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 SmAA10A 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 x 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.
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