Peroxide-Driven Hydroxylation of Small Alkanes Catalyzed by an Artificial P450BM3 Peroxygenase System

We report the selective hydroxylation of small alkanes with H2O2 catalyzed by an artificial P450 peroxygenase system generated from engineered cytochrome P450BM3 variants in assistance with dual-functional small molecule (DFSM), in which DFSM acts as a general acid–base co-catalyst for activating H2O2. This peroxygenase system exhibited comparable catalytic turnover number (TON) to the fungal peroxygenase AaeUPO, the only known H2O2-dependent natural alkane hydroxylase. Moreover, when compared with evolved/engineered NADPH-dependent P450 variants, the current system yielded similar or even better product formation rates (PFRs) but lower total TONs. The substitution of the highly conserved T268 with amino acids having hydrophobic side chains was identified to play critical roles in improving the hydroxylation activity of the DFSM-facilitated P450BM3 peroxygenase system, which is distinct from NADPH-dependent P450 enzymes. These results offer useful insights into how to tune the catalytic functions and chemistry of P450 peroxygenases.