Crucial gating residues govern the enhancement of peroxygenase activity in an engineered cytochrome P450 -demethylase.

P450-catalyzed -demethylation reactions have recently attracted particular attention because of their potential applications in lignin bioconversion. We recently enabled the peroxygenase activity of CYP199A4, a NADH-dependent cytochrome P450 monooxygenase from , by engineering a hydrogen peroxide (HO) tunnel. In this report, we reveal by crystallography and molecule dynamics simulations that key residues located at one of the water tunnels in CYP199A4 play a crucial gating role, which enhances the peroxygenase activity by regulating the inflow of HO. These results provide a more complete understanding of the mechanism by which monooxygenase is converted into peroxygenase activity through the HO tunnel engineering (HTE) strategy. Furthermore, a library of engineered CYP199A4 peroxygenases was constructed to explore their application potentials for -demethylation of various methoxy-substituted benzoic acid derivatives. The engineered CYP199A4 peroxygenases showed good functional group tolerance and preferential -demethylation at the - or -position, indicating potential -demethylation of H- and G-type lignin monomers. This work reveals the feasibility of the HTE strategy in creating P450 peroxygenase from a mechanistic perspective, laying the foundation for developing an effective P450 -demethylase applicable in lignin bioconversion.