Structure and Function of IscA, a Potential Iron Delivery Protein for Iron-Sulfur Cluster Assembly

铁硫簇合物是生物中最普遍存在的氧化还原辅因子之一。在整个进化过程中，铁硫簇已经成为各种生理过程的组成部分，如能量转换，糖代谢，固氮，血红素和生物素的生物合成，DNA合成和DNA修复，以及基因表达的调控。由基因簇iscSUA-hscBA-fdx编码的至少六种蛋白质（IscS、IscU、IscA、HscB、HscA和铁氧还蛋白）已被鉴定为细菌中铁硫簇的一般生物发生所必需的。在真核生物中也发现了这6种蛋白的同源物，表明铁硫簇组装机制高度保守。已经表明，IscS，半胱氨酸脱硫酶，为铁硫簇提供硫，并且IscU是IscS介导的铁硫簇组装的支架，并最终将铁硫簇转移到靶蛋白。然而，铁硫簇合物的铁供体在很大程度上仍然是难以捉摸的。本项目旨在验证一个假设，即IscA的主要功能是募集细胞内铁并为IscU中的铁-硫簇组装提供铁，并且IscA介导的铁转移到IscU是通过蛋白质-蛋白质相互作用协调的。本研究有两个具体的目的：1）通过结合生物化学、生物物理学和X射线晶体学方法来确定IscA的铁结合特性，以确定对铁结合重要的氨基酸残基，并确定铁中心的氧化还原特性。2)通过研究IscA与IscU和IscS的蛋白质-蛋白质相互作用，探讨IscA向IscU中铁硫簇转移的机制。这些结果将为铁硫簇的一般生物成因提供基础知识，这是一个从细菌到人类都保守的过程。此外，了解铁硫簇生物成因的铁传递将提供细胞内铁如何在其他生物过程中利用的分子细节。更广泛的影响：该项目预计将吸引和留住高质量的研究生和本科生。学生不仅将学习多学科方法来进行拟议的研究，而且还将通过积极的研究活动培养对他们未来在学术界或工业界的职业生涯至关重要的态度。将特别努力招募积极性高的少数民族和女研究生加入研究小组。研究项目将进一步融入PI和Co-PI在其机构教授的生物化学课程，并应大大有利于机构生物化学课程。

Iron-sulfur clusters are one of the most ubiquitous redox co-factors in biology. Throughout evolution, iron-sulfur clusters have become integral part of diverse physiological processes such as energy conversion, sugar metabolism, nitrogen fixation, heme and biotin biosynthesis, DNA synthesis and DNA repair, and regulation of gene expression. At least six proteins (IscS, IscU, IscA, HscB, HscA and Ferredoxin) encoded by a gene cluster iscSUA-hscBA-fdx have been identified as essential for the general biogenesis of iron-sulfur clusters in bacteria. The homologs of the six proteins have also been found in eukaryotic organisms, indicating that the iron-sulfur cluster assembly mechanism is highly conserved. It has been shown that IscS, a cysteine desulfurase, provides sulfur for iron-sulfur clusters, and that IscU is a scaffold for the IscS-mediated assembly of iron-sulfur clusters and eventually transfers iron-sulfur clusters to target proteins. However, the iron donor for iron-sulfur clusters largely remains elusive. This project aims to test a hypothesis that the primary function of IscA is to recruit intracellular iron and deliver iron for the assembly of iron-sulfur clusters in IscU, and that the IscA-mediated iron transfer to IscU is coordinated through the protein-protein interactions. The research has two specific aims: 1) To define the iron binding properties of IscA by combining the biochemical, biophysical and X-ray crystallographic approaches to identify the amino acid residues that are important for the iron binding and to determine the redox properties of the iron center. 2) To investigate the mechanism of iron transfer from IscA to the iron-sulfur clusters in IscU by exploring the protein-protein interactions of IscA with IscU and IscS. The results will provide fundamental knowledge on the general biogenesis of iron-sulfur clusters, a process that is conserved from bacteria to humans. In addition, understanding iron delivery for the biogenesis of iron-sulfur clusters will offer the molecular details on how intracellular iron may be utilized in other biological processes.Broader Impacts: The project is expected to attract and retain high quality graduate and undergraduate students. The students will not only learn the multidisciplinary methodology to conduct the proposed research, but also develop the attitude crucial to their future career in academia or industries through vigorous research activities. Special efforts will be taken to recruit highly motivated minority and woman graduate students to the research group. The research projects will further be integrated into the biochemistry courses that the PI and Co-PI have been teaching at their institution, and should greatly benefit the institutional biochemistry curricula.