Spectroscopic and computational insights into the cofactor activation mechanisms of cobalamin-dependent methionine synthase

The geometric and electronic structures of the cob(I)alamin (Co 1+Cbl) and base-off cob(II)alamin (Co2+Cbl) forms of the vitamin B₁₂ cofactor were characterized using a combined spectroscopic and computational approach. Electronic absorption, circular dichroism, magnetic circular dichroism, resonance Raman, and electron paramagnetic resonance spectroscopic data were utilized to critically evaluate density functional theory calculations of these species. These studies successfully assigned several electronic and vibrational transitions of Co1+Cbl, revealed that significant configuration interaction mixing occurs among the electronic ground state and ligand field excited states of the four-coordinate form of base-off Co 2+Cbl, and indicated that the reduction of four-coordinate, base-off Co2+Cbl and the subsequent alkyl transfer to Co1+Cbl can occur in rapid succession because the electron and alkyl donors can approach the Co center from different directions.;The results of these combined spectroscopic and computational studies of the enzyme-free cobalamin cofactor served as the foundation for investigations of the molecular mechanism of cofactor activation employed by cobalamin-dependent methionine synthase (MetH), which catalyzes the conversion of homocysteine to methionine as the final step in the de novo biosynthesis of methionine. A combined spectroscopic and computational study of the H759G variant of MetH locked into the cofactor activation conformation indicated that the active site bound base-off Co2+Cbl species binds a water molecule exclusively to its β-face. The results of this study also revealed that the MetH active site is inaccessible to solvent if the enzyme is in a conformation relevant to catalytic turnover.;A detailed spectroscopic and computational investigation of the I690C/G743C and I690C/G743C/Y1139F mutants of truncated MetH revealed additional details of the strategy employed by MetH to activate Co2+Cbl for Co 2+ → Co1+ reduction. This strategy involves a significant lengthening, or perhaps complete rupture, of the Co-OH₂ bond of the cofactor, which causes a large stabilization of the Co 3d _z2-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".