Investigating the Substrate Oxidation Mechanism in Lytic Polysaccharide Monooxygenase: H₂O₂- versus O₂-Activation

Lytic polysaccharide monooxygenases (LPMOs) form a copper-dependent family of enzymes classified under the auxiliary activity (AA) superfamily. The LPMOs are known for their boosting of polysaccharide degradation through oxidation of the glycosidic bonds that link the monosaccharide subunits. This oxidation has been proposed to be dependent on either O₂ or H₂O₂ as cosubstrate. Theoretical investigations have previously supported both mechanisms, although this contrasts with recent experiments. A possible explanation is that the theoretical results critically depend on how the Cu active site is modeled. This has also led to different results even when employing only H₂O₂ as cosubstrate. In this paper, we investigate both the O₂- and H₂O₂-driven pathways, employing LsAA9 as the underlying LPMO and a theoretical model based on a quantum mechanics/molecular mechanics (QM/MM) framework. We ensure to consistently include all residues known to be important by using extensive QM regions of up to over 900 atoms. We also investigate several conformers that can partly explain the differences seen in previous studies. We find that the O₂-driven reaction is unfeasible, in contrast with our previous QM/MM calculations with smaller QM regions. Meanwhile, the H₂O₂-driven pathway is feasible, showing that for LsAA9, only H₂O₂ is a viable cosubstrate as proposed experimentally.