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

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.