Linear free energy relationship between reduction potential and photoreduction rate: studies on <i>Drosophila</i> cryptochrome

Cryptochromes are flavin adenine dinucleotide (FAD)-containing blue-light photoreceptors involved in the regulation of the circadian clock and may play a role in magnetic field sensing. The photochemistry of cryptochromes is based on the isoalloxazine moiety, which can be photoreduced and subsequently reoxidized by an electron acceptor such as oxygen, corresponding to a photo-switch between the dark and signaling state. We replaced the FAD cofactor of Drosophila cryptochrome with a series of FAD cofactors modified at the 7α or 8α positions, in order to modulate the chemical properties of the electron acceptor. These modifications were shown to alter the kinetics of the light-dependent reactions. Notably, 7-halogenated FADs form the signaling state more than six times faster compared to the natural FAD cofactor. The more positive reduction potentials as well as the increased intersystem crossing rates due to heavy halogen atoms were identified as reasons for the altered photochemistry. Both parameters show a linear dependence on the reaction kinetics, according to the Hammett relationship. With this knowledge, the photochemistry of cryptochromes may be modified in a defined way without changing its amino acid sequence.