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

IscA同源物参与铁硫簇生物合成。在非固氮蓝藻集胞藻PCC 6803中，存在两个IscA同源物，SLR 1417和SLR 1565（命名为IscA 1和IscA 2），其中只有IscA 2作为与含HEAT重复序列的蛋白SLR 1098（IaiH）的蛋白复合物存在。我们观察到，重组IscA 2/IaiH复合物的吸收光谱类似于单独的IscA 2的吸收光谱，尽管它更尖锐：在二硫苏糖醇的存在下，单独的IscA 2的[2Fe-2S]簇，而不是IscA 2/IaiH复合物的[2Fe-2S]簇，在加入ditbionite钠后变得还原不稳定。这意味着[2Fe-2S]簇的IscA 2部分通过IaiH的存在而稳定。在不存在二硫苏糖醇的情况下，IscA 2/IaiH复合物的[2Fe-2S]簇被连二亚硫酸钠去稳定化，表明IscA 2/IaiH复合物中的铁-硫簇的体内稳定性受细胞硫醇的氧化还原状态的影响。当任何一个 关于我们 将IscA 2中3个保守的半胱氨酸残基（[2Fe-2S]簇的潜在配体）替换为丝氨酸，可显著降低[2Fe-2S]簇的组装量和蛋白质复合物的组装量。半胱氨酸突变的IscA 2/IaiH复合物都含有一个类似[2Fe-2S]的簇，这表明与IscA 2结合的稳定的铁硫簇的组装是有效和稳定的复合物形成所必需的。来自拟南芥，与被提议用作分子支架的氰基己二醇NifU样蛋白具有高度序列相似性。AtCnfU-V在拟南芥的几种组织中组成型表达，而AtCnfU-IVb的表达在地上部分中是突出的。突变体拟南芥缺乏AtCnfU-V表现出一个矮小的表型与微弱的苍白giten叶和有显着受损的光系统I积累。突变体中的叶绿体也表现出铁氧还蛋白，一个主要的电子载体的基质，含有一个[2Fe-2S]集群，并在体外活性的铁硫簇插入到脱辅基-fenedoxin的量减少。当在E. AtG~ fU-V在体外可与脱辅基铁氧还蛋白形成[2Fe-2S]样簇的同源二聚体，该簇在体外可转移到脱辅基铁氧还蛋白上形成全铁氧还蛋白。我们认为AtCnfU作为叶绿体铁硫簇生物合成的分子支架具有重要的功能，因此是铁氧还蛋白和光系统I生物合成所必需的。少

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