pH-mediated manipulation of the histidine brace in LPMOs and generation of a tri-anionic variant, investigated by EPR, ENDOR, ESEEM and HYSCORE spectroscopy

Lytic Polysaccharide Monooxygenases (LPMOs) catalyze the oxidative depolymerization of polysaccharides at a monocopper active site, that is coordinated by the so-called histidine brace. In the past, this motif has sparked considerable interest, mostly due to its ability to generate and stabilize highly oxidizing intermediates during catalysis. We used a variety of advanced EPR techniques, including Electron Nuclear Double Resonance (ENDOR), Electron Spin Echo Envelope Modulation (ESEEM) and Hyperfine Sublevel Correlation (HYSCORE) spectroscopy in combination with isotopic labelling (¹⁵N, ²H) to characterize the active site of the bacterial LPMO <i>Sm</i>AA10A over a wide pH range (pH 4.0-pH 12.5). At elevated pH values, several ligand modifications are observed, including changes in the H _<i>x</i> O ligand coordination, but also regarding the protonation state of the histidine brace. At pH > 11.5, the deprotonation of the two remote nitrogen nuclei of the imidazole moieties and of the terminal amine is observed. These deprotonations are associated with major electronic changes, including increased σ-donor capabilities of the imidazolates and an overall reduced interaction of the deprotonated amine function. This observation highlights a potentially more significant role of the imidazole ligands, particularly for the stabilization of potent oxidants during turnover. The presented study demonstrates the application of advanced EPR techniques for a thorough characterization of the active site in LPMOs, which ultimately sets a foundation for and affords an outlook on future applications characterizing reaction intermediates.