Decoding the Ambiguous Electron Paramagnetic Resonance Signals in the Lytic Polysaccharide Monooxygenase from Photorhabdus luminescens br

Understanding the structure and function of lytic poly-saccharide monooxygenases (LPMOs), copper enzymes that degraderecalcitrant polysaccharides, requires the reliable atomistic interpretation ofelectron paramagnetic resonance (EPR) data on the Cu(II) active site.Among various LPMO families, the chitin-activePlAA10 shows an intriguingphenomenology with distinct EPR signals, a major rhombic and a minor axialsignal. Here, we combine experimental and computational investigations touncover the structural identity of these signals. X-band EPR spectra recordedat different pH values demonstrate pH-dependent population inversion: themajor rhombic signal at pH 6.5 becomes minor at pH 8.5, where the axialsignal dominates. This suggests that a protonation change is involved in the interconversion. Precise structural interpretations arepursued with quantum chemical calculations. Given that accurate calculations of Cug-tensors remain challenging for quantumchemistry, wefirst address this problem via a thorough calibration study. This enables us to define a density functional that achievesaccurate and reliable prediction ofg-tensors, giving confidence in our evaluation ofPlAA10 LPMO models. Large models wereconsidered that include all parts of the protein matrix surrounding the Cu site, along with the characteristic second-sphere features ofPlAA10. The results uniquely identify the rhombic signal with afive-coordinate Cu ion bearing two water molecules in addition tothree N-donor ligands. The axial signal is attributed to a four-coordinate Cu ion where only one of the waters remains bound, ashydroxy. Alternatives that involve decoordination of the histidine brace amino group are unlikely based on energetics andspectroscopy. These results provide a reliable spectroscopy-consistent view on the plasticity of the resting state inPlAA10 LPMO asa foundation for further elucidating structure-property relationships and the formation of catalytically competent species. Ourstrategy is generally applicable to the study of EPR parameters of mononuclear copper-containing metalloenzymes