Switching an Artificial P450 Peroxygenase into Peroxidase via Mechanism-Guided Protein Engineering

Exploring the catalytic promiscuity of enzymes is a longstanding challenge and a current topic of interest. Our group previously modified a cytochrome P450BM3 monooxygenase to perform peroxygenase activity with assistance from a rationally designed dual-functional small molecule (DFSM). However, the DFSM-facilitated P450-H2O2 system showed limited peroxidase activity. On the basis of a mechanistic analysis of the possible competitive oxidation pathways, the present work applies a protein engineering strategy of mutating redox-sensitive residues that enables the peroxygenase system to achieve efficient peroxidase activity. The engineered system exhibits efficient one-electron oxidation activity toward various substrates, including guaiacol, 2,6-dimethoxyphenol, o-phenylenediamine, and p-phenylenediamine. This system attains the best peroxidase activity of any P450 reported to date and rivals most natural peroxidases, suggesting significant potential for practical applications. This work provides insights and strategies relevant for expanding the catalytic promiscuity of P450s through combining the effects of protein engineering and exogenous molecules.