Investigating the Substrate Oxidation Mechanism in Lytic Polysaccharide Monooxygenase: HO- versus O-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 O or HO 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 HO as cosubstrate. In this paper, we investigate both the O- and HO-driven pathways, employing AA9 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 O-driven reaction is unfeasible, in contrast with our previous QM/MM calculations with smaller QM regions. Meanwhile, the HO-driven pathway is feasible, showing that for AA9, only HO is a viable cosubstrate as proposed experimentally.