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

IscA同系物参与铁硫簇生物合成。在非固氮蓝藻Synechocystis PCC6803中，存在两个IscA同源物SLR1417和SLR1565（指定为IscA1和IscA2），其中只有IscA2与含有heat - repeats的蛋白slr1098 （IaiH）作为蛋白复合体存在。我们观察到重组IscA2/IaiH复合物的吸收光谱类似于单独的IscA2，尽管它更清晰：在二硫代苏糖醇存在的情况下，IscA2的[2Fe-2S]簇，而不是IscA2/IaiH复合物的[2Fe-2S]簇，在添加双bionite钠后变得还原不稳定。这表明IaiH的存在稳定了[2Fe-2S]簇的IscA2片段。在没有二硫苏糖的情况下，IscA2/IaiH配合物的[2Fe-2S]簇被二亚硫酸钠破坏，这表明IscA2/IaiH配合物中铁-硫簇的体内稳定性受到细胞硫醇氧化还原状态的影响。当IscA2 （[2Fe-2S]簇的潜在配体）中超过3个保守半胱氨酸残基中的任何一个被丝氨酸取代时，在co/i细胞中[2Fe2S]簇和蛋白质复合物的组装量显著减少。存在的半胱氨酸突变的IscA2/IaiH复合物都含有一个[2Fe-2S]样簇，这表明一个稳定的铁硫簇结合在IscA2上的组装是有效和稳定的复合物形成所必需的。我们还从拟南芥中鉴定出两个叶绿体定位的nifu样蛋白，AtCnfU-V和AtCnfU-IVb，它们与一种被提议作为分子支架的蓝藻nifu样蛋白具有高度的序列相似性。AtCnfU-V在拟南芥的多个组织中均有组成性表达，而AtCnfU-IVb在空中部位的表达较为突出。缺乏AtCnfU-V的突变体拟南芥表现为矮化表型，叶片苍白，光系统I积累严重受损。突变体的叶绿体中铁氧还蛋白（一种含有[2Fe-2S]簇的基质的主要电子载体）的数量以及铁硫簇插入载铁氧还蛋白的体外活性均有所下降。AtG~f U-V在e.c cli细胞中表达后，形成了一个类似[2Fe-2S]簇的同二聚体，该簇可以在体外转移到载铁还蛋白中形成全氧铁还蛋白。我们认为AtCnfU在叶绿体中作为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. Less的生物发生所必需的

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