What Triggers the Cleavage of the Co–C5′ Bond in Coenzyme B12-Dependent Itaconyl-CoA Methylmalonyl-CoA Mutase?

Abstract

Adenosylcobalamin (AdoCbl)-dependent enzymes catalyze complex radical-mediated reactions initiated by homolytic cleavage of the organometallic Co–C5′ bond of the cofactor (AdoCbl) upon substrate binding. Several mechanistic proposals for the activation of the Co–C5′ bond were previously put forward, but none can fully explain the trillion-fold rate enhancement that is observed for cleavage of the Co–C5′ bond in enzymes as compared to AdoCbl in solution. Thus, how the arrival of a substrate triggers the cleavage of the Co–C5′ bond in these enzymes remains an open question. Recently, two crystal structures for AdoCbl-dependent methylmalonyl-CoA mutase (MCM) were reported and provide the opportunity to reinvestigate this long-standing issue. We investigated the activation of the Co–C5′ bond using substrate-free MCM and inhibitor-bound MCM (inhibitor = itaconyl-CoA, I-CoA) by computing the bond dissociation energy (BDE) by utilizing the ONIOM-based quantum mechanics/molecular mechanics (QM/MM) method. The BDE potential energy curves revealed that there is an insensitivity of the Co–C5′ BDE to substrate binding and the energy barrier for cleavage for substrate-free MCM and MCM–[I-CoA] is nearly identical (22.2 vs 22.7 kcal/mol, respectively). This observation puts into question previously reported hypotheses that substrate-induced conformational changes reduce the BDE and trigger the cleavage of the Co–C5′ bond. The present QM/MM results also indicate that the energy barrier for homolytic cleavage of the Co–C5′ bond in both substrate-free MCM and MCM–[I-CoA] is lower than the energy barrier for the isolated cofactor. While the protein environment clearly reduces the barrier for the Co–C5′ bond cleavage, the reduction to the energy barrier is too small to fully explain the trillion-fold rate acceleration. Hence, we also explored the role of Tyr105 as a redox center and the feasibility of a proton-coupled electron transfer (PCET) mechanism. It was found that the [AdoCbl]•–/TyrO• diradical state is the lowest electronic state, implying that the Tyr105 residue can play a crucial role in triggering the cleavage of the Co–C5′ bond in MCM–[I-CoA] by facilitating an ET to the AdoCbl cofactor via a PCET mechanism.