Understanding the molecular basis for controlled H 2 O 2 activation is of fundamental importance for peroxide-driven catalysis by metalloenzymes. In addition to O 2 activation in the presence of stoichiometric reductants, an increasing number of metalloenzymes are found to activate the H 2 O 2 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-Lphenylalanine (Im-C6-Phe), which enables an efficient peroxygenase activity for P450BM3. Our computational investigations show that the H 2 O 2 activations by P450BM3 are highly dependent on the substrate and the DFSM. In the absence of both the substrate and the DFSM, H 2 O 2 activation via the O−O homolysis mechanism is significantly inhibited by the H-bonding network from the proximal H of H 2 O 2 . However, the presence of the substrate expels the solvation waters and disrupts the H-bonding network from the proximal H of H 2 O 2 , 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 H 2 O 2 , 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 H 2 O 2 is the key to control of the H 2 O 2 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 H 2 O 2 activations.

No abstract

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 M.aq ) 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.