H-Bonding Networks Dictate the Molecular Mechanism of H2O2 Activation by P450

Understanding the molecular basis for controlled H2O2 activation is of fundamental importance for peroxide-driven catalysis by metalloenzymes. In addition to O2 activation in the presence of stoichiometric reductants, an increasing number of metalloenzymes are found to activate the H2O2 cosubstrate for oxidative transformations in the absence of stoichiometric reductants. Herein, we characterized the X-ray structure of the P450BM3 F87A mutant in complex with the dual-functional small molecule (DFSM) N-(ω-imidazolyl)-hexanoyl-l-phenylalanine (Im-C6-Phe), which enables an efficient peroxygenase activity for P450BM3. Our computational investigations show that the H2O2 activations by P450BM3 are highly dependent on the substrate and the DFSM. In the absence of both the substrate and the DFSM, H2O2 activation via the O–O homolysis mechanism is significantly inhibited by the H-bonding network from the proximal H of H2O2. However, the presence of the substrate expels the solvation waters and disrupts the H-bonding network from the proximal H of H2O2, thus remarkably favoring homolytic O–O cleavage toward Cpd I formation. However, the presence of the DFSM forms a proton channel between the imidazolyl group of the DFSM and the proximal H of H2O2, thus enabling a heterolytic O–O cleavage and Cpd I formation that is greatly favored over the homolysis mechanism. Meanwhile, our simulations demonstrate that the H-bonding network from the distal H of H2O2 is the key to control of the H2O2 activation in the homolytic route. These findings are in line with all available experimental data and highlight the key roles of H-bonding networks in dictating H2O2 activations.