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

[Image: see text] Understanding the structure and function of lytic polysaccharide monooxygenases (LPMOs), copper enzymes that degrade recalcitrant polysaccharides, requires the reliable atomistic interpretation of electron paramagnetic resonance (EPR) data on the Cu(II) active site. Among various LPMO families, the chitin-active PlAA10 shows an intriguing phenomenology with distinct EPR signals, a major rhombic and a minor axial signal. Here, we combine experimental and computational investigations to uncover the structural identity of these signals. X-band EPR spectra recorded at different pH values demonstrate pH-dependent population inversion: the major rhombic signal at pH 6.5 becomes minor at pH 8.5, where the axial signal dominates. This suggests that a protonation change is involved in the interconversion. Precise structural interpretations are pursued with quantum chemical calculations. Given that accurate calculations of Cu g-tensors remain challenging for quantum chemistry, we first address this problem via a thorough calibration study. This enables us to define a density functional that achieves accurate and reliable prediction of g-tensors, giving confidence in our evaluation of PlAA10 LPMO models. Large models were considered that include all parts of the protein matrix surrounding the Cu site, along with the characteristic second-sphere features of PlAA10. The results uniquely identify the rhombic signal with a five-coordinate Cu ion bearing two water molecules in addition to three N-donor ligands. The axial signal is attributed to a four-coordinate Cu ion where only one of the waters remains bound, as hydroxy. Alternatives that involve decoordination of the histidine brace amino group are unlikely based on energetics and spectroscopy. These results provide a reliable spectroscopy-consistent view on the plasticity of the resting state in PlAA10 LPMO as a foundation for further elucidating structure–property relationships and the formation of catalytically competent species. Our strategy is generally applicable to the study of EPR parameters of mononuclear copper-containing metalloenzymes.