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Analysis on Molecular Mechanism of Iron-Sulfur Cluster Biosynthesis in Cyanobacteria and in Chloroplasts

蓝藻和叶绿体铁硫簇生物合成的分子机制分析

基本信息

批准号：
14580626
负责人：
NAKAI Masato
金额：
$ 2.3万
依托单位：
Osaka University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2002
资助国家：
日本
起止时间：
2002 至 2003
项目状态：
已结题

项目摘要

IscA homologues are involved in iron-sulfur cluster biosynthesis. In the non-nitrogen-fixing cyanobacterium Synechocystis PCC6803, there are two IscA homologues, SLR1417 and SLR1565 (designated IscA1 and IscA2), of which only IscA2 exists as a protein complex with the HEAT-repeat-containing protein, SLR1O98 (IaiH). We observed that the absorption spectrum of the recombinant IscA2/IaiH complex resembles that of IscA2 alone, although it is sharper: In the presence of dithiothreitol, the [2Fe-2S] cluster of IscA2 alone, but not of the IscA2/IaiH complex, became reductively labile upon the addition of sodium ditbionite. This implies that the IscA2 moiety of the [2Fe-2S] cluster is stabilized by the presence of IaiH. The [2Fe-2S] cluster of the IscA2/IaiH complex was destabilized by sodium dithionite in the absence of dithiothreitol, suggesting that the in vivo stability of the iron-sulfur cluster in the IscA2/IaiH complex is influenced by the redox state of cellular thiols. When any one of … More three conserved cysteine residues in IscA2, potential ligands for the [2Fe-2S] cluster, was replaced with serine, the amount of assembled [2Fe2S] cluster and protein complex was significantly reduced inE co/i cells. The cysteine mutated IscA2/IaiH complexes that were present all contained a [2Fe-2S]-like cluster suggesting that the assembly of a stable iron-sulfur cluster bound to IscA2 is required for efficient and stable complex formation.We also identified two chloroplast-localized NifU-like proteins, AtCnfU-V and AtCnfU-IVb, from Arabidopsis thaliana with high sequence similarity to a cyanobaderial NifU-like protein that was proposed to serve as a molecular scaffold. AtCnfU-V is constitutively expressed in several tissues of Arabidopsis, whereas the expression of AtCnfU-IVb is prominent in the aerial parts. Mutant Arabidopsis lacking AtCnfU-V exhllited a dwarf phenotype with faint pale-giten leaves and had drastically impaired photosystem I accumulation. Chloroplasts in the mutants also showed a decrease in both the amount of ferredoxin, a major electron carrier of the stroma that contains a [2Fe-2S] cluster, and in the in vitro activity of iron-sulfur cluster insertion into apo-fenedoxin. When expressed in E. ccli cells, AtG~f U-V formed a homodimer canying a [2Fe-2S]-like cluster, and this cluster could be transferred to apo-ferredoxin in vitro to form holo-ferredoxin. We propose that AtCnfU has an important function as a molecular scaffold for iron-sulfur cluster biosynthesis in chlotoplasts and thereby is required for biogenesis of ferredoxin and photosystem I. Less

ISCA同系物参与了铁-硫簇的生物合成。在不固氮的聚球藻PCC6803中，存在两个ISCA同源物SLR1417和SLR1565(分别命名为IscA1和IscA2)，其中只有IscA2与热重复蛋白SLR1O98(IaiH)形成蛋白质复合体。我们观察到，重组IscA2/IaiH复合体的吸收光谱与IscA2单独的吸收光谱相似，但更加尖锐：在二硫苏糖醇的存在下，只有IscA2的[2Fe-2S]簇，而不是IscA2/IaiH复合体的[2Fe-2S]簇，在加入双亚硫酸钠后变得还原不稳定。这意味着[2Fe-2S]团簇的IscA2部分通过IaiH的存在而稳定下来。在没有二硫苏糖醇的情况下，IscA2/IaiH复合体的[2Fe-2S]团簇被二亚硫酸钠破坏，表明IscA2/IaiH复合体中铁-硫团簇的体内稳定性受细胞内硫醇的氧化还原状态的影响。当…中的任何一个[2Fe-2S]簇的潜在配体IscA2中3个保守的半胱氨酸残基被丝氨酸取代，在Co/I细胞中组装的[2Fe2S]簇和蛋白质复合体的数量显著减少。半胱氨酸突变的IscA2/IaiH复合体都含有一个类似[2Fe-2S]的簇，这表明与IscA2结合的稳定的铁硫簇是形成高效稳定复合体所必需的。我们还从拟南芥中鉴定了两个叶绿体定位的类NifU蛋白AtCnfU-V和AtCnfU-IVb，它们与一个氰基类NifU蛋白具有很高的序列相似性，被认为是一种分子支架。AtCnfU-V在拟南芥的几个组织中呈结构性表达，而AtCnfU-IVb在地上部分的表达显著。缺失AtCnfU-V的拟南芥突变体表现为矮生型，叶片苍白，光系统I的积累受到严重损害。突变体中的叶绿体也表现出铁氧还蛋白(一种含有[2Fe-2S]簇的基质的主要电子载体)的数量和铁-硫簇插入脱脂蛋白的体外活性降低。当在E.CCLI细胞中表达时，ATG~f U-V形成一个同源二聚体，形成一个类似[2Fe-2S]的簇，该簇可以在体外转移到脱铁还蛋白上形成全铁还蛋白。我们认为AtCnfU是叶绿体中铁-硫簇生物合成的重要分子支架，是铁还蛋白和光系统I生物发生所必需的。