ASSEMBLY AND FUNCTION OF BIOLOGICAL IRON-SULFUR CLUSTERS

生物铁硫簇的组装和功能

• 批准号: 6692650
• 负责人: MICHAEL K. JOHNSON
• 金额: $ 16.29万
• 依托单位: UNIVERSITY OF GEORGIA
• 依托单位国家: 美国
• 财政年份: 2001
• 资助国家: 美国
• 起止时间: 2001-01-01 至 2004-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6692650
• 关键词: NAD(P)H oxidoreductase; biosynthesis; biotin; carbon sulfur lyase; chemical group; chemical kinetics; chemical structure function; crystallization; cysteine; enzyme mechanism; ferredoxin; iron; iron sulfur protein; metal metabolism; molecular assembly /self assembly; molecular genetics; nitrogen fixation; nitrogenase; oxidation reduction reaction; spectrometry; structural biology; sulfur; sulfurtransferase; thioredoxin

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.

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，并构成 最古老、最普遍、结构最多样化的类别之一 生物假肢组。尽管他们的主要作用是调解 生物电子传输，已知铁硫中心构成 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，对于细胞铁稳态和调节至关重要 铁硫簇的作用仍然知之甚少。长期目标 该项目的核心是对簇生物合成的分子水平理解和 生物铁硫簇在二硫化物中的新作用 还原，引发 S-腺苷甲硫氨酸依赖性自由基反应 酶，并为生物素和硫辛酸的生物合成提供硫。 最终，这将增强对铁稳态的理解和 与铁超载和铁缺陷或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实现大规模 目标酶和蛋白质的表达和/或位点特异性变化， 生化和酶测定以及生物物理学的应用 光谱技术（电子顺磁共振、吸收、磁 圆二色性、共振、吸收、磁圆二色性、 共振拉曼、穆斯堡尔和质谱），可以探测性质和 催化循环过程中铁或铁硫中心的详细性质或 簇生物合成。需要研究的具体系统包括 与固氮特异性和一般铁硫有关的蛋白质 维氏固氮菌中的簇生物合成，生物素合酶 大肠杆菌和铁氧还蛋白：来自叶绿体的硫氧还蛋白还原酶。这 目标是建立NifU/NifS和IscU/IscS介导的机制 铁硫簇生物合成，确定铁硫簇的作用 铁氧还蛋白：硫氧还蛋白还原酶介导还原性裂解 活性位点二硫化物，表征簇转变，即 负责为生物素生物合成提供硫，确定 铁硫簇介导的还原裂解机制 生物素合酶中的S-腺苷甲硫氨酸，并开发了一种体外催化 生物素生物合成系统。