Photochemical and mutational analysis of the FMN-binding domains of the plant blue light receptor, phototropin
Photochemical and mutational analysis of the FMN-binding domains of the plant blue light receptor, phototropin
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DOI：
10.1021/bi000585
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发表时间：
2000-08-08
期刊：
影响因子：
2.9
通讯作者：
Briggs, WR
中科院分区：
文献类型：
--
作者：
Salomon, M;Christie, JM;Briggs, WR
植物光感受器趋光蛋白是一种被UV-A/蓝光激活的自磷酸化丝氨酸-苏氨酸蛋白激酶。两个域，LOV 1和LOV 2，PAS域超家族的成员，通过向光蛋白介导光敏感。异源表达研究表明，这两个域的功能作为FMN结合位点。尽管迄今为止已经鉴定出三种植物蓝光光感受器cry 1、cry 2和phototropin，但迄今为止尚未描述这些光感受器光激活背后的光化学反应。在此，我们证明了燕麦向光素的LOV结构域经历了一个独立的光循环，其特征在于在响应于光的蓝光吸收损失和在黑暗中的蓝光吸收形式的自发恢复。光反应的速率常数和量子效率表明，LOV 1表现出比LOV 2更低的光敏性。两个LOV域所产生的光产物的光谱性质是无关的光还原黄素和黄素蛋白，但与黄素-半胱氨酰加合物的那些是一致的。黄素-硫醇加合物通常是在黄素蛋白催化的蛋白质二硫化物还原过程中形成的短寿命反应中间体。通过定点诱变，我们已经确定了几个氨基酸残基内的推定的发色团结合位点的LOV 1和LOV 2似乎是重要的FMN结合和/或光化学反应性。其中Cys 39在LOV结构域的光化学反应中起重要作用。用Ala取代Cys 39消除了两个LOV结构域的光化学反应。因此，我们提出，光感由phototropin LOV域发生通过形成一个稳定的加合物之间的FMN发色团和Cys 39。
The plant photoreceptor phototropin is an autophosphorylating serine-threonine protein kinase activated by UV-A/blue light. Two domains, LOV1 and LOV2, members of the PAS domain superfamily, mediate light sensing by phototropin. Heterologous expression studies have shown that both domains function as FMN-binding sites. Although three plant blue light photoreceptors, cry1, cry2, and phototropin, have been identified to date, the photochemical reactions underlying photoactivation of these light sensors have not been described so far. Herein, we demonstrate that the LOV domains of Avena sativa phototropin undergo a self-contained photocycle characterized by a loss of blue light absorbance in response to light and a spontaneous recovery of the blue light-absorbing form in the dark. Rate constants and quantum efficiencies for the photoreactions indicate that LOV1 exhibits a lower photosensitivity than LOV2. The spectral properties of the photoproduct produced for both LOV domains are unrelated to those found for photoreduced flavins and flavoproteins, but are consistent with those of a flavin-cysteinyl adduct. Flavin-thiol adducts are generally short-lifetime reaction intermediates formed during the flavoprotein-catalyzed reduction of protein disulfides. By site-directed mutagenesis, we have identified several amino acid residues within the putative chromophore binding site of LOV1 and LOV2 that appear to be important for FMN binding and/or the photochemical reactivity. Among those is Cys39, which plays an important role in the photochemical reaction of the LOV domains. Replacement of Cys39 with Ala abolished the photochemical reactions of both LOV domains. We therefore propose that light sensing by the phototropin LOV domains occurs via the formation of a stable adduct between the FMN chromophore and Cys39.