ASSEMBLY AND FUNCTION OF BIOLOGICAL IRON-SULFUR CLUSTERS

描述：(改编自申请人的摘要)铁-硫团簇是 存在于120多种不同类型的酶或蛋白质中，并构成 最古老、最普遍、结构最多样化的一类 生物假体组。尽管他们的主要角色是调解 生物电子传输，铁-硫中心已知构成 许多酶的活性部位，并具有重要的结构和 监管角色。然而，生物铁硫的功能多样性 集群的定义尚未完全明晰，集群的机制也尚未完全明晰 生物合成，这是细胞铁稳态和调节的核心 对于铁-硫团簇的作用，人们仍然知之甚少。长期目标 这个项目的主要内容是从分子水平上理解簇的生物合成和 生物铁-硫团簇在二硫化物中新出现的作用 S-腺苷甲硫氨酸依赖的还原和引发自由基反应 酶，为生物素和硫辛酸的生物合成提供硫。 最终，这将导致对铁的动态平衡和 与铁超载和铁缺乏或抑制有关的人类疾病 呼吸链酶。 这一方法涉及到使用分子生物学技术来影响大规模 靶标酶和蛋白质的表达和/或特定部位的变化， 生化和酶分析以及生物物理学的应用 光谱技术(电子顺磁共振、吸收、磁性 圆二色、共振、吸收、磁性圆二色、 共振拉曼、穆斯堡尔和质谱学)，可以探测自然和 铁或铁硫中心在催化循环过程中的详细性质 团簇生物合成。将被调查的具体系统包括 与固氮有关的蛋白质--特异铁和一般铁-硫 棕色固氮菌的簇状生物合成、生物素合成酶 大肠杆菌和叶绿体中的铁氧还蛋白：硫氧还蛋白还原酶。这个 目的是建立NifU/NIFS和IscU/ISCS介导的机制 铁硫簇的生物合成，决定了铁硫簇的作用 铁氧还蛋白：硫氧还蛋白还原酶在介导细胞还原切割中的作用 活性中心二硫化物，表征了簇的转化，即 负责为生物素生物合成提供硫磺，确定 铁-硫团簇介导的还原裂解机理 S-腺苷蛋氨酸在生物素合成酶中的应用 生物素生物合成系统。

DESCRIPTION: (Adapted from applicant's abstract) Iron-sulfur clusters are present in more than 120 different types of enzymes or proteins and constitute one of the most ancient, ubiquitous and structurally diverse classes of biological prosthetic groups. Although their primary role lies in mediating biological electron transport, iron-sulfur centers are known to constitute the active sites of numerous enzymes and to have important structural and regulatory roles. However, the functional diversity of biological iron-sulfur clusters has yet to be fully defined, and the mechanism of cluster biosynthesis, which is central to cellular iron homeostasis and the regulatory roles of iron-sulfur clusters, is still poorly understood. The long-term goal of this project is a molecular-level understanding of cluster biosynthesis and of the newly emerging roles of biological iron-sulfur clusters in disulfide reduction, initiating radical reactions in S-adenosylmethionine-dependent enzymes, and providing the sulfur for biosynthesis of biotin and lipoic acid. Ultimately this will lead to enhanced understanding of iron homeostasis and human diseases related to iron overload and defects or inhibition of respiratory chain enzymes. 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, and the application of biophysical spectroscopic techniques (electron paramagnetic resonance, absorption, magnetic circular dichroism, resonance, absorption, magnetic circular dichroism, resonance Raman, Mossbauer and mass spectrometry) that can probe the nature and detailed properties of iron or iron-sulfur centers during catalytic cycling or cluster biosynthesis. The specific systems to be investigated include the proteins involved with nitrogen-fixation-specific and general iron-sulfur cluster biosynthesis in Azotobacter vinelandii, biotin synthase from Escherichia coli and ferredoxin:thioredoxin reductase from chloroplasts. The objectives are to establish the mechanism of NifU/NifS- and IscU/IscS-mediated iron-sulfur cluster biosynthesis, determine the role of the iron-sulfur cluster in ferredoxin:thioredoxin reductase in mediating reductive cleavage of the active-site disulfide, characterize the cluster transformation that is responsible for providing the sulfur for biotin biosynthesis, determine the mechanism of iron-sulfur cluster-mediated reductive cleavage of S-adenosylmethionine in biotin synthase, and develop an in vitro catalytic system for biotin biosynthesis.