The blue light using flavin(BLUF)domain is one of nature’s smallest photoswitching protein domains,yet a cross-species photoactivation mechanism is lacking.Its photoactivation involves an intricate bidirectional prot...The blue light using flavin(BLUF)domain is one of nature’s smallest photoswitching protein domains,yet a cross-species photoactivation mechanism is lacking.Its photoactivation involves an intricate bidirectional proton-coupled electron transfer(PCET)reaction;however,the key reverse PCET route remains largely elusive,with its elementary steps undissected.Here,we resolved the light-state photoreaction cycles of the BLUF domains in 3 species,i.e.,AppA from Rhodobacter sphaeroides,OaPAC from Oscillatoria acuminata,and SyPixD from Synechocystis sp.PCC6803,with a unified kinetic model.Using mutant design and femtosecond spectroscopy,we captured the spectroscopic snapshots of a key proton-relay intermediate in all species,revealing that the light-state photoreaction cycle consists of 4 elementary steps including a forward concerted electron-proton transfer(CEPT),a 2-step proton rocking,and a reverse CEPT.We emphasize that the last reverse CEPT step(1.5 to 3.7 ps)is shared by both the light-state and dark-state photocycles and is essential to the photoswitching functionality.展开更多
基金supported by the National Key Research and Development Program of China(grant 2020YFA0509700)National Natural Science Foundation of China(grant 31971233).
文摘The blue light using flavin(BLUF)domain is one of nature’s smallest photoswitching protein domains,yet a cross-species photoactivation mechanism is lacking.Its photoactivation involves an intricate bidirectional proton-coupled electron transfer(PCET)reaction;however,the key reverse PCET route remains largely elusive,with its elementary steps undissected.Here,we resolved the light-state photoreaction cycles of the BLUF domains in 3 species,i.e.,AppA from Rhodobacter sphaeroides,OaPAC from Oscillatoria acuminata,and SyPixD from Synechocystis sp.PCC6803,with a unified kinetic model.Using mutant design and femtosecond spectroscopy,we captured the spectroscopic snapshots of a key proton-relay intermediate in all species,revealing that the light-state photoreaction cycle consists of 4 elementary steps including a forward concerted electron-proton transfer(CEPT),a 2-step proton rocking,and a reverse CEPT.We emphasize that the last reverse CEPT step(1.5 to 3.7 ps)is shared by both the light-state and dark-state photocycles and is essential to the photoswitching functionality.
