Electrochemical Monitoring of Heterogeneous Peroxygenase Reactions Unravels LPMO Kinetics

Biological conversion of plant biomass depends on peroxygenases and peroxidases acting on insoluble polysaccharides and lignin. Among these are cellulose- and hemicellulose-degrading lytic polysaccharide monooxygenases (LPMOs), which have revolutionized our concept of biomass degradation. Major obstacles limiting mechanistic and functional understanding of these unique peroxygenases are their complex and insoluble substrates and the hard-to-measure H₂O₂ consumption, resulting in the lack of suitable kinetic assays. We report a versatile and robust electrochemical method for real-time monitoring and kinetic characterization of LPMOs and other H₂O₂-dependent interfacial enzymes based on a rotating disc electrode for the sensitive and selective quantitation of H₂O₂ at biologically relevant concentrations. The H₂O₂ sensor works in suspensions of insoluble substrates as well as in homogeneous solutions. Our characterization of multiple LPMOs provides unprecedented insights into the substrate specificity, kinetics, and stability of these enzymes. High turnover and total turnover numbers demonstrate that LPMOs are fast and durable biocatalysts.