Serial femtosecond X‐ray crystallography of an anaerobically formed catalytic intermediate of copper amine oxidase

The mechanisms by which enzymes promote catalytic reactions efficiently through their structural changes remain to be fully elucidated. Recent progress in serial femtosecond X‐ray crystallography (SFX) using X‐ray free‐electron lasers (XFELs) has made it possible to address these issues. In particular, mix‐and‐inject serial crystallography (MISC) is promising for the direct observation of structural changes associated with ongoing enzymic reactions. In this study, SFX measurements using a liquid‐jet system were performed on microcrystals of bacterial copper amine oxidase anaerobically premixed with a substrate amine solution. The structure determined at 1.94 Å resolution indicated that the peptidyl quinone cofactor is in equilibrium between the aminoresorcinol and semiquinone radical intermediates, which accumulate only under anaerobic single‐turnover conditions. These results show that anaerobic conditions were well maintained throughout the liquid‐jet SFX measurements, preventing the catalytic intermediates from reacting with dioxygen. These results also provide a necessary framework for performing time‐resolved MISC to study enzymic reaction mechanisms under anaerobic conditions. Serial femtosecond X‐ray crystallography was performed using a liquid‐jet system on bacterial copper amine oxidase microcrystals anaerobically premixed with an amine substrate. The structure determined at 1.94 Å resolution revealed catalytic intermediates in two reduced forms, confirming that anaerobic conditions were well maintained throughout the measurements.