Absorption and fluorescence spectroscopic characterization of cryptochrome 3 from Arabidopsis thaliana

The blue light photoreceptor cryptochrome 3 (cry3) from Arabidopsis thaliana was characterized at room temperature in vitro in aqueous solution by optical absorption and emission spectroscopic studies. The protein non-covalently binds the chromophores flavin adenine dinucleotide (FAD) and N5,N10-methenyl-5,6,7,8-tetrahydrofolate (MTHF). In the dark-adapted state of cry3, the bound FAD is present in the oxidized form (FADox, ca. 38.5%), in the semiquinone form (FADH, ca. 5%), and in the fully reduced neutral form (FADredH2) or fully reduced anionic form (FADredH−, ca. 55%). Some amount of FAD (ca. 1.5%) in the oxidized state remains unbound probably caused by chromophore release and/or denaturation. Förster-type energy transfer from MTHF to FADox is observed. Photo-excitation reversibly modifies the protein conformation causing a slight rise of the MTHF absorption strength and an increase of the MTHF fluorescence efficiency (efficient protein conformation photo-cycle). Additionally there occurs reversible reduction of bound FADox to FADredH2 (or FADredH−, FADox photo-cycle of moderate efficiency), reversible reduction of FADH to FADredH2 (or FADredH−, FADH photo-cycle of high efficiency), and modification of re-oxidable FADredH2 (or FADredH−) to permanent FADredH2 (or FADredH−) with low quantum efficiency. Photo-excitation of MTHF causes the reversible formation of a MTHF species (MTHF′, MTHF photo-cycle, moderate quantum efficiency) with slow recovery to the initial dark state, and also the formation of an irreversible photoproduct (MTHF″).