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Vitamin B 12 -dependent enzymes catalyze several difficult radical reactions. There are fundamental open questions that need to be addressed to fully understand the formation of highly reactive radical species, its dynamics, and interaction with the substrate and enzyme. In this work, ab initio molecular dynamics was performed within a QM/MM framework on a reduced AdoCbl cofactor, which was taken as a post proton-coupled electron transfer initial step for the activation of the AdoCbl-dependent methylmalonyl CoA mutase enzyme. The calculated free-energy profile reveals two possible pathways, stepwise (I) and concerted (II) for the reductive Co-C cleavage and subsequent H-abstraction. The computed activation barrier from metadynamics for both the pathways is comparable (78.5 and 76.2 kJ/mol, respectively); however, the concerted pathway may be preferred kinetically because it avoids the formation of a high-energy radical intermediate with possibly a larger recrossing rate. Our results are consistent with the previous conductor hypothesis, indicating the explicit role of cob(II)alamin in stabilizing the radical intermediate involved in the H-atom transfer.

Detailed theoretical and X-ray based structural analysis has been carried out in order to unmask the role of the tyrosine residue (Y) in the activation of the Co-C bond in AdoCbl-dependent mutases. In particular, methylmalonyl-CoA mutase (MCM) and glutamate mutase (GLM) enzymes have been studied; in the case of MCM, the significance of the Y89 residue has been analyzed extensively. Three different theoretical platforms encompassing the DFT, CASSCF/QDPT2, and QM/MM frameworks have been employed to elucidate the energetics of the AdoCbl-Y(-) complex while taking into account a varied degree of structural complexity. The diradical state, [AdoCbl](*-)-Y*, has been found to be the lowest electronic state of the AdoCbl-Y(-) complex, providing strong evidence that electron transfer from the Y89 residue to the cofactor is feasible. Crystallographic analysis of the active sites of MCM and GLM enzymes reveals that substrate binding can play a critical role in displacing the hydroxyl proton of the Y residue (Y89 in the case of MCM enzyme and Y181 in the case of GLM enzyme) that will facilitate the electron transfer (ET), hence making the activation process a case of proton-coupled electron transfer (PCET). PCET-inspired enzymatic catalysis implies that the cleavage of the Co-C bond takes place via one-electron reduced form of the AdoCbl cofactor (i.e., [AdoCbl](*-)), rather than its neutral analogue, thus providing an efficient mode of cleavage that can help in understanding the origin of the catalytic effect in such enzymes.

Density functional theory (DFT)-based normal mode calculations have been carried out on models for B12-cofactors to assign reported isotope-edited resonance Raman spectra, which isolate vibrations of the organo-Co group. Interpretation is straightforward for alkyl-Co derivatives, which display prominent Co-C stretching vibrational bands. DFT correctly reproduces Co-C distances and frequencies for the methyl and ethyl derivatives. However, spectra are complex for adenosyl derivatives, due to mixing of Co-C stretching with a ribose deformation coordinate and to activation of modes involving Co-C-C bending and Co-adenosyl torsion. Despite this complexity, the computed spectra provide a satisfactory re-assignment of the experimental data. Reported trends in adenosyl-cobalamin spectra upon binding to the methylmalonyl CoA mutase enzyme, as well as on subsequent binding of substrates and inhibitors, provide support for an activation mechanism involving substrate-induced deformation of the adenosyl ligand.

A combined density functional theory (DFT) and molecular mechanics (MM) approach was applied to investigate the relationship between the structure of a free coenzyme B12, and bound to methylmalonyl-CoA mutase. It was found that, upon coenzyme binding to apoenzyme, the Co-C bond remains intact, while the C-Naxial bond becomes slightly elongated and labilized. The labilization of the Co-Naxial bond that takes place in coenzyme B12-dependent enzymes is most likely necessary for fine-tuning of the cobalt-nitrogen (axial base) distance. The controlling of this distance is important to inhibit abiological site reaction involving heterolysis of the Co-C bond but is not important for biologically relevant Co-C bond homolysis.