Computational Study of Cobalamin-Dependent Epoxyqueuosine Reductase: Formation of a Key Organometallic Intermediate...

QueG is a cobalamin-dependent enzyme that catalyzes the epoxide reduction of queuosine (Q). The quantum chemical cluster approach has been performed to investigate the mechanism of the QueG-catalyzed reaction. Our calculations reveal a nucleophilic attack mechanism involving the formation of an intermediate with an unusual organometallic Co-C bond. Initially, single electron reduction generates a highly reactive Co(I) species, which acts as a "super-nucleophile". This intermediate attacks the epoxide carbon, concurrently promoting the formation of the unique "stable yet fragile" Co-C bond and the heterolytic cleavage of one C-O bond. Subsequently, a proton-coupled electron transfer (PCET) process drives the concerted heterolysis of the Co-C bond and the remaining C-O bond, leading to water formation and C═C bond generation. The rate-determining step is the Co-C bond formation, with a calculated barrier of 15.3 kcal/mol, highlighting its critical catalytic role. Furthermore, our findings suggested that Tyr105, instead of Tyr238 and Asp134, serves as the proton transfer shuttle during catalysis. Additionally, our calculations further validate that Asp134 acts as a reaction facilitator in the catalytic process, which mechanistically explains why mutating Asp134 results in a substantial decline in catalytic activity. These insights advance the understanding of Class III cobalamin-dependent enzymes and provide a broader perspective on prior research.