼30 μg (A) or ∼140 μg (B) cell extract, and either 35 μM Fd (A) or 150 μM MTZ (B). The reduction was started by CoM-S-S-CoB and followed photometrically at 390 nm (A) or 320 nm (B). After 30 s both Fd and MTZ were reduced to 100%.

Fig. 2.

Ferredoxin (Fd) reduction with 100% H₂ catalyzed by (A) [FeFe]-hydrogenase from Clostridium pasteurianum and by (B) the MvhADG/HdrABC complex from M. marburgensis in the presence of CoM-S-S-CoB. The 1.5-mL anaerobic cuvettetes contained 0.75 mL assay mixture containing 1.6 M potassium phosphate (pH 7) and where indicated Fd from C. pasteurianum (20 μM), [FeFe]-hydrogenase (∼1 unit), MvhADG/HdrABC complex (∼0.5 unit), CoM-S-S-CoB (0.5 mM), and/or sodium dithionite (0.5 mM). The gas phase was 100% H₂ at 1.2 bar and the temperature was 40 °C. The reactions were started by the addition of [FeFe]-hydrogenase (A, b) or dithionite (A, c) or CoM-S-S-CoB (B, b) followed by dithionite (B, c) and were completed within in a few seconds. The absorbance difference between the assay cuvettete and a cuvettete containing only phosphate buffer was recorded. A 0.5-mM dithionite solution in 1.6 M potassium phosphate (pH 7) showed an absorbance maximum near 320 nm with a ΔA₃₂₀ of 1.2; at 380 nm ΔA was 0.

Fig. 3.

Ferredoxin (Fd) reduction with 100% H₂ at limiting concentrations of CoM-S-S-CoB catalyzed by the MvhADG/HdrABC complex from M. marburgensis. (A) Time course with 12 nmol CoM-S-S-CoB (33 nmol Fd), 24 nmol CoM-S-S-CoB (33 nmol Fd), and 36 nmol CoM-S-S-CoB (45 nmol Fd). (B) Amounts of Fd reduced versus the amounts of CoM-S-S-CoB added. The assays were performed at 40 °C in 1.5-mL anaerobic cuvettetes containing 0.75 mL assay mixture with 1.6 M potassium phosphate (pH 7), MvhADG/HdrABC complex (∼0.1 unit), Fd from C. pasteurianum, and CoM-S-S-CoB in the amounts indicated. The reduction was started by enzyme protein and followed photometrically at 390 nm.

Fig. 4.

Metronidazole (MTZ) reduction with 100% H₂ at limiting concentrations of CoM-S-S-CoB catalyzed by the MvhADG/HdrABC complex from M. marburgensis. (A) Time course with 90 nmol MTZ and 72 nmol CoM-S-S-CoB. (B) Amounts of MTZ reduced versus the amounts of CoM-S-S-CoB added. The assays were performed at 60 °C in 1.5-mL anaerobic cuvettetes containing 0.75 mL assay mixture with 1.6 M potassium phosphate (pH 7), MvhADG/HdrABC complex (∼0.1 unit), MTZ (90 nmol), and CoM-S-S-CoB as indicated. The reduction was started by enzyme protein and followed photometrically at 320 nm.

Fig. 5.

Reactions, coenzymes, and enzymes involved in CO₂ reduction with 4 H₂ to CH₄ in M. marburgensis. Reduced ferredoxin (Fd), which is required for CO₂ reduction to formylmethanofuran (CHO-MFR), is regenerated in the MvhADG/HdrABC catalyzed reaction. If the latter is not completely coupled, then less reduced Fd is regenerated than required. In this case the energy-converting hydrogenases (EhaA-T and/or EhbA-Q) that catalyze the sodium motive force-driven Fd reduction with H₂ are proposed to step in. Eha and Ehb mainly have an anabolic function in providing the reduced Fd required for the reduction of CO₂ to CO and of acetyl-CoA + CO₂ to pyruvate. In M. marburgensis there are several ferredoxins that are considered to form a pool into which electrons are fed and from which electrons can be taken out (Discussion) (9). MFR, methanofuran; H₄MPT, tetrahydromethanopterin; CH≡H₄MPT⁺, methenyl-H₄MPT⁺; CH₂ = H₄MPT, methylene-H₄MPT; CH₃-H₄MPT, methyl-H₄MPT; MtrA-H, methyl transferase; FwdA-DFGH/FwdAFmdBCE, formyl-methanofuran dehydrogenase; AhaA-IK, A₁A₀ ATP synthase.