Coupled second sphere residues boost H2O2 production in Lytic Polysaccharide Monooxygenases: a computational study in an AA11 LPMO

Lytic polysaccharide monooxygenases (LPMOs) are copper-dependent enzymes that play a central role in biomass deconstruction for sustainable biofuel production. Although H₂O₂ is recognized as the preferred co-substrate, the mechanism of its formation remains incompletely understood. We show the experimental correlation between enhanced H₂O₂ production and the presence of Glu-Tyr pair in the secondary coordination sphere for many LPMOs. We investigate the structural basis using QM/MM calculations on an AA11 enzyme. Multiple proton- and electron-transfer pathways were evaluated, highlighting proton transfer as a key determinant of H₂O₂ formation. The results show that Glu facilitates proton shuttling from Tyr to the Cu-bound hydroperoxo intermediate, promoting H₂O₂ release. Solvation-corrected thermodynamic analyses reveal the importance of first-shell solvent reorganization during dioxygen intermediate dissociation, while Local Energy Decomposition identifies residue-specific non-covalent interactions throughout catalysis. These findings provide mechanistic insights and design principles for engineering more efficient LPMOs and bio-inspired catalysts.