Mechanistic Investigation on the C–C Bond Cleavage of α-Hydroxy Ketone Catalyzed by Engineering P450 Monooxygenase Enzyme F182L-CYP199A4

In addition to the typical oxygen insertion reaction, some cytochrome P450 also catalyze the C–C cleavage, and most P450 employs Cpd I to trigger lyase reactions. There is also evidence to support an alternative mechanism, in which a species earlier than the formation of Cpd I was suggested to perform the lyase reaction. To understand the detailed reaction mechanism, we performed quantum mechanics/molecular mechanics (QM/MM) calculations to explore the cleavage mechanism of mutated CYP199A4 (F182L-CYP199A4). Our calculation results reveal that the peroxoanion (Fe(III)–O–O^2–) rather than oxo-ferrous (Fe(III)–O–O^1–) and Cpd I is responsible for initiating the reaction. The Fe(III)–O–O^2– nucleophilic directly attacks the carbonyl of the substrate, which first generates an unstable peroxo intermediate, followed by the Baeyer–Villiger-type oxidative cleavage of the C–C bond. During the reaction, complex electronic and structural rearrangements are involved. In the active site of F182L-CYP199A4, the formation of Cpd I is successfully slowed down owing to the absence of mediated water to promote two steps of protonation of Fe-coordinated dioxygen. In addition, Fe(III)–O–O^2– shows higher reactivity than Fe(III)–O–O^1– toward C–C cleavage. In general, the fact that CYP can also employ the peroxoanion to execute the C–C bond cleavage further enriches the catalytic chemistry of P450.