Assembly of Biological Iron-Sulfur Clusters

描述（由申请人提供）： 铁硫簇存在于200多种不同类型的酶或蛋白质中，是最古老、最普遍、结构最多样的一类生物辅基。因此，铁硫生物合成过程是几乎所有生命形式所必需的，并且在原核生物和真核生物中非常保守。已经出现了三种不同类型的铁-硫簇组装机制，称为NIF、ISC和SUF系统，并且在每种情况下，总体机制都涉及半胱氨酸脱硫酶介导的支架蛋白上的瞬时簇的组装以及随后将预先形成的簇或簇片段转移到载脂蛋白。然而，在任何情况下，都不能在分子水平上理解组装或转移机制。该项目的长期目标是使用NIF、ISC和SUF系统在分子水平上理解铁硫簇生物合成。阐明铁硫簇生物合成的机制对于理解细胞铁稳态以及与线粒体呼吸链中的铁过载和缺陷相关的人类疾病至关重要。该方法涉及使用分子生物学技术来实现目标酶和蛋白质的大规模表达和/或位点特异性变化、生物化学和酶测定、X射线晶体学以及生物物理光谱技术的应用（电子顺磁共振，吸收，磁性和自然圆二色性，共振拉曼，和穆斯堡尔谱），可以探测在簇生物合成或转移到受体蛋白质期间铁或铁-硫中心的性质和详细性质。本研究的目的是建立四种已知的铁硫簇支架蛋白的簇结合形式的结构和簇组装和转移的分子机制，确定每种类型的支架蛋白在铁硫蛋白成熟中的作用和特异性，并表征用于调节铁硫簇生物合成的机制。公共卫生相关性：铁硫簇对人类健康的重要性主要源于它们在铁稳态中的关键作用以及它们参与大量酶和蛋白质，特别是线粒体呼吸链中的酶和蛋白质。铁硫簇生物发生的分子水平的理解是至关重要的，了解各种人类疾病，包括贫血，肌病和共济失调，所产生的铁硫簇组装蛋白的缺陷。

DESCRIPTION (provided by applicant): Project Summary Iron-sulfur clusters are present in more than 200 different types of enzymes or proteins and constitute one of the most ancient, ubiquitous and structurally diverse classes of biological prosthetic groups. Hence the process of iron-sulfur biosynthesis is essential to almost all forms of life and is remarkably conserved in prokaryotic and eukaryotic organisms. Three distinct types of iron-sulfur cluster assembly machinery have emerged, termed the NIF, ISC and SUF systems, and in each case the overall mechanism involves cysteine desulfurase-mediated assembly of transient clusters on scaffold proteins and subsequent transfer of preformed clusters or cluster fragments to apo proteins. However, in no case is the assembly or transfer mechanism understood at the molecular level. The long-term goal of this project is a molecular- level understanding of iron-sulfur cluster biosynthesis using the NIF, ISC and SUF systems. Elucidating the mechanism of iron-sulfur cluster biosynthesis is central to understanding cellular iron homeostasis and thereby human diseases associated with iron-overload and defects in the mitochondrial respiratory chain. The approach involves using molecular biology techniques to effect large scale expression and/or site-specific changes in the target enzymes and proteins, biochemical and enzymatic assays, X-ray crystallography, and the application of biophysical spectroscopic techniques (electron paramagnetic resonance, absorption, magnetic and natural circular dichroism, resonance Raman, and M"ssbauer) that can probe the nature and detailed properties of iron or iron-sulfur centers during cluster biosynthesis or transfer to acceptor proteins. The objectives are to establish the structure of cluster-bound forms and the molecular mechanism of cluster assembly and transfer for each the four known types of iron-sulfur cluster scaffold proteins, identify the roles and specificity of each type of scaffold protein in the maturation of iron-sulfur proteins, and characterize the mechanisms used to regulate iron-sulfur cluster biosynthesis. PUBLIC HEALTH RELEVANCE: The importance of iron-sulfur clusters to human health stems largely from their crucial role in iron homeostasis and their involvement in a large number of enzymes and proteins, particularly those in the mitochondrial respiratory chain. A molecular-level understanding of iron-sulfur cluster biogenesis is crucial for understanding a variety of human diseases involving anemias, myopathies and ataxias that arise from defects in iron-sulfur cluster assembly proteins.