Song S-H, Dick B, Penzkofer A, Pokorny R, Batschauer A, Essen L-O
Institut für Physikalische und Theoretische Chemie, Universität Regensburg, 93053 Regensburg, Germany.
J Photochem Photobiol B. 2006 Oct 2;85(1):1-16. doi: 10.1016/j.jphotobiol.2006.03.007. Epub 2006 May 24.
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 (FAD(ox), ca. 38.5%), in the semiquinone form (FADH., ca. 5%), and in the fully reduced neutral form (FAD(red)H2) or fully reduced anionic form (FAD(red)H-, 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 FAD(ox) 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 FAD(ox) to FAD(red)H2 (or FAD(red)H-, FAD(ox) photo-cycle of moderate efficiency), reversible reduction of FADH. to FAD(red)H2 (or FAD(red)H-, FADH. photo-cycle of high efficiency), and modification of re-oxidable FAD(red)H2 (or FAD(red)H-) to permanent FAD(red)H2 (or FAD(red)H-) 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'').
通过光吸收和发射光谱研究，在室温下于体外水溶液中对来自拟南芥的蓝光光感受器隐花色素3（cry3）进行了表征。该蛋白质非共价结合发色团黄素腺嘌呤二核苷酸（FAD）和N5，N10-亚甲基-5，6，7，8-四氢叶酸（MTHF）。在cry3的暗适应状态下，结合的FAD以氧化形式（FAD(ox)，约38.5%）、半醌形式（FADH·，约5%）以及完全还原的中性形式（FAD(red)H2）或完全还原的阴离子形式（FAD(red)H-，约55%）存在。一些处于氧化态的FAD（约1.5%）未结合，这可能是由于发色团释放和/或变性所致。观察到从MTHF到FAD(ox)的福斯特型能量转移。光激发可逆地改变蛋白质构象，导致MTHF吸收强度略有上升以及MTHF荧光效率增加（高效蛋白质构象光循环）。此外，还发生结合的FAD(ox)可逆还原为FAD(red)H2（或FAD(red)H-，中等效率的FAD(ox)光循环）、FADH·可逆还原为FAD(red)H2（或FAD(red)H-，高效的FADH·光循环）以及可再氧化的FAD(red)H2（或FAD(red)H-）转变为永久性FAD(red)H2（或FAD(red)H-），量子效率较低。MTHF的光激发导致形成一种MTHF物种（MTHF'，MTHF光循环，中等量子效率），其缓慢恢复到初始暗状态，同时还形成一种不可逆光产物（MTHF''）。
