Diverse Mechanisms for the Aromatic Hydroxylation: Insights into the Mechanisms of the Coumarin Hydroxylation by CYP2A6

Different P450 isoforms may catalyze different types of reactions on the same substrate due to differences in their protein environments. To uncover how the spatial environment within the enzyme regulates substrate reactivity, we conducted quantum mechanics/molecular mechanics (QM/MM) simulations on the CYP2A6-catalyzed 7-hydroxylation of coumarin. The results revealed that water molecules can flexibly enter the active site of CYP2A6. In the absence of water molecules, the NIH shift mechanism was found to be the most favorable reaction pathway, leading to the keto intermediate that further undergoes the isomerization to form the C7-hydroxylated product. However, when water molecules are present at the active site, the N-protonation route can be facilitated by the active site waters and thus becomes the preferred one. Both the NIH mechanism and the N-protonation can rationalize the 1,2-H shift for the aromatic hydroxylation reactions. This study highlights that P450s can employ diverse and flexible mechanisms for aromatic hydroxylation, offering deeper insight into the mechanisms of P450-catalyzed aromatic hydroxylation reactions.