Understanding the Multifaceted Mechanism of Compound I Formation in Unspecific Peroxygenases through Multiscale Simulations

Abstract

Unspecific peroxygenases (UPOs) can selectively oxyfunctionalize unactivated hydrocarbons by using peroxides under mild conditions. They circumvent the oxygen dilemma faced by cytochrome P450s and exhibit greater stability than the latter. As such, they hold great potential for industrial applications. A thorough understanding of their catalysis is needed to improve their catalytic performance. However, it remains elusive how UPOs effectively convert peroxide to Compound I (CpdI), the principal oxidizing intermediate in the catalytic cycle. Previous computational studies of this process primarily focused on heme peroxidases and P450s, which have significant differences in the active site from UPOs. Additionally, the roles of peroxide unbinding in the kinetics of CpdI formation, which is essential for interpreting existing experiments, have been understudied. Moreover, there has been a lack of free energy characterizations with explicit sampling of protein and hydration dynamics, which is critical for understanding the thermodynamics of the proton transport (PT) events involved in CpdI formation. To bridge these gaps, we employed multiscale simulations to comprehensively characterize the CpdI formation in wild-type UPO from Agrocybe aegerita (AaeUPO). Extensive free energy and potential energy calculations were performed in a quantum mechanics/molecular mechanics setting. Our results indicate that substrate-binding dehydrates the active site, impeding the PT from H2O2 to a nearby catalytic base (Glu196). Furthermore, the PT is coupled with considerable hydrogen bond network rearrangements near the active site, facilitating subsequent O–O bond cleavage. Finally, large unbinding free energy barriers kinetically stabilize H2O2 at the active site. These findings reveal a delicate balance among PT, hydration dynamics, hydrogen bond rearrangement, and cosubstrate unbinding, which collectively enable efficient CpdI formation. Our simulation results are consistent with kinetic measurements and offer new insights into the CpdI formation mechanism at atomic-level details, which can potentially aid the design of next-generation biocatalysts for sustainable chemical transformations of feedstocks.

Computational Characterization of the Reactivity of Compound I in Unspecific Peroxygenases

“…Of particular significance is Glu196: when its charge is neutralized, the energy barrier increases by over 6 kcal/mol. Notably, our recent study reveals that this residue is also an essential catalytic base that facilitates CpdI’s formation via the mixed homolysis–heterolysis mechanism . This underscores the multifaceted roles of Glu196 in both the preparatory and the oxidative phases of the Aae UPO’s catalytic cycle.…”

“…The hydroxylation of n -alkanes by WT Aae UPO exhibits enantioselectivity in addition to regioselectivity . Specifically, for n -heptane, the enantiomeric excess (ee) of the R enantiomer is 62.2% for 2-alcohols and 99.9% for 3-alcohols . To decipher the molecular mechanisms underlying this enantioselectivity, we first analyzed the distribution of the angle α Fe–O–H in the CpdI state from selected conformations in classical MD simulations (Figure ).…”

“…To this end, QM/MM calculations are indispensable for distinguishing the reactivities of different sites in the substrate. Building upon our prior research and combining both free energy simulations and potential energy calculations at stationary points, we determined that the HAT step emerges as the rate-limiting step of the overall catalytic process. Importantly, our results emphasize that distinct kinetics for HAT across different types of H atoms largely dictate the observed selectivity, showing a marked preference toward the R enantiomers of 2- and 3-alcohols.…”

“…Our analysis delves into the key factors determining the enantio- and regioselectivity of the hydroxylation and elucidates the role of protein electrostatics in enzyme activity. Together with our preceding study, this study offers a comprehensive characterization of the entire catalytic cycle of Aae UPO.…”

“…Although recent studies have employed molecular simulations to understand the catalytic selectivity , and facilitate the directed evolution of UPOs, − these simulations fell short of systematic quantum mechanical (QM) characterizations of substrate reactivities in realistic enzyme environments. Notably, no previous studies characterized the free energy profiles of UPO-catalyzed reactions at QM level of theory, which are essential for understanding the chemical reactivity in condensed-phase biological systems. − As established in our prior study, relying solely on potential energy calculations can be misleading due to their sensitivity to the initial conditions (ICs) in intricate biological systems. Also, potential energy calculations often fail to accurately incorporate the entropic contributions from conformational fluctuations of the system.…”