Methanogenic archaea inhabiting anaerobic environments play a crucial role in the global biogeochemical material cycle. The most universal electrogenic reaction of their methane-producing energy metabolism is catalyzed by N ⁵-methyl-tetrahydromethanopterin: coenzyme M methyltransferase (MtrABCDEFGH), which couples the vectorial Na⁺ transport with a methyl transfer between the one-carbon carriers tetrahydromethanopterin and coenzyme M via a vitamin B₁₂ derivative (cobamide) as prosthetic group. We present the 2.08 Å cryo-EM structure of Mtr(ABCDEFG)₃ composed of the central Mtr(ABFG)₃ stalk symmetrically flanked by three membrane-spanning MtrCDE globes. Tetraether glycolipids visible in the map fill gaps inside the multisubunit complex. Putative coenzyme M and Na⁺ were identified inside or in a side-pocket of a cytoplasmic cavity formed within MtrCDE. Its bottom marks the gate of the transmembrane pore occluded in the cryo-EM map. By integrating Alphafold2 information, functionally competent MtrA-MtrH and MtrA-MtrCDE subcomplexes could be modeled and thus the methyl-tetrahydromethanopterin demethylation and coenzyme M methylation half-reactions structurally described. Methyl-transfer-driven Na⁺ transport is proposed to be based on a strong and weak complex between MtrCDE and MtrA carrying vitamin B₁₂, the latter being placed at the entrance of the cytoplasmic MtrCDE cavity. Hypothetically, strongly attached methyl-cob(III)amide (His-on) carrying MtrA induces an inward-facing conformation, Na⁺ flux into the membrane protein center and finally coenzyme M methylation while the generated loosely attached (or detached) MtrA carrying cob(I)amide (His-off) induces an outward-facing conformation and an extracellular Na⁺ outflux. Methyl-cob(III)amide (His-on) is regenerated in the distant active site of the methyl-tetrahydromethanopterin binding MtrH implicating a large-scale shuttling movement of the vitamin B₁₂-carrying domain.

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