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

铁-硫(Fe-S)蛋白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，以及对调节过程的感知。它们含有复杂程度不同的铁和硫原子团簇，如[2Fe-2S]、[3Fe-4S]和[4Fe-3S]团簇。我们最近发现了两个独立的体系，称为ISC和SuF，它们对于Fe-S团簇的生物合成是必不可少的，尽管对其机制细节仍知之甚少。在这项研究中，我们采用了遗传、生化和结构方法来阐明Fe-S团簇组装的复杂机制。我们在ISC机制中分离并分析了一株缺乏HSCA和HSCB的突变株产生的14个伪回复突变，发现了位于ISCU的各种抑制突变。我们还发现，嗜热菌Aquifex aeolicus的ISCU同源物异常地以稳定的形式携带中间Fe-S簇，通过引入几个突变进一步稳定了该簇。纯化和生化表征表明，SufD和SuFC_2SufD_2齐聚物以及SufB_1SufC_2SufD_1络合物是二聚体形式的SufD和SuFC_2SufD_2，表明低聚物状态的动态变化参与了反应。突变研究也鉴定了SufD功能的必需残基。我们测定了E.coliyfhJ(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中心结构。我们还发现了一个盐桥，它似乎触发了SUFC的构象变化。SufD的结构揭示了一种前所未有的β-螺旋二聚体。SuFC和SufD之间的对接模型表明，SuFC在改变SufD的寡聚状态方面发挥了作用。

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.