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 2 O 2 is the most efficient cosubstrate for LPMOs. Understanding the reaction mechanism of LPMOs with H 2 O 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 2 O 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 2 O 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 2 O 2 and xyloglucan interact with the enzyme and investigate transient species that form uncoupled pathways of NcLPMO9C. Our study shows how the H 2 O 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 2 O 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.

Fatty acid photodecarboxylase (FAP) is a promising target for the production of biofuels and fine chemicals. It contains a flavin adenine dinucleotide cofactor and catalyzes the blue-light-dependent decarboxylation of fatty acids to generate the corresponding alkane. However, little is known about the catalytic mechanism of FAP, or how light is used to drive enzymatic decarboxylation. Here, we have used a combination of time-resolved and cryogenic trapping UV–visible absorption spectroscopy to characterize a red-shifted flavin intermediate observed in the catalytic cycle of FAP. We show that this intermediate can form below the “glass transition” temperature of proteins, whereas the subsequent decay of the species proceeds only at higher temperatures, implying a role for protein motions in the decay of the intermediate. Solvent isotope effect measurements, combined with analyses of selected site-directed variants of FAP, suggest that the formation of the red-shifted flavin species is directly coupled with hydrogen atom transfer from a nearby active site cysteine residue, yielding the final alkane product. Our study suggests that this cysteine residue forms a thiolate-flavin charge-transfer species, which is assigned as the red-shifted flavin intermediate. Taken together, our data provide insights into light-dependent decarboxylase mechanisms catalyzed by FAP and highlight important considerations in the (re)design of flavin-based photoenzymes.

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The unique light-driven enzyme protochlorophyllide oxidoreductase (POR) is an important model system for understanding how light energy can be harnessed to power enzyme reactions. The ultrafast photochemical processes, essential for capturing the excitation energy to drive the subsequent hydride- and proton-transfer chemistry, have so far proven difficult to detect. We have used a combination of time-resolved visible and IR spectroscopy, providing complete temporal resolution over the picosecond–microsecond time range, to propose a new mechanism for the photochemistry. Excited-state interactions between active site residues and a carboxyl group on the Pchlide molecule result in a polarized and highly reactive double bond. This so-called “reactive” intramolecular charge-transfer state creates an electron-deficient site across the double bond to trigger the subsequent nucleophilic attack of NADPH, by the negatively charged hydride from nicotinamide adenine dinucleotide phosphate. This work provides the crucial, missing link between excited-state processes and chemistry in POR. Moreover, it provides important insight into how light energy can be harnessed to drive enzyme catalysis with implications for the design of light-activated chemical and biological catalysts.