On the pH Dependency of the Catalysis by a Lytic Polysaccharide Monooxygenase from the Fungus <i>Trichoderma reesei</i>

Lytic polysaccharide monooxygenases (LPMOs) catalyze the oxidative cleavage of glycosidic bonds in industrially important polysaccharides like cellulose. The activity of these monocopper enzymes depends on the presence of H2O2 cosubstrate and reductant. Besides the polysaccharide peroxygenase reaction, LPMOs catalyze reductant oxidase and peroxidase side reactions. The multiplicity and interplay between different LPMO reactions hamper the interpretation of the kinetic data, which is best reflected by the scarcity of the studies of the pH dependency of the LPMO catalysis. Here, we studied the pH dependency of the reductant oxidase/peroxidase as well as the cellulose peroxygenase reactions of TrAA9A, an LPMO from the fungus Trichoderma reesei. The pH dependency of reductant oxidase/peroxidase reaction was governed by the rate-limiting step (reduction of LPMO-Cu(II) or reoxidation of LPMO-Cu(I)) depending on the experimental conditions. The pH dependency of the kcat and Km(H2O2) of the cellulose peroxygenase reaction was best described by the single base catalysis with pKa around 3.5–4.0. Experiments made in D2O showed isotope effects but only at pD values below 5.0. The conserved second coordination sphere histidine (His163) is the most probable candidate responsible for shaping the pH dependency of the TrAA9A peroxygenase reaction. We propose that the double protonation of His163 at acidic pH alters its conformation to the catalytically incompetent one. However, the little pH dependency of the kcat/Km(H2O2) of the cellulose peroxygenase reaction suggests that at very low H2O2 concentrations, TrAA9A may be efficient catalyst also in the acidic conditions that prevail in fungal habitats.