Absorption and fluorescence spectroscopic characterisation of the circadian blue-light photoreceptor cryptochrome from Drosophila melanogaster (dCry) J. Shirdel a , P. Zirak a , A. Penzkofer a, * , H. Breitkreuz b , E. Wolf b a Institut II – Experimentelle und Angewandte Physik, Universität Regensburg, Universitätsstrasse 31, D-93053 Regensburg, Germany b Max-Planck-Institute of Molecular Physiology, Department of Structural Biology, Otto-Hahn-Strasse 11, D-44227 Dortmund, Germany article info Article history: Received 19 February 2008 Accepted 12 June 2008 Available online 18 June 2008 Keywords: Drosophila cryptochrome dCry Fluorescence quantum yield Fluorescence lifetime Photo-cycle dynamics Flavin adenine dinucleotide (FAD) Oxidized FAD Anionic FAD semiquinone 5,10-Methenyltetrahydrofolate (MTHF) 10-Formyldihydrofolate (10-FDHF) Electron transfer Photo-interconversion abstract 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 dinucle- otide (FAD) cofactor of dCry is identified to be present in its oxidized form (FAD ox ), and the 5,10-methe- nyltetrahydrofolate (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 FAD ox in dCry causes a reductive electron transfer to the formation of anionic FAD semiqui- none (FAD Å ), and photo-excitation of the generated FAD Å causes an oxidative electron transfer to the back formation of FAD ox . In light adapted dCry a photo-induced equilibrium between FAD ox and FAD Å 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 Å to FAD ox in the dark with a time constant of 1.6 min at room tem- perature is found. Ó 2008 Elsevier B.V. All rights reserved. 1. Introduction Cryptochromes are blue-light sensitive flavoproteins (for re- views see [1–14]) that are related to photolyases [1,12–15], but do not have the DNA repair mechanism of photolyases. Crypto- chromes were found and investigated in the plant Arabidopsis tha- liana (cry1 [16–27], cry2 [24,25,28–30], cry3 also called cry-DASH [31–35]), in green algae [36,37], ferns [38], mosses [39], fruit flies (Drosophila melanogaster [40–48]), monarch butterflies (Danaus plexippus [48–51]), mosquitos (Anopheles gambiae [48]), Chinese oak silk moths (Antherea pernyl [48]), birds (migrating garden war- bler [52]), mice [44,53], humans [54,55], and bacteria (Synechocys- tis sp. PCC 6803 cry-DASH [56,57], Vibrio cholerae cry-DASH [12,58], and others [12,59]). They regulate growth and develop- ment in plants, regulate circadian rhythms in plants and animals, act as chemical magneto receptors in migratory birds, and are functioning in bacteria and algae. The animal cryptochromes divide into two broad groups: the type 1 cryptochromes which are circadian photoreceptors, and the type 2 cryptochromes which function as core clock proteins [48,60,61]. Drosophila cryptochrome (dCry), which is investigated herein, is a type 1 animal cryptochrome. It is involved in the syn- chronization of the Drosophila circadian clock with the environ- mental light-dark cycle [40–47]. In a recent paper successful expression of dCry from Sf21 insect cells was reported [46]. The purified photoreceptor was investi- gated in aqueous solution at pH 8. It was shown, that dCry contains flavin adenine dinucleotide (FAD) as a blue-light absorbing chro- mophore as well as 5,10-methenyltetrahydrofolate (MTHF). The photoreaction mechanism of dCry was studied in detail [46]. The photo-cyclic reduction of the oxidized cofactor FAD ox in the dCry receptor state to the anionic semireduced form FAD Å in the puta- tive dCry signalling state was observed. In this paper, absorption and emission spectroscopic studies on dCry in aqueous solution at pH 8 are continued. The redox state composition of FAD in dark-adapted dCry is determined showing that FAD is practically exclusively present in its oxidized form (FAD ox ). The folic acid derivative MTHF is found to have been con- verted to 10-formyldihydrofolate (10-FDHF). In absorption spec- troscopic studies photo-induced formation of FAD Å from FAD ox , and of FAD ox from FAD Å is found (photo-interconversion of FAD ox and FAD Å ). The quantum efficiencies of anionic semiquinone for- mation (FAD Å ) from FAD ox and of FAD ox from FAD Å are calculated from excitation intensity dependent absorption spectra changes. The recovery of FAD Å to FAD ox in the dark is monitored at three 0301-0104/$ - see front matter Ó 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.chemphys.2008.06.006 * Corresponding author. Tel.: +49 941 943 2107; fax: +49 941 943 2754. E-mail address: alfons.penzkofer@physik.uni-regensburg.de (A. Penzkofer). Chemical Physics 352 (2008) 35–47 Contents lists available at ScienceDirect Chemical Physics journal homepage: www.elsevier.com/locate/chemphys