Absorption and fluorescence spectroscopic characterization of cryptochrome 3 from Arabidopsis thaliana.
Absorption and fluorescence spectroscopic characterization of cryptochrome 3 from Arabidopsis thaliana.
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DOI：
10.1016/j.jphotobiol.2006.03.007
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发表时间：
2006-10
期刊：
影响因子：
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通讯作者：
Sang-Hun Song;Bernhard Dick;A. Penzkofer;Richard Pokorny;A. Batschauer;L. Essen
中科院分区：
文献类型：
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作者：
Sang-Hun Song;Bernhard Dick;A. Penzkofer;Richard Pokorny;A. Batschauer;L. Essen
通过光学吸收和发射光谱研究，在室温下在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-次甲基-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（FADox，约38.5％）、半醌形式（FADH，约5％）和完全还原的中性形式（FADredH2）或完全还原的阴离子形式（FADredH−，约55％）存在。一定量的氧化态 FAD（约 1.5%）仍未结合，可能是由于发色团释放和/或变性造成的。观察到从 MTHF 到 FADoxis 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，并增加 MTHF 荧光效率（有效的蛋白质构象光循环）。此外，还会发生结合的FADox到FADredH2（或FADredH−，中等效率的FADox光循环）的可逆还原，FADH可逆还原为FADredH2（或FADredH−，高效率的FADH光循环），以及将可再氧化的FADredH2（或FADredH−）修饰为永久FADredH2（或 FADredH−) 具有低量子效率。 MTHF 的光激发导致 MTHF 物质（MTHF′、MTHF 光循环、中等量子效率）的可逆形成，并缓慢恢复到初始暗态，并且还形成不可逆光产物（MTHF”）。
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 (FADox, ca. 38.5%), in the semiquinone form (FADH, ca. 5%), and in the fully reduced neutral form (FADredH2) or fully reduced anionic form (FADredH−, 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 FADoxis 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 FADoxto FADredH2(or FADredH−, FADoxphoto-cycle of moderate efficiency), reversible reduction of FADH to FADredH2(or FADredH−, FADH photo-cycle of high efficiency), and modification of re-oxidable FADredH2(or FADredH−) to permanent FADredH2(or FADredH−) 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″).