ASSEMBLY AND FUNCTION OF BIOLOGICAL IRON-SULFUR CLUSTERS

描述：（改编自申请人的摘要）铁硫簇是 存在于120多种不同类型的酶或蛋白质中， 最古老、最普遍、结构最多样的一类 生物假体虽然他们的主要作用在于调解 生物电子传递，铁硫中心是已知的构成 许多酶的活性位点，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 集群尚未完全定义，集群的机制 生物合成，这是核心的细胞铁稳态和调节 铁硫簇的作用，仍然知之甚少。远景目标 该项目的主要目的是在分子水平上理解簇生物合成， 生物铁硫簇在二硫化物中的新出现的作用， 还原，引发S-腺苷甲硫氨酸依赖性 酶，并为生物素和硫辛酸的生物合成提供硫。 最终，这将导致对铁稳态的更好理解， 与铁过载和铁缺乏或铁抑制有关的人类疾病 呼吸链酶 该方法涉及使用分子生物学技术来影响大规模 靶酶和蛋白的表达和/或位点特异性变化， 生物化学和酶测定，以及生物物理 光谱技术（电子顺磁共振、吸收、磁性 圆二色性，共振，吸收，磁性圆二色性， 共振拉曼，穆斯堡尔和质谱），可以探测性质和 在催化循环期间铁或铁-硫中心的详细性质，或 簇生物合成需要调查的具体系统包括 与固氮特异性和一般铁硫有关的蛋白质 棕色固氮菌中的簇生物合成，来自的生物素合成酶 大肠杆菌与铁氧还蛋白：叶绿体硫氧还蛋白还原酶。的 目的是建立NifU/NifS-和IscU/IscS-介导的细胞凋亡机制， 铁硫簇的生物合成，确定铁硫簇的作用 在铁氧还蛋白：硫氧还蛋白还原酶介导的还原性切割的 活性位点二硫化物，表征簇转化，即 负责为生物素生物合成提供硫，确定 铁硫团簇介导的还原裂解机理 S-腺苷甲硫氨酸在生物素合成酶，并制定了在体外催化 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.