Flavin coenzymes are universally found in biological redox reactions. DNA photolyases, with their flavin chromophore (FAD), utilize blue light for DNA repair and photoreduction. The latter process involves two single-electron transfers to FAD with an intermittent protonation step to prime the enzyme active for DNA repair. Here we use time-resolved serial femtosecond X-ray crystallography to describe how light-driven electron transfers trigger subsequent nanosecond-to-microsecond entanglement between FAD and its Asn/Arg-Asp redox sensor triad. We found that this key feature within the photolyase-cryptochrome family regulates FAD re-hybridization and protonation. After first electron transfer, the FAD^•− isoalloxazine ring twists strongly when the arginine closes in to stabilize the negative charge. Subsequent breakage of the arginine–aspartate salt bridge allows proton transfer from arginine to FAD^•−. Our molecular videos demonstrate how the protein environment of redox cofactors organizes multiple electron/proton transfer events in an ordered fashion, which could be applicable to other redox systems such as photosynthesis.

黄素辅酶普遍存在于生物氧化还原反应中。DNA 光解酶及其黄素发色团 (FAD) 利用蓝光进行 DNA 修复和光还原。后一个过程涉及到 FAD 的两个单电子转移，其中间断质子化步骤以启动对 DNA 修复具有活性的酶。在这里，我们使用时间分辨的串行飞秒 X 射线晶体学来描述光驱动电子转移如何触发 FAD 与其 Asn/Arg-Asp 氧化还原传感器三元组之间的后续纳秒到微秒纠缠。我们发现光解酶-隐花色素家族中的这一关键特征可调节 FAD 再杂交和质子化。第一次电子转移后，FAD ^•−当精氨酸靠近以稳定负电荷时，异恶嗪环强烈扭曲。随后精氨酸-天冬氨酸盐桥的断裂允许质子从精氨酸转移到 FAD ^•−。我们的分子视频展示了氧化还原辅因子的蛋白质环境如何以有序的方式组织多个电子/质子转移事件，这可能适用于其他氧化还原系统，例如光合作用。