Song Sang-Hun, Oztürk Nuri, Denaro Tracy R, Arat N Ozlem, Kao Ya-Ting, Zhu Haisun, Zhong Dongping, Reppert Steven M, Sancar Aziz
Department of Biochemistry and Biophysics, University of North Carolina School of Medicine, Chapel Hill, North Carolina 27599, USA.
J Biol Chem. 2007 Jun 15;282(24):17608-12. doi: 10.1074/jbc.M702874200. Epub 2007 Apr 25.
The monarch butterfly (Danaus plexippus) cryptochrome 1 (DpCry1) belongs in the class of photosensitive insect cryptochromes. Here we purified DpCry1 expressed in a bacterial host and obtained the protein with a stoichiometric amount of the flavin cofactor in the two-electron oxidized, FAD(ox), form. Exposure of the purified protein to light converts the FAD(ox) to the FAD*(-) flavin anion radical by intraprotein electron transfer from a Trp residue in the apoenzyme. To test whether this novel photoreduction reaction is part of the DpCry1 physiological photocycle, we mutated the Trp residue that acts as the ultimate electron donor in flavin photoreduction. The mutation, W328F, blocked the photoreduction entirely but had no measurable effect on the light-induced degradation of DpCry1 in vivo. In light of this finding and the recently published action spectrum of this class of Crys, we conclude that DpCry1 and similar insect cryptochromes do not contain flavin in the FAD(ox) form in vivo and that, most likely, the [see text] photoreduction reaction is not part of the insect cryptochrome photoreaction that results in proteolytic degradation of the photopigment.
黑脉金斑蝶（Danaus plexippus）的隐花色素1（DpCry1）属于光敏昆虫隐花色素类别。在此，我们纯化了在细菌宿主中表达的DpCry1，并获得了具有化学计量的黄素辅因子的蛋白质，其处于双电子氧化的FAD(ox)形式。将纯化后的蛋白质暴露于光下，通过脱辅基酶中一个色氨酸残基的蛋白质内电子转移，将FAD(ox)转化为FAD*(-)黄素阴离子自由基。为了测试这种新的光还原反应是否是DpCry1生理光循环的一部分，我们对在黄素光还原中作为最终电子供体的色氨酸残基进行了突变。该突变W328F完全阻断了光还原，但对体内DpCry1的光诱导降解没有可测量的影响。鉴于这一发现以及最近发表的这类隐花色素的作用光谱，我们得出结论，DpCry1和类似的昆虫隐花色素在体内不含有FAD(ox)形式的黄素，并且很可能，[见正文]光还原反应不是导致光色素蛋白水解降解的昆虫隐花色素光反应的一部分。
