Spectroscopic and computational insights into the mechanisms employed by ATP:Corrinoid adenosyltransferases for coenzyme B12

It has been hypothesized previously that the Co2+corrinoid → Co1+corrinoid reduction step in coenzyme B12 biosynthesis is facilitated by ATP:corrinoid adenosyltransferases (ATRs), which drastically weaken the bond between the axial ligand and the cobalt center, so as to generate unusual, essentially square-planar Co2+ species. The geometric and electronic properties of these unprecedented Co2+corrinoid species, as well as the mechanism employed by the PduO-type ATR from Lactobacillus reuteri (LrPduO) were studied using electronic absorption, circular dichroism (CD), magnetic CD, electron paramagnetic resonance, and resonance Raman (rR) spectroscopic techniques. Our results reveal that LrPduO converts both Co2+ cobalamin (Co2+Cbl) and Co2+cobinamide (Co2+Cbi+) to four-coordinate Co2+corrinoids and that the hydrophobicity and crowdedness of the enzyme active site are essential for the stability of four-coordinate Co2+corrinoids relative to their "unactivated" five-coordinate counterparts.;The addition of a flavin-containing reducing system to samples containing Co2+corrinoids bound to the wild-type (WT) LrPduO/ATP complex was found to trigger an instantaneous reduction of the "activated" four-coordinate Co2+corrinoids to Co1+corrinoids, whereas the five-coordinate fraction was reduced much more slowly. Our results revealed further that the WT LrPduO/ATP complex facilitates the adenosylation of Co1+corrinoids by increasing the nucleophilicity of the Co1+ center.;To assign the dominant features in the rR spectra of Co2+corrinoids bound to the WT LrPduO/ATP complex, the relative rR intensities of the relevant corrin-based vibrational modes were calculated using density functional theory-based approaches. Our results indicate that Co2+ corrinoid binding to the WT LrPduO/ATP complex largely suppresses nuclear motion along the C5···C15 axis, suggesting that the mechanism of Co2+/1+corrinoid "activation" by WT LrPduO also involves the formation of non-covalent interactions between the corrin ring and the enzyme active site.;Lastly, our spectroscopic methodology was applied to EutT, a member of a structurally and evolutionarily unrelated and largely uncharacterized third family of ATRs. Remarkably, EutT utilizes the same strategy as the other ATRs to accomplish the thermodynamically challenging Co2+→Co 1+ reduction, converting the five-coordinate Co2+Cbl species to a four-coordinate form. However, unlike LrPduO, EutT cannot "activate" Co2+Cbi+, which implies that the binding of the 5,6-dimethylbenzimidazole base of Co2+Cbl to EutT induces a major conformational change of the enzyme active site.