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 H ₂O ₂ is the most efficient cosubstrate for LPMOs. Understanding the reaction mechanism of LPMOs with H ₂O ₂ 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 H ₂O ₂ 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 H ₂O ₂-driven catalytic cycles of a fungal LPMO. Using rapid mixing stopped-flow spectroscopy, alongside electron paramagnetic resonance and UV-Vis spectroscopy, we reveal how H ₂O ₂ and xyloglucan interact with the enzyme and investigate transient species that form uncoupled pathways of NcLPMO9C. Our study shows how the H ₂O ₂ 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 H ₂O ₂ 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.