A novel photoreaction mechanism for the circadian blue light photoreceptor Drosophila cryptochrome.
Cryptochromes are flavoproteins that are evolutionary related to the DNA photolyases but lack DNA repair activity. Drosophila cryptochrome (dCRY) is a blue light photoreceptor that is involved in the synchronization of the circadian clock with the environmental light-dark cycle. Until now, spectroscopic and structural studies on this and other animal cryptochromes have largely been hampered by difficulties in their recombinant expression. We have therefore established an expression and purification scheme that enables us to purify mg amounts of monomeric dCRY from Sf21 insect cell cultures. Using UV-visible spectroscopy, mass spectrometry, and reversed phase high pressure liquid chromatography, we show that insect cell-purified dCRY contains flavin adenine dinucleotide in its oxidized state (FAD(ox)) and residual amounts of methenyltetrahydrofolate. Upon blue light irradiation, dCRY undergoes a reversible absorption change, which is assigned to the conversion of FAD(ox) to the red anionic FAD(.) radical. Our findings lead us to propose a novel photoreaction mechanism for dCRY, in which FAD(ox) corresponds to the ground state, whereas the FAD(.) radical represents the light-activated state that mediates resetting of the Drosophila circadian clock.
Berndt
Alex
A
Max Planck Institute of Molecular Physiology, Department of Structural Biology, Otto-Hahn-Strasse 11, 44227 Dortmund, Germany.
Kottke
Tilman
T
Breitkreuz
Helena
H
Dvorsky
Radovan
R
Hennig
Sven
S
Alexander
Michael
M
Wolf
Eva
E
D000818:Animals; D002460:Cell Line; D002940:Circadian Rhythm; D056931:Cryptochromes; D004260:DNA Repair; D004255:Deoxyribodipyrimidine Photo-Lyase / Q000737:chemistry; D004330:Drosophila / Q000737:chemistry / Q000378:metabolism; D029721:Drosophila Proteins / Q000737:chemistry* / Q000378:metabolism; D005182:Flavin-Adenine Dinucleotide / Q000737:chemistry* / Q000378:metabolism; D005420:Flavoproteins / Q000737:chemistry* / Q000378:metabolism; D010084:Oxidation-Reduction; D010777:Photochemistry; D017956:Photoreceptor Cells, Invertebrate / Q000737:chemistry* / Q000378:metabolism
D056931:Cryptochromes; D029721:Drosophila Proteins; D005420:Flavoproteins; D005182:Flavin-Adenine Dinucleotide; D004255:Deoxyribodipyrimidine Photo-Lyase