The flavoprotein AppA is a blue-light photoreceptor that functions as an antirepressor ofphotosynthesis gene expression in the purple bacterium Rhodobacter sphaeroides. Heterologous expressionstudies show that FAD binds to a 156 amino acid N-terminal domain of AppA and that this domain isitself photoactive. A pulse of white light causes FAD absorption to be red shifted in a biphasic processwith a fast phase occurring in <1 μs and a slow phase occurring at approximately 5 ms. The absorbanceshift was spontaneously restored over a 30 min period, also in a biphasic process as assayed by fluorescencequenching and electronic absorption analyses. Site-directed replacement of Tyr21 with Leu or Phe abolishedthe photochemical reaction implicating involvement of Tyr21 in the photocycle. Nuclear magnetic resonanceanalysis of wild-type and mutant proteins also indicates that Tyr21 forms π−π stacking interactions withthe isoalloxazine ring of FAD. We propose that photochemical excitation of the flavin results instrengthening of a hydrogen bond between the flavin and Tyr 21 leading to a stable local conformationalchange in AppA.