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J. Biol. Chem., Vol. 282, Issue 17, 13011-13021, April 27, 2007

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A Novel Photoreaction Mechanism for the Circadian Blue Light Photoreceptor Drosophila Cryptochrome^*

Alex Berndt¹, Tilman Kottke^¶2, Helena Breitkreuz, Radovan Dvorsky, Sven Hennig, Michael Alexander, and Eva Wolf³

From the Max Planck Institute of Molecular Physiology, Department of Structural Biology, Otto-Hahn-Strasse 11, 44227 Dortmund, Germany, IBI-2, Structural Biology, Research Center Jülich, 52425 Jülich, Germany, and the ^¶Bielefeld University, Department of Chemistry, Universitätsstrasse 25, 33615 Bielefeld, Germany

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 radical. Our findings lead us to propose a novel photoreaction mechanism for dCRY, in which FAD_ox corresponds to the ground state, whereas the radical represents the light-activated state that mediates resetting of the Drosophila circadian clock.

Received for publication, September 15, 2006 , and in revised form, January 31, 2007.

^* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Present address: MRC, Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, United Kingdom.

² Supported by Helmholtz-Gemeinschaft Grant VH-NG-014.

³ Supported by Deutsche Forschungsgemeinschaft Grant FOR526-WO-695/3. To whom correspondence should be addressed. Tel.: 49-231-1332162; Fax: 49-231-1332199; E-mail: eva.wolf{at}mpi-dortmund.mpg.de.

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