Microbial methane formation from organic matter plays a major role in the global carbon cycle, contributing to more than half of the annual methane production worldwide¹. In nature, the conversion of organic substrates into CH₄ and CO₂ is catalyzed by strictly anaerobic, methanogenic archaea in syntrophic association with fermenting bacteria. The latter thereby produce the methanogen"s substrates formate, H₂, CO₂ and acetate from short-chain fatty acids or alcohols. For many years, the endergonic electron transfer from β-oxidation-derived electrons via an electron-transferring flavoprotein (ETF, E⁰' ≈ −10 mV) to CO₂ (E' ≈ −290 mV at formate concentrations < 10 µM) remained hypothetical. In a recent study, we identified a key enzyme in the model organism Syntrophus aciditrophicus, a membrane-bound FeS cluster- and heme b-containing ETF:(methyl)menaquinone oxidoreductase (EMO). This enzyme enables the proton motive force-driven reduction of CO₂ via a reverse redox loop between EMO and membrane-bound formate dehydrogenase with methylmenaquinone (MMK, E⁰' = −156 mV) serving as membrane-bound electron carrier².

Until recently, structural data of EMO and its interaction partners was lacking. Based on experiments evaluated by blue native PAGE, we observed the formation of a complex between enriched ETF and EMO in vitro. Using cryo electron microscopy, we obtained several 3D structures of the EMO-ETF complex and EMO alone, with atomic resolutions down to 2.7 Å. The data suggests the presence of two non-cubane [4Fe−4S] clusters, to date exclusively known from B subunits of heterodisulfide reductases (Hdr) of methanogenic archaea³, in the cytoplasm-facing soluble domain of EMO. In addition, we observed several conformational states of the flavin-containing region of ETF while bound to the HdrB-like domain of EMO, providing first insights into the interaction between flavin cofactor and non-cubane [4Fe−4S] clusters. Given the high abundance of emo genes in a variety of (M)MK-containing organisms, including the pathogen Mycobacterium tuberculosis², knowledge of this interaction is essential for understanding the endergonic electron transfer during β-oxidation in these prokaryotes.

1 Evans, P. et al., Nat Rev Microbiol 17, 219-232 (2019)

2 Agne, M. et al., PNAS 118, 40 (2021)

3 Wagner, T. et al., Science 357, 699-703 (2017)