Emerging Strategies for Modifying Cytochrome P450 Monooxygenases into Peroxizymes.

ConspectusCytochrome P450 monooxygenase is a versatile oxidizing enzyme with great potential in synthetic chemistry and biology. However, the dependence of its catalytic function on the nicotinamide cofactor NAD(P)H and redox partner proteins limits the practical catalytic application of P450 . An alternative to expensive cofactors is low-cost HO, which can be used directly to exploit the catalytic potential of P450s. However, the peroxide shunt pathway is generally inefficient at driving P450 catalysis compared to normal NAD(P)H-dependent activity. Over the last few decades, the scientific community has made continuous efforts to use directed evolution or site-directed mutagenesis to modify P450 monooxygenases into their peroxizyme modes─peroxygenase and peroxidase. Despite significant progress, obtaining efficient P450 peroxizymes remains a huge challenge. Here, we summarize our efforts to modulate peroxizyme activity in P450 monooxygenases and exploit their catalytic applications in challenging selective C-H oxidation, oxygenation, and oxyfunctionalization over the past seven years. We first developed a dual-functional small molecule (DFSM) strategy for transforming P450BM3 monooxygenase into peroxygenase. In this strategy, the typical DFSM, such as -(ω-imidazolyl)-hexanoyl-l-phenylalanine (Im-C6-Phe), binds to the P450BM3 protein with an anchoring group at one end and plays a general acid-base catalytic role in the activation of HO with an imidazolyl group at the other end. Compared with the O-O homolysis mechanism in the absence of DFSM, the addition of DFSM efficiently enables the heterolytic O-O cleavage of the adduct Fe-O-OH, thus being favored for the formation of active species compound I, which has been demonstrated by combining crystallographic and theoretical calculations. Furthermore, protein engineering showed the unique catalytic performance of DFSM-facilitated P450 peroxygenase for the highly difficult selective oxidation of C-H bonds. This catalytic performance was demonstrated during the chemoselective hydroxylation of gaseous alkanes, regioselective -demethylation of aryl ethers, highly ()-enantioselective epoxidation of styrene, and regio- and enantiomerically diverse hydroxylation of alkylbenzenes. Second, we demonstrated that DFSM-facilitated P450BM3 peroxygenase could be effectively switched to an efficient peroxidase mode through mechanism-guided protein engineering of redox-sensitive residues. Utilizing the peroxidase function of P450 enabled the direct nitration of unsaturated hydrocarbons including phenols, aromatic amines, and styrene derivatives, which was not only the P450-catalyzed direct nitration of phenols and aromatic amines for the first time but also the first example of the direct biological nitration of olefins. Finally, we report an HO tunnel engineering strategy to enable peroxygenase activity in several different P450 monooxygenases for the first time, providing a general approach for accessing engineered P450 peroxygenases. In this Account, we highlight the emerging strategies we have developed for producing practical P450 peroxizyme biocatalysts. Although the DFSM strategy is primarily applied to P450BM3 to date, both strategies of redox-sensitive residue engineering and HO tunnel engineering show great potential to extend to other P450s. These strategies have expanded the scope of applications of P450 chemistry and catalysis. Additionally, they provide a unique solution to the challenging selective oxidation of inert C-H bonds in synthetic chemistry.