| Sumario: | CobA from Salmonella enterica is a member of an enzymatic system responsible for the de novo biosynthesis of adenosylcobalamin (AdoCbl), catalyzing the formation of the essential Co—C bond by transferring the adenosyl group from a molecule of ATP to a transient Cocorrinoid species generated in the enzyme active site. A particularly fascinating aspect of this reaction is that the flavodoxin in vivo reducing agent that serves as the electron donor to CobA possesses a reduction potential that is considerably more positive than that of the Co couple of the corrinoid substrate. To explore how CobA may overcome this challenge, we have employed electronic absorption, magnetic circular dichroism, and electron paramagnetic resonance (EPR) spectroscopies to probe the interaction between Co- and Cocorrinoids and the enzyme active site. Our data reveal that while Cocorrinoids interact only weakly with CobA, Cocorrinoids undergo partial conversion to a new paramagnetic species that can be obtained in nearly quantitative yield when CobA is preincubated with the co-substrate ATP. This "activated" species is characterized by a distinct set of ligand field transitions in the near-IR spectral region and EPR parameters that are unprecedented for Cocorrinoids. Analysis of these data on the basis of qualitative spectral correlations and density functional theory computations reveals that this unique Cocorrinoid species possesses an essentially square-planar Co center that lacks any significant axial bonding interactions. Possible implications of these findings for the mechanism of Co → Co reduction employed by CobA and Co—C bond-forming enzymes in genera! are explored.
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