Enabling Peroxygenase Activity in Cytochrome P450 Monooxygenases by Engineering Hydrogen Peroxide Tunnels

Given prominent physicochemical similarities between H₂O₂ and water, we report a new strategy for promoting the peroxygenase activity of P450 enzymes by engineering their water tunnels to facilitate H₂O₂ access to the heme center buried therein. Specifically, the H₂O₂-driven activities of two native NADH-dependent P450 enzymes (CYP199A4 and CYP153A) increase significantly (by >183-fold and >15-fold, respectively). Additionally, the amount of H₂O₂ required for an artificial P450 peroxygenase facilitated by a dual-functional small molecule to obtain the desired product is reduced by 95%-97.5% (with ∼95% coupling efficiency). Structural analysis suggests that mutating the residue at the bottleneck of the water tunnel may open a second pathway for H₂O₂ to flow to the heme center (in addition to the natural substrate tunnel). This study highlights a promising, generalizable strategy whereby P450 monooxygenases can be modified to adopt peroxygenase activity through H₂O₂ tunnel engineering, thus broadening the application scope of P450s in synthetic chemistry and synthetic biology.