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同源物与铁硫簇生物合成有关。在非氮固定蓝细菌Syechocystis PCC6803中，有两个ISCA同源物SLR1417和SLR1565（指定为ISCA1和ISCA2），其中仅作为一种蛋白质作为一种蛋白质，具有蛋白质复合物，带有热重复蛋白质的蛋白质，SLR1O98（iiaih）。我们观察到，重组ISCA2/IAIH复合物的抽象光谱单独类似于ISCA2，尽管它是更锐利的：在存在二硫代硫醇的存在下，仅[2FE-2S] ISCA2群，但isca2/iaih综合体的却不较少，但在sodium ditbionite ditbionite上添加了较少的标签。这意味着[2FE-2S]簇的ISCA2部分通过IAIH的存在稳定。在没有二硫代硫代醇的情况下，ISCA2/IAIH复合物的[2FE-2S]簇通过钠矿会不稳定，这表明ISCA2/IAIH复合物中铁硫簇的体内稳定性受到细胞硫醇的氧化还原状态的影响。当…更多的三个配置半胱氨酸保留在ISCA2中时，[2FE-2S]簇的潜在配体被丝氨酸取代时，组装的[2FE2S]簇和蛋白质复合物的量显着降低了CO/I细胞。半胱氨酸突变的ISCA2/IAIH复合物均包含[2FE-2S]类似的聚类，这表明与ISCA2结合的稳定的铁硫簇组成是有效且稳定的复杂形成所必需的。与蓝核nifu样蛋白相似，该蛋白被提议用作分子支架。 ATCNFU-V在拟南芥的几个组织中始终表达，而ATCNFU-IVB的表达在空中部分中很突出。缺乏ATCNFU-V的突变拟南芥具有淡淡的浅叶叶的矮人表型，并累积了光化系统。突变体中的叶绿体也显示出含有[2FE-2S]簇的基质的主要电子载体的量，以及铁硫簇插入到脱甲基蛋白中的体外活性。当在E. ccli细胞中表达时，ATG〜F U-V形成了类似[2FE-2S]类簇的同型二聚体，并且可以在体外转移到Apo-Ferredoxin中，形成全蛋白毒素。我们建议ATCNFU具有重要的功能，它是Chlotoplasts中铁硫簇生物合成的分子支架，因此是费雷德蛋白和光系统的生物发生所必需的。