Reactivity of O₂ versus H₂O₂ with polysaccharide monooxygenases

Enzymatic conversion of polysaccharides into lower-molecular-weight, soluble oligosaccharides is dependent on the action of hydrolytic and oxidative enzymes. Polysaccharide monooxygenases (PMOs) use an oxidative mechanism to break the glycosidic bond of polymeric carbohydrates, thereby disrupting the crystalline packing and creating new chain ends for hydrolases to depolymerize and degrade recalcitrant polysaccharides. PMOs contain a mononuclear Cu(II) center that is directly involved in C-H bond hydroxylation. Molecular oxygen was the accepted cosubstrate utilized by this family of enzymes until a recent report indicated reactivity was dependent on H₂O₂ Reported here is a detailed analysis of PMO reactivity with H₂O₂ and O₂, in conjunction with high-resolution MS measurements. The cosubstrate utilized by the enzyme is dependent on the assay conditions. PMOs will directly reduce O₂ in the coupled hydroxylation of substrate (monooxygenase activity) and will also utilize H₂O₂ (peroxygenase activity) produced from the uncoupled reduction of O₂ Both cosubstrates require Cu reduction to Cu(I), but the reaction with H₂O₂ leads to nonspecific oxidation of the polysaccharide that is consistent with the generation of a hydroxyl radical-based mechanism in Fenton-like chemistry, while the O₂ reaction leads to regioselective substrate oxidation using an enzyme-bound Cu/O₂ reactive intermediate. Moreover, H₂O₂ does not influence the ability of secretome from Neurospora crassa to degrade Avicel, providing evidence that molecular oxygen is a physiologically relevant cosubstrate for PMOs.