Computational Characterization of the Reactivity of Compound I in Unspecific Peroxygenases

Unspecific peroxygenases (UPOs) are emerging as promising biocatalysts for selective oxyfunctionalization of unactivated C–H bonds. However, their potential in large-scale synthesis is currently constrained by suboptimal chemical selectivity. Improving the selectivity of UPOs requires a deep understanding of the molecular basis of their catalysis. Recent molecular simulations have sought to unravel UPO’s selectivity and inform their design principles. However, most of these studies focused on substrate-binding poses. Few researchers have investigated how the reactivity of CpdI, the principal oxidizing intermediate in the catalytic cycle, influences selectivity in a realistic protein environment. Moreover, the influence of protein electrostatics on the reaction kinetics of CpdI has also been largely overlooked. To bridge this gap, we used multiscale simulations to interpret the regio- and enantioselective hydroxylation of the n-heptane substrate catalyzed by Agrocybe aegerita UPO (AaeUPO). We comprehensively characterized the energetics and kinetics of the hydrogen atom-transfer (HAT) step, initiated by CpdI, and the subsequent oxygen rebound step forming the product. Notably, our approach involved both free energy and potential energy evaluations in a quantum mechanics/molecular mechanics (QM/MM) setting, mitigating the dependence of results on the choice of initial conditions. These calculations illuminate the thermodynamics and kinetics of the HAT and oxygen rebound steps. Our findings highlight that both the conformational selection and the distinct chemical reactivity of different substrate hydrogen atoms together dictate the regio- and enantio-selectivity. Building on our previous study of CpdI’s formation in AaeUPO, our results indicate that the HAT step is the rate-limiting step in the overall catalytic cycle. The subsequent oxygen rebound step is swift and retains the selectivity determined by the HAT step. We also pinpointed several polar and charged amino acid residues whose electrostatic potentials considerably influence the reaction barrier of the HAT step. Notably, the Glu196 residue is pivotal for both the CpdI’s formation and participation in the HAT step. Our research offers in-depth insights into the catalytic cycle of AaeUPO, which will be instrumental in the rational design of UPOs with enhanced properties.