Multiscale Modeling of Lytic Polysaccharide Monooxygenases in Oxidized and Reduced Redox States

Abstract Lytic polysaccharide monooxygenases (LPMOs) are copper-dependent oxidative enzymes whose catalytic activity depends on redox cycling of a single copper ion in the active site, coordinated by the conserved histidine brace motif. Although the static coordination geometry of the active site has been characterized crystallographically, the dynamic response of the histidine brace to Cu(I)/Cu(II) cycling and the role of second-sphere residues in modulating this response remain poorly understood at the atomistic level. Here, we address this gap using a multiscale computational approach combining molecular dynamics (MD) simulations, QM/MM optimization, and EPR parameter calculations, applied to two representative LPMOs: NcAA9C (AA9, axial tyrosine) and BaAA10A (AA10, axial phenylalanine). The structural response to the change in oxidation state is seen almost entirely in the exogenous ligands rather than in the histidine brace itself, with the oxidized form showing markedly higher and longer-lived water coordination. In BaAA10A, the equatorial water is anchored by a second-sphere glutamate with no counterpart in NcAA9C. QM/MM electrostatic perturbation analysis identifies Asp76 (NcAA9C) and Glu68 (BaAA10A) as the dominant stabilizing residues of the catalytic core, acting through water-mediated hydrogen-bond bridges that are more pronounced in the Cu(II) state. Computed EPR parameters respond to the displacement of the copper site from a planar geometry, and the equatorial water position was shown to have the dominant effect, differentiating the two LPMOs.