Theoretical study of the <i>in situ</i> formation of H₂O₂ by lytic polysaccharide monooxygenases: the reaction mechanism depends on the type of reductant

Lytic polysaccharide monooxygenases (LPMOs) are a unique group of monocopper enzymes that exhibit remarkable ability to catalyze the oxidative cleavage of recalcitrant carbohydrate substrates, such as cellulose and chitin, by utilizing O₂ or H₂O₂ as the oxygen source. One of the key challenges in understanding the catalytic mechanism of LPMOs lies in deciphering how they activate dioxygen using diverse reductants. To shed light on this intricate process, we conducted in-depth investigations using quantum mechanical/molecular mechanical (QM/MM) metadynamics simulations, molecular dynamics (MD) simulations, and density functional theory (DFT) calculations. Specifically, our study focuses on elucidating the <i>in situ</i> formation mechanism of H₂O₂ by LPMOs in the presence of cellobiose dehydrogenase (CDH), a proposed natural reductant of LPMOs. Our findings reveal a proton-coupled electron transfer (PCET) process in generating the Cu(ii)-hydroperoxide intermediate from the Cu(ii)-superoxide intermediate. Subsequently, a direct proton transfer to the proximal oxygen of Cu(ii)-hydroperoxide results in the formation of H₂O₂ and LPMO-Cu(ii). Notably, this mechanism significantly differs from the LPMO/ascorbate system, where two hydrogen atom transfer reactions are responsible for generating H₂O₂ and LPMO-Cu(i). Based on our simulations, we propose a catalytic mechanism of LPMO in the presence of CDH and the polysaccharide substrate, which involves competitive binding of the substrate and CDH to the reduced LPMOs. While the CDH-bound LPMOs can activate dioxygen to generate H₂O₂, the substrate-bound LPMOs can employ the H₂O₂ generated from the LPMO/CDH system to perform the peroxygenase reactions of the polysaccharide substrate. Our work not only provides valuable insights into the reductant-dependent mechanisms of O₂ activation in LPMOs but also holds implications for understanding the functions of these enzymes in their natural environment.