Photocycle dynamics of the E149A mutant of cryptochrome 3 from Arabidopsis thaliana |
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Authors: | P. Zirak A. Penzkofer J. Moldt R. Pokorny A. Batschauer L.-O. Essen |
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Affiliation: | aInstitut II, Experimentelle und Angewandte Physik, Universität Regensburg, Universitätstrasse 31, D-93053 Regensburg, Germany;bFachbereich Biologie, Pflanzenphysiologe/Photobiologie, Philipps-Universität, Karl-von-Frisch-Strasse 8, D-35032 Marburg, Germany;cFachbereich Chemie, Philipps-Universität Marburg, Hans-Meerwein-Strasse, D-35032 Marburg, Germany |
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Abstract: | The E149A mutant of the cryDASH member cryptochrome 3 (cry3) from Arabidopsis thaliana was characterized in vitro by optical absorption and emission spectroscopic studies. The mutant protein non-covalently binds the chromophore flavin adenine dinucleotide (FAD). In contrast to the wild-type protein it does not bind N5,N10-methenyl-5,6,7,8-tetrahydrofolate (MTHF). Thus, the photo-dynamics caused by FAD is accessible without the intervening coupling with MTHF. In dark adapted cry3-E149A, FAD is present in the oxidized form (FADox), semiquinone form (FADH), and anionic hydroquinone form (FADredH−). Blue-light photo-excitation of previously unexposed cry3-E149A transfers FADox to the anionic semiquinone form (FAD−) with a quantum efficiency of about 2% and a back recovery time of about 10 s (photocycle I). Prolonged photo-excitation leads to an irreversible protein re-conformation with structure modification of the U-shaped FAD and enabling proton transfer. Thus, a change in the photocycle dynamics occurs with photo-conversion of FADox to FADH, FADH to FADredH−, and thermal back equilibration in the dark (photocycle II). The photocycle dynamics of cry3-E149A is compared with the photocycle behaviour of wild-type cry3 and other photo-sensory cryptochromes. |
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Keywords: | Cryptochrome cry3 Cryptochrome mutant Arabidopsis thaliana Blue-light photoreceptor, DNA-photolyase, flavin adenine dinucleotide (FAD) Photocycle dynamics Photo-induced charge transfer, FAD reduction and oxidation |
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