Participation of redox-active residues in polysaccharide monooxygenase catalysis

Polysaccharide monooxygenases (PMOs) degrade carbohydrate polymers via an oxidative mechanism involving activation of O₂ or H₂O₂ at a mononuclear copper active site. The reaction with O₂ requires the timely delivery of two electrons to the copper during each catalytic cycle. Large polymeric substrates might occlude the active site from reductant access, which raises questions of whether the substrate stays associated with the PMO during catalysis and whether there is an alternate path for electrons to reach the active site. Previous work provided evidence for electron transfer through residues Y168 (the axial tyrosine) and Y62 in an AA9 PMO, MtPMO9E. Results reported here further investigate these redox-active residues by comparing the Y62F/Y168F double variant (2F) to the wild-type (WT) enzyme. Oxidase activity and oxygenase activity were compared using three different reductants: cysteine, ascorbate, and cellobiose dehydrogenase. Unlike cysteine and ascorbate, cellobiose dehydrogenase is comparatively large and thought to be the native electron donor to fungal PMOs. The oxidase and oxygenase activities of 2F are significantly decreased compared to WT, but the peroxygenase activity is unaffected. The slow oxygenase activity of 2F suggests the PMO is preferentially reduced via the redox-active residues, although some reduction may occur directly at the active site when the substrate is present. Engineering tyrosine residues into an AA9 PMO that natively lacks the redox-active residues increases oxidase activity. The oxygenase reaction is first-order in the reductant, suggesting the rate-limiting step involves electron transfer between the reductant and the redox-active residues. These observations underscore the importance of redox-active residues in O₂ utilization by PMOs.