Experimental estimation of copper-site geometry reproducibility in biologically relevant redox and saccharide-bound states of a model lytic polysaccharide monooxygenase.

Lytic polysaccharide monooxygenases (LPMOs) are copper-dependent enzymes classified into the auxiliary activity (AA) families of the CAZy database. They oxidatively cleave glycosidic bonds in recalcitrant polysaccharides, playing a key role in biomass degradation and contributing to the virulence of some pathogens. The redox state of the active-site copper and its coordination geometry are central to LPMO catalysis, yet the subtle structural consequences of redox and substrate-binding transitions remain insufficiently resolved. In previous work, a comparative X-ray crystallographic analysis of a model AA9 LPMO (LsAA9A) was conducted under four distinct conditions at 100 K: Cu(II), Cu(I) and the corresponding saccharide-bound states, with the Cu(I) state generated by X-ray photoreduction. In this study, LsAA9A crystals were chemically reduced with or without saccharide substrate prior to low-dose X-ray data collection to minimize radiation damage. Copper-coordination distances and angles were determined precisely through triplicate structure determinations (each from an independent crystal) for each condition, revealing small but reproducible geometry differences across key states in the LPMO catalytic pathway. In order to identify the most significant differences, statistical evaluation using one-way analysis of variance (ANOVA), followed by Tukey-Kramer post hoc tests and pairwise t-tests, was carried out. Within the assumptions made, statistically significant differences in the coordinated Cu-His1 N