Difference in the Charge-Separation Energetics between Distinct Conformers in the PixD Photoreceptor.

Blue light using flavin (BLUF) domain proteins are photoreceptors in various organisms. The PixD BLUF domain can adopt two conformations, W91 out and W91 in , with Trp91 either proximal or distal to flavin (FMN). Using a quantum mechanical/molecular mechanical/polarizable continuum model approach, the energetics of charge-separated and biradical states in the two conformations were investigated. In the W91 out conformation, the charge-separated state (FMN •- ) is more stable than the photoexcited state (FMN*), whereas it is less stable due to an electrostatic repulsive interaction with the Ser28 side chain in the W91 in conformation. This leads to a lower activation energy for the charge separation in the W91 out conformation, resulting in a faster charge separation compared to that in the W91 in conformation. In the W91 out conformation, the radical state (FMNH • ) is more stable than FMN •- and forms from FMN •- , leading to reorientation of the Gln50 side chain adjacent to FMN and formation of a hydrogen bond between Gln50 and FMN. Subsequently, a signaling state forms through charge recombination. In contrast, in the W91 in conformation, FMN •- cannot proceed further, returning to the dark-adapted state, as FMNH • is less stable. Thus, formation of the signaling state exclusively occurs in the W91 out conformation.