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.