Investigation of Water Splitting Reaction by a Multicopper Oxidase through X‐ray Absorption Nanospectroelectrochemistry

Abstract This study introduces an experimental setup based on X‐ray absorption nanospectroelectrochemisty (XA‐NSEC) to investigate the water oxidation reaction (WOR) in a copper‐containing oxidoreductase enzyme. A 3 GeV fourth‐generation synchrotron light source with a high optical demagnification and an X‐ray beam with nanometer‐scale dimensions is used. This technique is particularly beneficial in bioelectrocatalysis as spatial heterogeneities are usually present in enzyme‐modified electrodes. Through XA‐NSEC, the WOR catalyzed by a bilirubin oxidase is investigated as a fortuitous side reaction, demonstrating an inverse proton dependence relative to the native catalytic activity in addition to its main reaction. This approach enables the in situ detection of a high‐valent copper character of the enzyme active site during the water oxidation bioelectrocatalysis and obtention of thermodynamic parameters. The results allow the understanding of the WOR bioelectrocatalytic mechanism and might promote the subsequent development of bioinspired catalysts, while also providing tools to engineer mutant multicopper oxidases with higher performance toward WOR.