The Energetics and Sodium‐Ion Dependence of N 5 ‐Methyltetrahydromethanopterin:Coenzyme M Methyltransferase Studied with Cob(I)Alamin as Methyl Acceptor and Methylcob(III)Alamin as Methyl Donor

N5 ‐Methyltetrahydromethanopterin: coenzyme M methyltransferase from methanogenic Archaea is a membrane‐associated enzyme complex that uses a methyl‐transfer reaction to drive an energy‐conserving sodium‐ion pump. Methyl transfer occurs in two steps, first from N 5 ‐methyl‐tetrahydromethanopterin (CH 3 ‐H 4 MPT) to an enzyme‐bound cob(I)amide prosthetic group, and secondly from the methylated cobamide to coenzyme M (H‐S‐CoM). In this study, we report that methyltransferase can also use exogenous cob(I)alamin and methylcob(III)alamin as methyl acceptor and methyl donor, respectively. The enzyme catalyzes methylcob(III)alamin formation from CH 3 ‐H 4 MPT and cob(I)alamin (reaction a), and methyl‐coenzyme M formation from methyl‐cob(III)alamin and H‐S‐CoM (reaction b). Both reactions were shown to be reversible. Reaction a was catalyzed at approximately the same rate (3 U/mg) and reaction b at approximately 10% the rate (0.3 U/mg) of the physiological reaction, namely methyl transfer from CH 3 ‐H 4 MPT to H‐S‐CoM. The free energy changes (Δ G °′ )associated with reactions a and b were both between –10 kJ/mol and ‐20 kJ/mol, consistent with a free energy change of approximately–30 kJ/mol determined for the physiological reaction. Reaction b but not reaction a was sodium‐ion dependent. Assuming that methylation of exogenous cob(I)alamin and demethylation of exogenous methylcob(III)alamin mimic methylation and demethylation of the enzyme‐bound corrinoid prosthetic group, it can be inferred that methyl transfer from the methylated cobamide prosthetic group to H‐S‐CoM is a site of coupling with sodium‐ion translocation.