Spectroscopic study of the cobalamin-dependent methionine synthase in the activation conformation: effects of the Y1139 residue and S-adenosylmethionine on the B12 cofactor.

The cobalamin-dependent methionine synthase (MetH) from Escherichia coli is a modular enzyme that catalyzes a methyl-group transfer from methyltetrahydrofolate to homocysteine via a methylcob(III)alamin (MeCbl) intermediate, generating tetrahydrofolate and methionine (Met). Once every ~2000 turnovers, the cobalamin cofactor is converted to the inactive cob(II)alamin (Co2+Cbl) form, from which MeCbl has to be recovered in order for MetH to re-enter the catalytic cycle. A particularly puzzling aspect of this reactivation process is that it requires the reduction of the Co2+Cbl species to cob(I)alamin (Co1+Cbl) by flavodoxin, a reaction that would appear to be endergonic based upon the corresponding reduction potentials. To explore how MetH may overcome this apparent thermodynamic challenge, we have prepared the I690C/G743C variant of a C-terminal fragment of MetH (MetHCT) so as to lock the enzyme into the activation conformation without perturbing any of the residues in the vicinity of the active site. A detailed spectroscopic characterization of this species and the I690C/G743C/Y1139F MetHCT triple mutant reveals that the strategy employed by MetH to activate Co2+Cbl for Co2+ → Co1+ reduction likely involves (i) an axial ligand switch to generate a five-coordinate species with an axially coordinated water molecule and (ii) a significant lengthening, or perhaps complete rupture, of the Co–OH2 bond of the cofactor, thereby causing a large stabilization of the Co 3dz2-based “redox-active” molecular orbital. The lengthening of the Co–OH2 bond is mediated by the Y1139 active-site residue and becomes much more dramatic when the S-adenosylmethionine substrate is present in the enzyme active site. This substrate requirement provides MetH a means to suppress deleterious side reactions involving the transiently formed Co1+Cbl “supernucleophile”.