Co2+cobalmain (Co2+Cbl) is implicated in the catalytic cycles of all adenosylcobalamin (AdoCbl)-dependent enzymes, as in each case catalysis is initiated through homolytic cleavage of the cofactor's Co-C bond. The rate of Co-C bond homolysis, while slow for the free cofactor, is accelerated by 12 orders of magnitude when AdoCbl is bound to the protein active site, possibly through enzyme-mediated stabilization of the post-homolysis products. As an essential step toward the elucidation of the mechanism of enzymatic Co-C bond activation, we employed electronic absorption (Abs), magnetic circular dichroism (MCD), and resonance Raman spectroscopies to characterize the electronic excited states of Co2+Cbl and Co 2+cobinamide (Co2+Cbl+, a cobalamin derivative that lacks the nucleotide loop and 5,6-dimethyl-benzimazole (DMB) base and instead binds a water molecule in the lower axial position). Although relatively modest differences exist between the Abs spectra of these two Co 2+corrinoid species, MCD data reveal that substitution of the lower axial ligand gives rise to dramatic changes in the low-energy region where Co2+-centered ligand field transitions are expected to occur. Our quantitative analysis of these spectral changes within the framework of time-dependent density functional theory (TD-DFT) calculations indicates that corrin-based π→π* transitions, which dominate the Co 2+corrinoid Abs spectra, are essentially insulated from perturbations of the lower ligand environment. Contrastingly, the Co2+-centered ligand field transitions, which are observed here for the first time using MCD spectroscopy, are extremely sensitive to alterations in the Co2+ ligand environment and thus may serve as excellent reporters of enzyme-induced perturbations of the Co2+ state. The power of this combined spectroscopic/computational methodology for studying Co2+corrinoid/ enzyme active site interactions is demonstrated by the dramatic changes in the MCD spectrum as Co2+Cbl+ binds to the adenosyltransferase CobA.
ASJC Scopus subject areas
- General Chemistry
- Colloid and Surface Chemistry