Spectroscopic and Theoretical Studies of Lytic Polysaccharide Monooxygenases

Lytic polysaccharide monooxygenases (LPMOs) are monometallic copper enzymes able to depolymerize polysaccharides through an oxidative mechanism, using O2 or H2O2 as co-substrates, involving hydroxylation at the C1 or C4 carbon of the polysaccharide chain and leading to subsequent cleavage of the glycosidic bond. In this work we present a multi-spectroscopic and theoretical study of the enzyme/substrate complex, using an LPMO from the auxiliary activity (AA) family 9 and cellohexaose as substrate. We were able to characterize the active site electronic structure in which the Cu ligand environment forces the Cu(II) semi occupied molecular orbital (SOMO) in a particular orientation which allows the formation of a covalent bond with exogenous ligands in the equatorial plane, with potential implications for O2 activation during catalytic turnover of the enzyme. A similar study was performed also with an AA11 LPMO that showed a very similar active site electronic structure as compared to the AA9 LPMO and therefore suggesting similar O2 reactivity with between the two families. Furthermore, when hydrogen peroxide is used as a co-substrate by LPMOs instead of O2, the rate of reaction is high but it is accompanied by rapid inactivation of the enzymes, presumably through protein oxidation. Herein, we present a multi-spectroscopic study, augmented with mass spectrometry and density functional theory calculations, to show that the product of reaction of an AA9 LPMO with H2O2 at higher pHs is a singlet Cu(II)−tyrosyl radical species, which is inactive for the oxidation of polysaccharide substrates. The Cu(II)−tyrosyl radical center entails the formation of significant Cu(II)−(•OTyr) overlap, which in turn requires that the plane of the SOMO of the Cu(II) is orientated toward the tyrosyl radical. We propose from the Marcus cross-relation that the active site tyrosine is part of a “hole-hopping” charge-transfer mechanism formed of a pathway of conserved tyrosine and tryptophan residues, which can protect the protein active site from inactivation during uncoupled turnover.