Powering methanogenesis from fatty acids by a twin-heme-mediated reverse redox-loop

The conversion of organic matter into methane is central to the global carbon cycle and engineered biogas production. In this process, syntrophic bacteria oxidize fatty acid fermentation products to acetate, coupled to the generation of H ₂ or formate, which are subsequently utilized by methanogenic archaea. During fatty acid β-oxidation, a membrane-bound electron-transferring flavoprotein (ETF):methylmenaquinone (MMK) oxidoreductase complex (EMO) has been proposed to drive endergonic electron transfer from reduced ETF to CO ₂ through a reverse redox loop, with its mechanistic basis remaining unresolved. Here we report cryo-electron microscopy structures of EMO and the EMO-ETF complex from Syntrophus aciditrophicus at 2.0 and 3.0 Å resolution, respectively. Complex formation induces substantial conformational rearrangements in ETF, positioning its flavin for efficient electron transfer to non-cubane [4Fe:4S] and [4Fe:5S] clusters. The membrane-integral domain harbors three heme b cofactors, including a specialized twin-heme unit that mediates proton-motive-force-driven MMK reduction. The structural and functional similarity of EMOs to heterodisulfide reductases, together with their broad distribution across bacteria and archaea, suggests an evolutionary link between methanogenesis and fatty acid β-oxidation, illustrating how ancient redox systems were repurposed for new metabolic functions.