Time-resolved X-ray crystallography allows time-dependent structural changes to be visualized as they evolve within protein crystals and can yield unique structural insights into the course of a biochemical reaction (1). Serial crystallography (2) is now routinely used for time-resolved X-ray diffraction studies of macromolecules at X-ray free electron laser and synchrotron radiation facilities. As the field grows, biological reactions that are not naturally light sensitive will be increasingly studied using time-resolved serial crystallography (1). In this work, a laser-flash was used to release photocaged oxygen and thereby initiate the reduction of oxygen to water in microcrystals of cytochrome c oxidase (3). Our structural results suggest how this reaction may be coupled to gating the uptake of protons from the cytoplasm and to the release of protons to the periplasm. Similar interdisciplinary experiments will allow other biological reactions to become accessible to time-resolved diffraction.

1. G. Branden, R. Neutze, Advances and challenges in time-resolved macromolecular crystallography. Science 373, eaba0954 (2021).
2. H. N. Chapman et al., Femtosecond X-ray protein nanocrystallography. Nature 470, 73 (2011).
3. R. Andersson et al., Serial femtosecond crystallography structure of cytochrome c oxidase at room temperature. Sci Rep 7, 4518 (2017).​

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