Light Activates Copper Radical Oxidases

Abstract Copper radical oxidases (CROs) form a functionally diverse family of enzymes catalyzing a broad range of oxidation reactions, converting mainly primary alcohols into aldehydes, making them attractive catalysts in several industrial applications. To be catalytically competent, all known fungal CROs need to undergo a priming oxidoreduction at their active site, which is usually carried out by peroxidases, chemical oxidants, or electrochemical approaches. Here, we discovered that electromagnetic waves, namely, ultraviolet (UV) light, can be used to prime CROs. Using as model the alcohol oxidase from the fungal phytopathogen Colletotrichum graminicola, we show that the UV-light intensity, wavelength range, and illumination mode (continuous vs discontinuous) have an impact on the enzymatic rate. Using electron paramagnetic resonance spectroscopy, we observed that UV-light irradiation induces the appearance of a radical species in the vicinity of the copper center. Furthermore, fluorescent probe assays show that the light-induced activation does not proceed via hydroxyl radicals generated in solution, suggesting a more direct mechanism. Site-directed mutagenesis pinpointed a copper-facing tryptophan adjacent to the Cys-Tyr cofactor as pivotal in the photoactivation process. Importantly, we show that this photoactivation mechanism also applies to other CRO members with different substrate specificities, including the archetypal galactose 6-oxidase as well as an aryl alcohol oxidase. Beyond presenting an alternative way of activating these biocatalysts, using light as an additive-free source of energy, our study raises questions regarding the chemistry at play in CROs and the potential importance of such photoinduced process in nature.