Absorption and fluorescence spectroscopic characterisation of the circadian blue-light photoreceptor cryptochrome from Drosophila melanogaster (dCry)

The absorption and fluorescence behaviour of the circadian blue-light photoreceptor cryptochrome from Drosophila melanogaster (dCry) in a pH 8 aqueous buffer solution is studied. The flavin adenine dinucleotide (FAD) cofactor of dCry is identified to be present in its oxidized form (FADox), and the 5,10-methenyltetrahydrofolate (MTHF) cofactor is found to be hydrolyzed and oxidized to 10-formyldihydrofolate (10-FDHF). The absorption and the fluorescence behaviour of dCry is investigated in the dark-adapted (receptor) state, the light-adapted (signalling) state, and under long-time violet light exposure. Photo-excitation of FADox in dCry causes a reductive electron transfer to the formation of anionic FAD semiquinone (FAD−), and photo-excitation of the generated FAD− causes an oxidative electron transfer to the back formation of FADox. In light adapted dCry a photo-induced equilibrium between FADox and FAD− exists. The photo-cycle dynamics of signalling state formation and recovery is discussed. Quantum yields of photo-induced signalling state formation of about 0.2 and of photo-induced back-conversion of about 0.2 are determined. A recovery of FAD− to FADox in the dark with a time constant of 1.6 min at room temperature is found.