Essential anaplerotic role for the energy-converting hydrogenase Eha in hydrogenotrophic methanogenesis

Abstract

Despite decades of study, electron flow and energy conservation in methanogenic Archaea are still not thoroughly understood. For methanogens without cytochromes, flavin-based electron bifurcation has been proposed as an essential energy-conserving mechanism that couples exergonic and endergonic reactions of methanogenesis. However, an alternative hypothesis posits that the energy-converting hydrogenase Eha provides a chemiosmosis-driven electron input to the endergonic reaction. In vivo evidence for both hypotheses is incomplete. By genetically eliminating all nonessential pathways of H(2) metabolism in the model methanogen Methanococcus maripaludis and using formate as an additional electron donor, we isolate electron flow for methanogenesis from flux through Eha. We find that Eha does not function stoichiometrically for methanogenesis, implying that electron bifurcation must operate in vivo. We show that Eha is nevertheless essential, and a substoichiometric requirement for H(2) suggests that its role is anaplerotic. Indeed, H(2) via Eha stimulates methanogenesis from formate when intermediates are not otherwise replenished. These results fit the model for electron bifurcation, which renders the methanogenic pathway cyclic, and as such requires the replenishment of intermediates. Defining a role for Eha and verifying electron bifurcation provide a complete model of methanogenesis where all necessary electron inputs are accounted for.

Fig. 1.

The methanogenic pathway. Eha, energy-converting hydrogenase A; Fdh, formate dehydrogenase; Fru and Frc, F₄₂₀-reducing hydrogenases; Ftr, formyl-MFR:H₄MPT formyltransferase; Fwd, formyl-MFR dehydrogenase; Hdr, heterodisulfide reductase; Hmd, H₂-dependent methylene-H₄MPT dehydrogenase; Mch, methenyl-H₄MPT cyclohydrolase; Mcr, methyl-CoM reductase; Mer, methylene-H₄MPT reductase; Mtd, F₄₂₀-dependent methylene-H₄MPT dehydrogenase; Mtr, methyl-H₄MPT-CoM methyltransferase; Vhu and Vhc, F₄₂₀-nonreducing (Hdr-associated) hydrogenases.

Fig. 2.

H₂ production by cell suspensions in the absence or presence of formate. Values in x axis are in minutes.

Fig. 3.

Requirements of the ∆3H₂ase (A) and ∆5H₂ase (B) mutants for H₂ and formate for growth. For growth of the ∆5H₂ase mutant on H₂ and formate, 14.3 μmoles of H₂ was added.

Fig. 4.

H₂ dose–response of ∆5H₂ase mutant. Clear bars, mineral medium; checkered bars, 10 mM acetate added; solid bars, 10 mM acetate and casamino acids (0.2% wt/vol) added. All cultures contained 200 mM formate. Five-milliliter cultures were incubated until stationary phase and OD₆₆₀ was measured.

Fig. 5.

Methanogenesis in cell extracts from CH₃-S-CoM and CO₂ using H₂ (solid line) or formate (dashed line) as the electron donor in cell extracts from (A) wild-type or (B) ∆6H₂ase mutant. Each reaction contained 300 nmols of CH₃-S-CoM and 200–350 μg of protein. Extracts with no CH₃-S-CoM added are represented by open diamonds.

Fig. 6.

CH₄ production by cell suspensions of the ∆6H₂ase mutant. Black diamonds, H₂ alone; open triangles, formate alone with (+) or without H₂ addition [8% (vol/vol) final concentration] at 60 min; black triangles, H₂ and formate.