Mapping the protonation states of the histidine brace in an AA10 lytic polysaccharide monooxygenase using CW-EPR spectroscopy and DFT calculations

The active site of the polysaccharide-degrading lytic polysaccharide monooxygenase (LPMO) enzyme features a single copper ion coordinated by a histidine brace. The primary coordination sphere of the copper contains several ligating atoms which are bonded to ionisable protons (e.g. OH2, NH2), the pK(a)s of which are unknown. Using a combination of CW-EPR X-band spectroscopy over a range of pH values and DFT calculations, we show that the active site of a chitin-active AA10 LPMO can exist in three different protonation states (pK(a1) = 8.7, pK(a2) similar to 11.5), representing the ionisation of the coordinating groups. The middle pH species (fully formed at pH similar to 10.5) is proposed to be Cu(ii)(His)(2)(OH)(2) (N2O2 coordination) with a decoordinated R-NH3+ group at the amino terminus. This species also sees a rotation of the SOMO equatorial plane from the canonical histidine brace plane, whereby the nominal Cu d(x(2) - y(2))-orbital has rotated some 45 degrees along the His-Cu(ii)-His axis, driven by the elongation and decoordination of the amino group. The highest pH species (>12) is proposed to exist as a Cu(ii)-azanide, in which the NH2 of the amino terminus has been deprotonated. The high pH means that this species is unlikely to be biologically relevant in the catalytic cycle of AA10 LPMOs.