Kinetic insights into the role of the reductant in H2O2-driven degradation of chitin by a bacterial lytic polysaccharide monooxygenase

Lytic polysaccharide monooxygenases (LPMOs) are monocopper enzymes that catalyze oxidative cleavage of glycosidic bonds in polysaccharides in the presence of an external electron donor (reductant). In the classical O-2-driven monooxygenase reaction, the reductant is needed in stoichiometric amounts. In a recently discovered, more efficient H2O2-driven reaction, the reductant would be needed only for the initial reduction (priming) of the LPMO to its catalytically active Cu(I) form. However, the influence of the reductant on reducing the LPMO or on H2O2 production in the reaction remains undefined. Here, we conducted a detailed kinetic characterization to investigate how the reductant affects H2O2-driven degradation of C-14-labeled chitin by a bacterial LPMO, SmLPMO10A (formerly CBP21). Sensitive detection of C-14-labeled products and careful experimental set-ups enabled discrimination between the effects of the reductant on LPMO priming and other effects, in particular enzyme-independent production of H2O2 through reactions with O-2. When supplied with H2O2, SmLPMO10A catalyzed 18 oxidative cleavages per molecule of ascorbic acid, suggesting a priming reduction reaction. The dependence of initial rates of chitin degradation on reductant concentration followed hyperbolic saturation kinetics, and differences between the reductants were manifested in large variations in their half-saturating concentrations (KmRapp). Theoretical analyses revealed that KmRapp decreases with a decreasing rate of polysaccharide-independent LPMO reoxidation (by either O-2 or H2O2). We conclude that the efficiency of LPMO priming depends on the relative contributions of reductant reactivity, on the LPMO's polysaccharide monooxygenase/peroxygenase and reductant oxidase/peroxidase activities, and on reaction conditions, such as O-2, H2O2, and polysaccharide concentrations.