Electrochemical Insight into the Copper Redox Chemistry and H ₂ O ₂ and O ₂ Reducing Capability of Two AA10 Lytic Polysaccharide Monooxygenases

Lytic polysaccharide monooxygenases ([L]-PMOs) are copper-containing enzymes that catalyse cleavage of the glycosidic bond, a process central to microbial biomass degradation. Here, we describe electrochemical methods used to investigate the Cu^2+/1+ redox chemistry and the polysaccharide-free catalytic activity of two AA10 LPMOs: <i>Cj</i>AA10B from <i>Cellvibrio japonicus</i> and <i>Cf</i>AA10 from <i>Cellulomonas fimi</i>. Immobilisation of these enzymes on the surface of a graphite electrode allows for direct electrochemical measurements of Cu^2+/1+ redox cycling as well as the ability of both LPMOs to reduce H₂O₂ vs O₂. These measurements can be advantageous when compared to biological dye assays as they provide direct kinetic measurements and allow for investigation over a wider range of environmental conditions. Values of <i>k</i> _cat and <i>K</i> _M- are reported for H₂O₂ and O₂ reduction by <i>Cj</i>AA10B and <i>Cf</i>AA10 from pH 5-7, with <i>Cf</i>AA10 consistently outperforming <i>Cj</i>AA10B. Both enzymes perform faster catalysis with H₂O₂ but when comparing the affinity-coupled specificity constant (<i>k</i> _cat/<i>K</i> _M), the LPMOs perform similarly with both H₂O₂ and O₂, suggesting both substrates are viable. We also note an increase in redox signals as pH is decreased that correlates with EPR data suggesting a second species is formed <pH 5, postulated to occur due to the protonation of a glutamate residue (p<i>K</i> _a ∼ 4.6). The increase in signal size with decreasing pH that is seen for the non-catalytic Cu^2+/1+ transition is interpreted in light of an increasing proportion of electroactive species at low pH; such a change in activity with pH is notably not observed in the presence of substrate (H₂O₂ or O₂). This suggests that substrate binding modulates the active site, disrupting the effect of protonation. These findings establish electrochemistry as a powerful tool for probing LPMO activity.