biostudies-literatureZhou YMinistry of Science and Technology of the People’s Republic of ChinaNational Natural Science Foundation of ChinaMinistry of Science and Technology of the People's Republic of China (Chinese Ministry of Science and Technology)National Natural Science Foundation of China (National Science Foundation of China)623https://www.ebi.ac.uk/biostudies/studies/S-EPMC10799861biostudies-literatureUnknown15(1)Blue light using flavin (BLUF) photoreceptors respond to light via one of nature's smallest photo-switching domains. Upon photo-activation, the flavin cofactor in the BLUF domain exhibits multi-phasic dynamics, quenched by a proton-coupled electron transfer reaction involving the conserved Tyr and Gln. The dynamic behavior varies drastically across different species, the origin of which remains controversial. Here, we incorporate site-specific fluorinated Trp into three BLUF proteins, i.e., AppA, OaPAC and SyPixD, and characterize the percentages for the W_out, W_inNH_in and W_inNH_out conformations using ¹⁹F nuclear magnetic resonance spectroscopy. Using femtosecond spectroscopy, we identify that one key W_inNH<sub>in<Nature communicationsOrigin of the multi-phasic quenching dynamics in the BLUF domains across the species.PMC10799861319712332020YFA0509700Zhang TDing BChen ZHuang JWang BKang XWZhong DTang SZou SZhou YZhou ZfalseOrigin of the multi-phasic quenching dynamics in the BLUF domains across the species.Blue light using flavin (BLUF) photoreceptors respond to light via one of nature's smallest photo-switching domains. Upon photo-activation, the flavin cofactor in the BLUF domain exhibits multi-phasic dynamics, quenched by a proton-coupled electron transfer reaction involving the conserved Tyr and Gln. The dynamic behavior varies drastically across different species, the origin of which remains controversial. Here, we incorporate site-specific fluorinated Trp into three BLUF proteins, i.e., AppA, OaPAC and SyPixD, and characterize the percentages for the W_out, W_inNH_in and W_inNH_out conformations using ¹⁹F nuclear magnetic resonance spectroscopy. Using femtosecond spectroscopy, we identify that one key W_inNH<sub>in<2024-01-01T00:00:00Z2024 Jan2026-06-03T01:41:58.818Z2024-10-15T23:59:45.15ZS-EPMC107998613824551810.1038/s41467-023-44565-5