Synthesis of chlorophyll b: localization of chlorophyllide a oxygenase and discovery of a stable radical in the catalytic subunit.
Synthesis of chlorophyll b: localization of chlorophyllide a oxygenase and discovery of a stable radical in the catalytic subunit.
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DOI：
10.1186/1471-2229-4-5
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发表时间：
2004-04-15
影响因子：
5.3
通讯作者：
Hoober JK
中科院分区：
文献类型：
--
作者：
Eggink LL;LoBrutto R;Brune DC;Brusslan J;Yamasato A;Tanaka A;Hoober JK
绿藻和植物叶绿体中稳定的光收集复合物（lhc）的组装需要叶绿素（Chl） b的合成，这一反应涉及到Chl a的7-甲基氧化成甲酰基。该反应使用分子氧，由叶绿素内酯加氧酶（CAO）催化。CAO的氨基酸序列预测了蛋白质中的单核铁和Rieske铁硫中心。Chl b的合成机制及其在叶绿体中的定位是了解LHC组装的重要步骤。在共聚焦荧光显微镜下，在豌豆幼叶中短暂表达的一种氧化钙-绿色荧光蛋白的荧光出现在成熟叶绿体的外围和类囊体膜上。然而，当莱茵衣藻（Chlamydomonas reinhardtii） cw15部分去核细胞的膜在蔗糖梯度上溶解时，免疫印迹分析仅在叶绿体包膜内膜上检测到全长CAO。CAO的电子顺磁共振谱在g = 4.3处有一个共振，分配给预测的单核铁中心。而不是预测的Rieske铁硫中心的光谱，在g = 2.057处观察到一个几乎对称的，大约100高斯的峰谷信号，对铁硫中心的温度特征很敏感。在g = 2.0042处，一个各向同性的9高斯峰谷信号揭示了蛋白质中一个非常稳定的自由基。加入125I-后的蛋白片段鉴定出一个保守的酪氨酸残基（衣藻中的tyr422和拟南芥中的tyr518）作为自由基种。自由基被叶绿素a猝灭，这表明它可能参与了酶促反应。在成熟叶绿体的叶绿体包膜和类囊体膜上发现了CAO，而在暗色生长的莱茵青霉细胞中只在包膜内膜上发现了CAO。这种定位进一步支持了叶绿体发育过程中包膜作为Chl b合成和lhc组装的初始位点。鉴定蛋白质中的酪氨酸自由基提供了对Chl b合成机制的深入了解。
Assembly of stable light-harvesting complexes (LHCs) in the chloroplast of green algae and plants requires synthesis of chlorophyll (Chl) b, a reaction that involves oxygenation of the 7-methyl group of Chl a to a formyl group. This reaction uses molecular oxygen and is catalyzed by chlorophyllide a oxygenase (CAO). The amino acid sequence of CAO predicts mononuclear iron and Rieske iron-sulfur centers in the protein. The mechanism of synthesis of Chl b and localization of this reaction in the chloroplast are essential steps toward understanding LHC assembly. Fluorescence of a CAO-GFP fusion protein, transiently expressed in young pea leaves, was found at the periphery of mature chloroplasts and on thylakoid membranes by confocal fluorescence microscopy. However, when membranes from partially degreened cells of Chlamydomonas reinhardtii cw15 were resolved on sucrose gradients, full-length CAO was detected by immunoblot analysis only on the chloroplast envelope inner membrane. The electron paramagnetic resonance spectrum of CAO included a resonance at g = 4.3, assigned to the predicted mononuclear iron center. Instead of a spectrum of the predicted Rieske iron-sulfur center, a nearly symmetrical, approximately 100 Gauss peak-to-trough signal was observed at g = 2.057, with a sensitivity to temperature characteristic of an iron-sulfur center. A remarkably stable radical in the protein was revealed by an isotropic, 9 Gauss peak-to-trough signal at g = 2.0042. Fragmentation of the protein after incorporation of 125I- identified a conserved tyrosine residue (Tyr-422 in Chlamydomonas and Tyr-518 in Arabidopsis) as the radical species. The radical was quenched by chlorophyll a, an indication that it may be involved in the enzymatic reaction. CAO was found on the chloroplast envelope and thylakoid membranes in mature chloroplasts but only on the envelope inner membrane in dark-grown C. reinhardtii cells. Such localization provides further support for the envelope membranes as the initial site of Chl b synthesis and assembly of LHCs during chloroplast development. Identification of a tyrosine radical in the protein provides insight into the mechanism of Chl b synthesis.