MANUEL MAESTRE-REYNAYang C.-HNango EHuang W.-CNgurah Putu E.P.GWu W.-JWang P.-HFranz-Badur SSaft MEmmerich H.-JWu H.-YLee C.-CHuang K.-FChang Y.-KLiao J.-HWeng J.-HGad WChang C.-WPang A.HSugahara MOwada SHosokawa YJoti YYamashita ATanaka RTanaka TLuo FTono KHsu K.-CKiontke SSchapiro ISpadaccini RRoyant AYamamoto JIwata SEssen L.-OBessho YTsai M.-D.2022-11-112022-11-11202217554330https://www.scopus.com/inward/record.uri?eid=2-s2.0-85127648859&doi=10.1038%2fs41557-022-00922-3&partnerID=40&md5=65afb613e670bbbcb95aa5e0e2d5c5a5https://scholars.lib.ntu.edu.tw/handle/123456789/624866Flavin 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. [Figure not available: see fulltext.] © 2022, The Author(s), under exclusive licence to Springer Nature Limited.[SDGs]SDG2[SDGs]SDG13arginine; deoxyribodipyrimidine photolyase; flavine adenine nucleotide; proton; quercetin; chemistry; crystallography; electron; electron transport; genetics; metabolism; oxidation reduction reaction; Arginine; Crystallography; Deoxyribodipyrimidine Photo-Lyase; Electron Transport; Electrons; Flavin-Adenine Dinucleotide; Flavins; Oxidation-Reduction; Protonsjournal article10.1038/s41557-022-00922-3353935542-s2.0-85127648859