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) are enzymes that catalyze the oxidative depolymerization of polysaccharides through activation of strong C-H bonds. Their active site is composed of a copper ion that is coordinated by the so-called histidine brace, consisting of two histidine residues that coordinate the metal ion in a T-shaped arrangement. The unique structure of this motif and its suggested ability to generate and stabilize highly oxidizing intermediates has sparked considerable interest, leading to various hypotheses on its role for LPMOs and LPMO-inspired biomimetic complexes. To gain detailed insights into the electronic and geometric structure of the histidine brace and test its chemical variability, we have used advanced electron paramagnetic resonance (EPR) techniques, in combination with isotopic labelling (15N, 2H) of the AA10 LPMO SmAA10A over a wide pH range (pH 4.0 - pH 12.5), in which the monocopper site remains intact and the protein does not exhibit permanent damage. Between pH 4 and 11.5, Electron Nuclear Double Resonance (ENDOR) spectroscopy identifies and discriminates between coordinating waters and hydroxo ions (pKa1 = 9.65) at an open-site of the histidine brace. Above pH 11.5, deprotonation of the two remote nitrogen nuclei of the coordinating imidazole moieties and of the coordinating N-terminal amine function was observed via ENDOR, Electron Spin Echo Envelope Modulation (ESEEM) and Hyperfine Sublevel Correlation (HYSCORE) spectroscopies. This is associated with major electronic changes in the histidine brace, including increased σ-donor capabilities of the imidazolates and an overall reduced interaction of the deprotonated amine function with the copper center. This observation highlights the possible larger role of the imidazole moieties, potentially stabilizing potent oxidants during turnover. The associated spectroscopic and electronic changes of the LPMO are discussed in reference with functional biomimetic complexes. 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.