Proton Transfer to Flavin Stabilizes the Signaling State of the Blue Light Receptor Plant Cryptochrome
Proton Transfer to Flavin Stabilizes the Signaling State of the Blue Light Receptor Plant Cryptochrome
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DOI：
10.1074/jbc.m114.606327
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发表时间：
2015-01-16
影响因子：
4.8
通讯作者：
Kottke, Tilman
中科院分区：
文献类型：
--
作者：
Hense, Anika;Herman, Elena;Kottke, Tilman
植物隐花色素调节高等植物的昼夜节律、开花时间和光形态建成，作为对蓝光的反应。在黑暗中，这些光感受器在光裂解酶同源区 (PHR) 中结合氧化的 FAD。吸收蓝光后，FAD 通过保守色氨酸三联体的电子转移和附近天冬氨酸的质子转移，转化为中性自由基状态（可能的信号状态）。在这里，我们通过 PHR 结构域上的红外和时间分辨紫外-可见光谱证明，用半胱氨酸替换天冬氨酸 Asp-396 可防止质子转移。自由基的寿命减少了6个数量级。如此短的寿命不允许驱动与信号转导相关的 C 端延伸构象变化。只有在 ATP 存在的情况下，野生型和突变体才能形成长寿命的自由基状态。然而，在突变体中，形成阴离子自由基而不是中性自由基，如之前在动物 I 型隐花色素中发现的那样。红外光谱实验表明，尽管缺乏质子转移，光诱导的 PHR 结构域构象变化在突变体中是保守的。在非光感 D-氨基酸氧化酶对阴离子自由基的光还原中未检测到这些变化。总之，阴离子自由基的形成足以在植物隐花色素中产生蛋白质反应。此外，在没有 ATP 的情况下，内部质子转移对于稳定信号状态是必需的。
Plant cryptochromes regulate the circadian rhythm, flowering time, and photomorphogenesis in higher plants as responses to blue light. In the dark, these photoreceptors bind oxidized FAD in the photolyase homology region (PHR). Upon blue light absorption, FAD is converted to the neutral radical state, the likely signaling state, by electron transfer via a conserved tryptophan triad and proton transfer from a nearby aspartic acid. Here we demonstrate, by infrared and time-resolved UV-visible spectroscopy on the PHR domain, that replacement of the aspartic acid Asp-396 with cysteine prevents proton transfer. The lifetime of the radical is decreased by 6 orders of magnitude. This short lifetime does not permit to drive conformational changes in the C-terminal extension that have been associated with signal transduction. Only in the presence of ATP do both the wild type and mutant form a long-lived radical state. However, in the mutant, an anion radical is formed instead of the neutral radical, as found previously in animal type I cryptochromes. Infrared spectroscopic experiments demonstrate that the light-induced conformational changes of the PHR domain are conserved in the mutant despite the lack of proton transfer. These changes are not detected in the photoreduction of the non-photosensory D-amino acid oxidase to the anion radical. In conclusion, formation of the anion radical is sufficient to generate a protein response in plant cryptochromes. Moreover, the intrinsic proton transfer is required for stabilization of the signaling state in the absence of ATP.