3.C.1 The Na⁺ Transporting Methyltetrahydromethanopterin:Coenzyme M Methyltransferase (NaT-MMM) Family

The NaT-MMM (EC 2.1.1.86) of Methanobacterium thermoautotrophicum has been purified and characterized, and the Na⁺ transport reaction has been studied in inverted membrane vesicles. Sequencing of the encoding genes revealed eight contiguous genes organized in an operon. The majority of the encoded subunits are probably integral membrane proteins. Thus, MtrA, B, F and G have 1 TMS; MtrC has 7; MtrD has 6; MtrE has 6; and MtrH has none (Gottschalk and Thauer, 2001). MtrE is believed to be the Na⁺-transporting subunit. MtrA bears a cob(I)amide prosthetic group which is methylated and demethylated in the catalytic cycle. Demethylation is the Na⁺-dependent step. Thus, methyl transfer from methylated MtrA to coenzyme M drives the electrogenic translocation of Na⁺. The overall methyl transfer reaction is strongly exergonic (ΔG°= -30 kJ/mol) (Gottschalk and Thauer, 2001).

The NaT-MMM complex probably contains iron/sulfur clusters. As noted above, the enzyme catalyzes two chemical reactions as follows:

(1) CH₃-H₄MPT + E:Co(I) → H₄MPT + E:CH₃-Co(III).

(2) E:CH₃-Co(III) + HS-CoM → E:Co(I) + CH₃-S-CoM.

Reaction 1 is reversible, but reaction 2 is essentially irreversible in the direction shown. Na⁺ efflux is probably coupled to the second of these two reactions.

The generalized transport reaction is therefore probably:

Na⁺ (in) + E:CH₃-Co(III) + HS-CoM → Na⁺ (out) + E:Co(I) + CH₃-S-CoM.

Methanogenic archaea inhabiting anaerobic environments are important for 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) 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 a prosthetic group. Aziz et al. 2024 presented 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 were structurally described. Methyl-transfer-driven Na⁺ transport may 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 (Aziz et al. 2024).