Mechanism of iron-sulfur cluster assembly : Structure and functional interaction of Isc proteins

铁硫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簇，如[2Fe-2S]、[3Fe-4S]和[4Fe-4S]团簇。对于这些簇的体内合成仍然知之甚少，人们认为这需要一个复杂的机制来编码我们的原核生物，由ISC(铁遭受簇)操纵子iscRSUA-hscBA-FDX-ORF3编码。我们采用遗传学和生物化学的方法来阐明铁-S除尘器组装的复杂机制。1.通过对大肠杆菌中ISC操纵子的系统突变分析，鉴定了ISC机制的必要成分(ISCS、ISCU、HSCB、HSCA和FDX)。2.重组ISCA的生化特征导致了不稳定的[2Fe-2S]簇的鉴定和与Fdx3的特异性相互作用。这表明Cys46位于[2Fe-2SJ簇附近的分子表面上。4.通过双杂交和下拉分析相结合的方法，阐明了ISC机制组件之间的蛋白质-蛋白质相互作用网络。5.我们分离了一个共价结合的ISCS/ISCU复合体，并鉴定了ISES的Cys328和LSCU的Cys63是二硫键形成的位置。6.我们分析了从一个缺乏ISC机制所有组件的突变页岩中分离到的自发超逆转物，这导致了参与Fe-S团簇组装的非ISC途径的suf ABCDSE(Suf)操纵子的确定。与sufBC同源的基因存在于广泛的细菌、古菌和叶绿体中，这表明这种类型的系统在自然界中几乎无处不在。

Iron-sulfur 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 vailiable complexity, such as [2Fe-2S], [3Fe-4S], and [4Fe-4S] clusters. Still very little is known about the in vivo synthesis of these clusters, which is believed to require a complex machinery encoded us prokaryotes by the isc (iron suffer cluster) operon, iscRSUA-hscBA-fdx-ORF3. We have undertaken genetic and biochemical approaches to elucidate the complex mechanism by which Fe-S dusters are assembled.1.Essential compornents of the ISC machinery (IscS, IscU, HscB, HscA, and Fdx) have been identified by a systematic mutational analysis of the isc operon in Eseherichia coli.2.Biochemical characterization of the recombinant IscA led to the identification of labile [2Fe-2S] cluster and specific interaction with Fdx.3.We determined the crystal structure of Fdx, which suggested the important role of Cys46 located on the molecular susface in the vicinity of [2Fe-2SJ cluster.4.Network of protein-protein interactions among the components of the ISC machinery has been elucidated by the combination of two-hybrid and pull-down analyses.5.We have isolated a covalently-bound IscS/IscU complex and identified that Cys328 of IseS and Cys63 of lscU are the sites of disulfide bridge formation.6.We have analyzed spontaneous psuedorevertants isolated from a mutant shale lacking all the components of the ISC machinery, which led to the identification of the sufABCDSE (suf) operon participating in the ISC-independent pathway for the assembly of Fe-S clusters. The genes homologous to sufBC are present in a wide range of bacteria, archaea and plastids, suggesting that this type of system is almost ubiquitous in nature.