Iron-sulfur cluster biosynthesis : Molucular mechanism of the assembly of intermediate cluster and its transfer to apo proteins.

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-4S]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，我们以ICSU为重点，它是ISC机制的中心关键角色，作为新生的Fe-S簇的支架，并采用遗传学、生化和结构方法来阐明Fe-S簇被组装并转移到靶蛋白的复杂机制。我们分离并分析了一株缺乏ISC机制的HSCA和HSCB的突变株产生的14个伪回复突变，发现负责的抑制突变都位于IscU。突变的IscU在二级结构水平上从WT IscU发生构象改变，这表明在野生型细胞中，HscA和HscB诱导IscU的结构变化，从而促进IscU向靶蛋白的簇状转移。与支架蛋白中出了名的不稳定的Fe-S簇中间体相比，我们发现来自嗜热细菌Aquifex aeolicus(AaIscU)的IscU同源物含有稳定的Fe-S簇。首次在厌氧条件下对AaIscU进行了纯化、表征和结晶。晶体结构表明，HALO-AaIscU是一个不对称的三聚体，在两个结构改变的原始体之间的界面上只有一个[2Fe-2S]团簇。不对称三聚体的结构为分子事件提供了有价值的见解：新生的Fe-S簇的组装、维护和转移与ISCU低聚态的转换紧密耦合。

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-4S] 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 focused on IcsU, a central key player of the ISC machinery working as a scaffold for a nascent Fe-S cluster, and undertaken genetic, biochemical and structural approaches to elucidate the complex mechanism by which the Fe-S clusters are assembled and transferred to target proteins.1. We have isolated and analyzed 14 psuedorevertants generated from a mutant strain lacking HscA and HscB of the ISC machinery, and found that the responsible suppressor mutations are all located in IscU. The mutated IscUs were conformationally altered from WT IscU at the secondary structure level, suggesting that, in wild-type cells, HscA and HscB induce the structural changes of IscU thereby facilitating the cluster transfer from IscU to target proteins.2. In contrast to the notoriously unstable Fe-S cluster intermediate in the scaffold proteins, we found that the IscU homolog from the hyperthermophilic bacterium Aquifex aeolicus (AaIscU) carries a stable Fe-S cluster. AaIscU was purified, characterized and crystallized for the first time under anaerobic conditions. The crystal structure has revealed that the holo-AaIscU is an asymmetric trimer carrying only one [2Fe-2S] cluster at the interface between two structurally altered protomers. The structure of the asymmetric trimer provides valuable insight into the molecular event: the assembly, maintenance and transfer of the nascent Fe-S cluster is tightly coupled to the conversion of the oligomeric state of IscU.