Electronic structure studies of the adenosylcobalamin cofactor in glutamate mutase.

Glutamate mutase (GM) is a cobalamin-dependent enzyme that catalyzes the reversible interconversion of L-glutamate and L-threo-3-methylaspartate via a radical-based mechanism. To initiate catalysis, the 5'-deoxyadenosylcobalamin (AdoCbl) cofactor's Co-C bond is cleaved homolytically to generate an adenosyl radical and Co2+ Cbl. In this work, we employed a combination of spectroscopic and computational tools to evaluate possible mechanisms by which the Co-C bond is activated for homolysis. Minimal perturbations to the electronic absorption (Abs), circular dichroism (CD), and magnetic CD (MCD) spectra of AdoCbl are observed upon formation of holoenzyme, even in the presence of substrate (or a substrate analogue), indicating that destabilization of the Co3+ Cbl "ground state" is an unlikely mechanism for Co-C bond activation. In contrast, striking alterations are observed in the spectroscopic data of the post-homolysis product Co2+ Cbl when bound to glutamate mutase in the presence of substrate (or a substrate analogue) as compared to unbound Co2+ Cbl. These enzymatic perturbations appear to most strongly affect the metal-to-ligand charge-transfer transitions of Co2+ Cbl, suggesting that the cofactor/active-site interactions give rise to a fairly uniform stabilization of the Co 3d orbitals. Remarkable similarities between the results obtained in this study and those reported previously for the related Cbl-dependent isomerase methylmalonyl-CoA mutase indicate that a common mechanism by which the cofactor's Co-C bond is activated for homolytic cleavage may be operative for all base-off/His-on Cbl-dependent isomerases.