Significance There are few detailed mechanistic models that describe how light-sensing proteins convert photochemistry into conformational signals. Combined computational and experimental investigations reveal how photoreduction of the Drosophila cryptochrome (dCRY) flavin induces protonation of a neighboring conserved His residue. Altered His hydrogen bonding then leads to conformational change in a key regulatory element of the protein. These data provide further evidence for involvement of a flavin anionic semiquinone in the dCRY signaling state and allow for the design of variants that can be used to separate dCRY biological functions or as tools for optogenetics.