Primary reactions of the LOV2 domain of phototropin, a plant blue-light photoreceptor
Primary reactions of the LOV2 domain of phototropin, a plant blue-light photoreceptor
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DOI：
10.1021/bi034022k
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发表时间：
2003-04-01
期刊：
影响因子：
2.9
通讯作者：
van Grondelle, R
中科院分区：
文献类型：
--
作者：
Kennis, JTM;Crosson, S;van Grondelle, R
向光蛋白是一类重要的植物光感受器激酶，它控制着植物的一系列生理反应，包括向光性、光诱导的叶绿体运动和光诱导的气孔开放。向光素的LOV 2结构域结合黄素单胞苷肽（FMN）分子，并经历涉及保守的半胱氨酸残基和FMN的C（4a）原子之间的光驱动共价加合物形成的光循环。该产物状态促进C-末端激酶活化和下游信号转导。在这里，我们报告的初级光物理学和光化学LOV 2域的光致蛋白I的一个静脉sativa（燕麦）和phy 3光感受器的铁线蕨capillus-veneris（马尾蕨）。与早期的报告一致[Swartz，T. E、等人（2001）J.Biol.Chem.276，36493-36500]，我们发现FMN三重态是发生光反应的活性物质。我们证明了三重态是LOV 2光循环中的主要光产物，以60%的效率产生。在飞秒到纳秒的时间尺度上，没有光谱上可区分的中间体先于FMN三重态，这表明它是直接通过单重态的系间穿越（ISC）形成的。我们的研究结果表明，大多数的FMN三联体在LOV 2结构域中存在的质子化的形式。我们提出了一个反应机制，涉及激发态质子转移，在纳秒级或更快的时间尺度上，从sulthydryl基团的保守的半胱氨酸的N5原子的FMN。该事件通过增加C（4a）的亲电性和随后由半胱氨酸的硫醇盐阴离子的亲核攻击来促进加合物形成。与溶液中的游离FMN的比较表明，LOV 2的蛋白质环境将FMN的ISC速率提高了2.4倍，从而提高了半胱氨酰-黄素加合物的产率和向光蛋白介导的信号传导过程的效率。
The phototropins constitute an important class of plant photoreceptor kinases that control a range of physiological responses, including phototropism, light-directed chloroplast movement, and light-induced stomatal opening. The LOV2 domain of phototropin binds a molecule of flavin mononucleotide (FMN) and undergoes a photocycle involving light-driven covalent adduct formation between a conserved cysteine residue and the C(4a) atom of FMN. This product state promotes C-terminal kinase activation and downstream signal transduction. Here, we report the primary photophysics and photochemistry of LOV2 domains of phototropin I of A vena sativa (oat) and of the phy3 photoreceptor of Adiantum capillus-veneris (maidenhair fern). In agreement with earlier reports [Swartz, T. E., et al. (2001) J. Biol. Chem. 276, 36493-36500], we find that the FMN triplet state is the reactive species from which the photoreaction occurs. We demonstrate that the triplet state is the primary photoproduct in the LOV2 photocycle, generated at 60% efficiency. No spectroscopically distinguishable intermediates precede the FMN triplet on the femtosecond to nanosecond time scale, indicating that it is formed directly via intersystem crossing (ISC) from the singlet state. Our results indicate that the majority of the FMN triplets in the LOV2 domain exist in the protonated form. We propose a reaction mechanism that involves excited-state proton transfer, on the nanosecond time scale or faster, from the sulthydryl group of the conserved cysteine to the N5 atom of FMN. This event promotes adduct formation by increasing the electrophilicity of C(4a) and subsequent nucleophilic attack by the cysteine's thiolate anion. Comparison to free FMN in solution shows that the protein environment of LOV2 increases the ISC rate of FMN by a factor of 2.4, thus improving the yield of the cysteinyl-flavin adduct and the efficiency of phototropin-mediated signaling processes.