Enhanced in situ H₂O₂ production explains synergy between an LPMO with a cellulose-binding domain and a single-domain LPMO

Lytic polysaccharide monooxygenases (LPMOs) are mono-copper enzymes that catalyze oxidative depolymerization of recalcitrant substrates such as chitin or cellulose. Recent work has shown that LPMOs catalyze fast peroxygenase reactions and that, under commonly used reaction set-ups, access to in situ generated H₂O₂ likely limits catalysis. Based on a hypothesis that the impact of a cellulose-binding module (CBM) on LPMO activity could relate to changes in in situ H₂O₂ production, we have assessed the interplay between CBM-containing ScLPMO10C and its truncated form comprising the catalytic domain only (ScLPMO10C_TR). The results show that truncation of the linker and CBM leads to elevated H₂O₂ production and decreased enzyme stability. Most interestingly, combining the two enzyme forms yields strong synergistic effects, which are due to the combination of high H₂O₂ generation by ScLPMO10C_TR and efficient productive use of H₂O₂ by the full-length enzyme. Thus, cellulose degradation becomes faster, while enzyme inactivation due to off-pathway reactions with excess H₂O₂ is reduced. These results underpin the complexity of ascorbic acid-driven LPMO reactions and reveal a potential mechanism for how LPMOs may interact synergistically during cellulose degradation.