Insights into the H₂O₂-driven catalytic mechanism of fungal lytic polysaccharide monooxygenases

Fungal lytic polysaccharide monooxygenases (LPMOs) depolymerise crystalline cellulose and hemicellulose, supporting the utilisation of lignocellulosic biomass as a feedstock for biorefinery and biomanufacturing processes. Recent investigations have shown that H₂O₂ is the most efficient cosubstrate for LPMOs. Understanding the reaction mechanism of LPMOs with H₂O₂ is therefore of importance for their use in biotechnological settings. Here, we have employed a variety of spectroscopic and biochemical approaches to probe the reaction of the fungal LPMO9C from N. crassa using H₂O₂ as a cosubstrate and xyloglucan as a polysaccharide substrate. We show that a single ‘priming’ electron transfer reaction from the cellobiose dehydrogenase partner protein supports up to 20 H₂O₂-driven catalytic cycles of a fungal LPMO. Using rapid mixing stopped-flow spectroscopy, alongside electron paramagnetic resonance and UV-Vis spectroscopy, we reveal how H₂O₂ and xyloglucan interact with the enzyme and investigate transient species that form uncoupled pathways of NcLPMO9C. Our study shows how the H₂O₂ cosubstrate supports fungal LPMO catalysis and leaves the enzyme in the reduced Cu⁺ state following a single enzyme turnover, thus preventing the need for external protons and electrons from reducing agents or cellobiose dehydrogenase and supporting the binding of H₂O₂ for further catalytic steps. We observe that the presence of the substrate xyloglucan stabilises the Cu⁺ state of LPMOs, which may prevent the formation of uncoupled side reactions.