Mechanism of iron-sulfur cluster biosynthesis : Analysis of a framework and the reaction mechanism for the two iron-sulfur cluster assembly systems.

铁硫蛋白（Fe-S）存在于几乎所有的生物体中，具有多种功能，包括电子传递、氧化还原和非氧化还原催化以及调控过程的传感等。它们含有复杂程度不同的铁和硫原子簇，如[2Fe-2S]、[3Fe-4S]和[4Fe-3S]簇。我们最近发现了两个独立的系统，称为ISC和SUF，必不可少的Fe-S簇的生物合成，虽然仍然知之甚少的机制细节。在这项研究中，我们采取了遗传，生物化学和结构的方法来阐明复杂的机制，其中的Fe-S集群是并行的。我们已经分离和分析了14个psuedoreventants产生的突变株缺乏HscA和HscB的ISC机制，揭示了各种抑制基因突变位于IscU。我们还发现，来自嗜热细菌Aquifex aeolicus的IscU同源物异常地携带稳定形式的中间Fe-S簇，其通过引入几个突变而进一步稳定。纯化和生化特性的SUF机制的组成部分已经揭示了二聚体形式的SufD和SufC_2SufD_2寡聚体以及SufB_1SufC_2SufD_1复合物，表明在反应中涉及的寡聚体状态的动态变化。通过诱变研究也鉴定了SufD功能的必需残基。我们已经确定了大肠杆菌yfhJ（IscX），SufA，SufC和SufD的晶体结构。SufA的结构揭示了由四个半胱氨酸残基组成的活性位点结构。我们还确定了一个盐桥，似乎引发的构象变化的SufC。SufD的结构揭示了前所未有的β-螺旋二聚体。SufC和SufD之间的对接模型表明SufC改变SufD的寡聚状态的作用。

Iron-sulfur (Fe-S) proteins are present in almost all living organisms and exhibit diverse functions, which include electron transport, redox and non-redox catalysis, and sensing for regulatory processes. They contain clusters of iron and sulfur atoms with variable complexity, such as [2Fe-2S], [3Fe-4S], and [4Fe-3S] clusters. We have recently found two independent systems, called ISC and SUF, essential for the biosynthesis of Fe-S clusters, although still very little is known about the mechanistic details. In this study we have undertaken genetic, biochemical and structural approaches to elucidate the complex mechanism by which the Fe-S clusters are assembled.1. We have isolated and analyzed 14 psuedorevertants generated from a mutant strain lacking HscA and HscB in the ISC machinery, which revealed various suppressor mutations located in IscU. We also found that the IscU homolog from the thermophilic bacterium Aquifex aeolicus exceptionally carries the intermediate Fe-S cluster in the stable form that was further stabilized by introduction of several mutations.2. Purification and biochemical characterization of the components of the SUF machinery have revealed dimeric form of SufD and the SufC_2SufD_2 oligomer as well as the SufB_1SufC_2 SufD_1 complex, suggesting that dynamic alteration of the oligomeric status is involved in the reaction. Essential residues for the SufD function have also been identified by mutagenic studies.3. We have determined the crystal structures of E.coli yfhJ (IscX), SufA, SufC, and SufD. The structure of SufA revealed the active-site architecture comprising of four cysteine residues. We have also identified a salt bridge that appears to trigger the conformational change of SufC. The structure of SufD revealed an unprecedented β-helix dimer. The docking model between SufC and SufD suggests the role of SufC to alter the oligomeric status of SufD.