Diverse Mechanisms\nfor the Aromatic Hydroxylation:\nInsights into the Mechanisms of the Coumarin Hydroxylation by CYP2A6

Different P450 isoforms may catalyze different types\nof reactions\non the same substrate due to differences in their protein environments.\nTo uncover how the spatial environment within the enzyme regulates\nsubstrate reactivity, we conducted quantum mechanics/molecular mechanics\n(QM/MM) simulations on the CYP2A6-catalyzed 7-hydroxylation of coumarin.\nThe results revealed that water molecules can flexibly enter the active\nsite of CYP2A6. In the absence of water molecules, the NIH shift mechanism\nwas found to be the most favorable reaction pathway, leading to the\nketo intermediate that further undergoes the isomerization to form\nthe C7-hydroxylated product. However, when water molecules are present\nat the active site, the N-protonation route can be facilitated by\nthe active site waters and thus becomes the preferred one. Both the\nNIH mechanism and the N-protonation can rationalize the 1,2-H shift\nfor the aromatic hydroxylation reactions. This study highlights that\nP450s can employ diverse and flexible mechanisms for aromatic hydroxylation,\noffering deeper insight into the mechanisms of P450-catalyzed aromatic\nhydroxylation reactions.