QM/MM Studies into the H2O2-Dependent Activity of Lytic Polysaccharide Monooxygenases: Evidence for the Formation of a Caged Hydroxyl Radical Intermediate

Lytic polysaccharide monooxygenases (LPMOs) are promising enzymes for the conversion of lignocellulosic biomass into biofuels and biomaterials. Classically considered oxygenases, recent work suggests that H 2 O 2 can, under certain circumstances, also be a potential substrate. Here we present a detailed mechanism of the activation of H 2 O 2 by a C4-acting LPMO using small-model DFT and QM/MM calculations. We show that there is an efficient mechanism to break the O–O bond of H 2 O 2, with a low barrier of 5.8 kcal/mol, via a one-electron transfer from the LPMO-Cu(I) site to form an HO • radical, stabilized by hydrogen bonding interactions. Our QM/MM calculations further show that the H-bonding machinery of the enzyme directs the HO • radical to abstract a hydrogen atom from the Cu(II)–OH unit rather than from the substrate in what is essentially a caged-radical reaction, thereby forming a Cu(II)-oxyl species. The Cu(II)-oxyl species then exclusively oxidizes the C4–H bond due to the suitable position of the substrate. Our calculations also suggest that the C4-hydroxylated intermediate can be efficiently hydrolyzed in water, and this process does not require enzymatic catalysis.