An Asynchronous, Concerted Mechanism for Cytochrome P450-Catalyzed Dehydrogenation: A Combined Deuterium Labeling and QM/MM Study

Cytochromes P450 (P450s) commonly catalyze hydroxylation but can also be responsible for dehydrogenation reactions, important in drug metabolism and biosynthesis; the mechanism of the latter transformation remains poorly understood. The well-characterized bacterial CYP199A4 catalyzes both hydroxylation and dehydrogenation of p-alkylbenzoic acids and thus provides an ideal model system in which to investigate the mechanism of P450-catalyzed aliphatic dehydrogenation. Through use of enantioselectively deuterated probes, metabolite analysis, protein crystallography, molecular dynamics simulations and QM/MM (ONIOM) modeling, CYP199A4-catalyzed dehydrogenation was found to be completely enantioselective and postulated to occur through an asynchronous proton coupled electron transfer. No definitive evidence of a cationic intermediate was uncovered but instead, the positioning of the substrate was postulated to be key in directing the chemoselectivity of the reaction i.e., dehydrogenation versus hydroxylation. This knowledge could be exploited to control dehydrogenation in other P450s and helps explain the common occurrence of P450-desaturated drug metabolites alongside hydroxylated ones.