Theoretical Insights into the Mechanism and Stereoselectivity of Olefin Cyclopropanation Catalyzed by Two Engineered Cytochrome P450 Enzymes

Engineered P450s can catalyze some non-natural reactions with high efficiency and excellent selectivity, such as the carbine transfer, nitrene transfer, C-H insertion, and C-H amination, opening alternative routes for sustainable production of chemicals. Recent experiments revealed that two engineered cytochrome P450 enzymes (P450(BM3)-CIS and P411(BM3)-CIS) show different efficiencies and stereoselectivities in the olefin cyclopropanation, but key factors that affect the activity remain unclear. In this work, both quantum mechanics (QM) and QM/molecular mechanics (MM) methods were employed to explore the catalytic reactions and selectivity of these two engineered cytochrome P450 enzymes. On the basis of our results, the cyclopropanation of styrene is suggested to mainly occur on the open-shell singlet (OSS) and triplet state surfaces, which contain two elementary steps. The reactive iron(III)-porphyrin carbene (IPC) radical first attacks the terminal alkenyl group of styrene to form a C-radical intermediate, which then undergoes a cyclization reaction affording the cyclopropanation products. Importantly, it is found that the stereoselectivity of cyclopropanations is elucidated only if considering the real protein environment, and the stereoselectivity is determined by multiple factors, such as the relative orientation of IPC to styrene, the binding affinity of the substrate, and the reaction barriers of rate-limiting steps. It is the enzymatic environment that makes the reaction highly stereoselective, which provides useful clues for designing whole-cell catalysts for non-natural chemical reactions.