Biosynthesis and Novel Functions of Fe-S Clusters

Fe-S团簇的生物合成和新功能

• 批准号: 6325357
• 负责人: Boi-Hanh V. Huynh
• 金额: $ 19.02万
• 依托单位: EMORY UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1992
• 资助国家: 美国
• 起止时间: 1992-09-30 至 2005-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6325357
• 关键词: Escherichia coli; Mossbauer spectrometry; bacterial proteins; cofactor; electron nuclear double resonance spectroscopy; electron spin resonance spectroscopy; enzyme activity; ferritin; ferroxidase; iron sulfur protein; metal complex; methane monooxygenase; oxidation reduction reaction; oxygen; protein biosynthesis; protein structure function; ribonucleotide reductase; stop flow technique

DESCRIPTION: (provided by applicant) Fe-S proteins are a group of functionally diverse proteins that contain prosthetic groups composed of Fe and inorganic sulfur of various structures, termed Fe-S clusters. In addition to the well-established role of electron transport, Fe-S proteins are involved in a diverse range of non-redox processes including sensing and regulatory functions. In this application, we propose to employ a combined spectroscopic/rapid-kinetic approach to investigate the biosynthesis of Fe-S clusters and to study the newly discovered functional role of Fe-S cluster in stabilizing radical intermediates. It has been established that a pair of the nitrogen fixation gene products, NifU and NifS, are essential for the assembly of the Fe-S clusters for the nitrogenase enzyme system. Homologs of NifS and NifU, termed IscS and IscU, respectively, are found in a wide spectrum of living organisms ranging from bacteria to human, and thus, have been proposed to be involved in the general assembly/repair of Fe-S clusters in biology. Here, experiments are proposed to investigate the mechanism of Fe-S biosynthesis and to establish the roles play by NifU/NifS and IscU/IscS in this important biological process. For the purpose of enhancing our understanding of Fe-S cluster functions, three functionally diverse proteins were chosen for the proposed studies: pyruvate formate-lyase activating enzyme (PFL-AE), ferredoxin: thioredoxin reductase (FTR) and biotin synthase. PFL-AE activates pyruvate formate lyase (PFL) by catalyzing the generation of a glycyl radical in PFL. FTR catalyzes the reductive cleavage of disulfide groups in thioredoxins for enzyme activations, and biotin synthase converts dethiobiotin to biotin. Evidence accumulated so far suggests that all three enzymes employ a 4Fe-4S cluster-mediated site-specific u(3)-S(2-) based chemistry for their respective functions. The proposed study is designed to evaluate the validity of this suggestion and to determine the detailed mechanistic steps involved in the catalytic cycles. The methods of choice for the proposed studies are Mossbauer and EPR spectroscopies, which are particularly suited for the study of Fe-containing proteins. Rapid freeze-quench kinetic techniques will be used to trap reaction intermediates for spectroscopic characterization and for kinetic investigations. Whenever possible, other complementary techniques, such as resonance Raman, ENDOR, and EXAFS will be used to obtain further structural information on the reaction intermediates. Site-specific variants will be engineered, produced and subjected to similar kinetic/spectroscopic investigations for the purpose of defining the functional roles of specific residues. Detailed mechanistic insights at a molecular level are expected to emerge from the proposed investigations.

描述：（申请人提供）Fe-S蛋白是一组功能性蛋白质 含有由铁和无机物组成的辅基的多种蛋白质 各种结构的硫，称为 Fe-S 簇。除了 Fe-S 蛋白具有明确的电子传递作用，参与 各种非氧化还原过程，包括传感和调节 功能。在此应用中，我们建议采用组合 光谱/快速动力学方法研究 Fe-S 的生物合成 团簇并研究新发现的 Fe-S 团簇在 稳定自由基中间体。已确定一对 固氮基因产物 NifU 和 NifS 对于组装至关重要 固氮酶系统的 Fe-S 簇。 NifS 的同系物和 NifU，分别称为 IscS 和 IscU，广泛存在于 从细菌到人类的生物体，因此，有人提出 参与生物学中Fe-S簇的组装/修复。 在这里，提出实验来研究 Fe-S 的机理 生物合成并确定 NifU/NifS 和 IscU/IscS 在此过程中发挥的作用 重要的生物过程。为了加深我们对 Fe-S簇功能，选择了三种功能不同的蛋白质 拟议的研究：丙酮酸甲酸裂解酶激活酶（PFL-AE）， 铁氧还蛋白：硫氧还蛋白还原酶（FTR）和生物素合酶。 PFL-AE 激活 丙酮酸甲酸裂解酶 (PFL) 通过催化甘氨酰自由基的产生 在 PFL 中。 FTR 催化二硫基团的还原裂解 硫氧还蛋白用于酶激活，生物素合酶转化脱硫生物素 到生物素。迄今为止积累的证据表明，所有三种酶均采用 4Fe-4S簇介导的位点特异性u(3)-S(2-)基化学 各自的职能。拟议研究的目的是评估有效性 该建议并确定涉及的详细机制步骤 催化循环。拟议研究选择的方法是 穆斯堡尔和 EPR 光谱，特别适合该研究 含铁蛋白质。将使用快速冷冻猝灭动力学技术 捕获反应中间体以进行光谱表征和 动力学研究。只要有可能，其他补充技术，例如 作为共振拉曼、ENDOR 和 EXAFS 将用于获得进一步的结构 反应中间体的信息。特定于站点的变体将是 设计、生产并经历类似的动力学/光谱 旨在确定特定职能角色的调查 残留物。分子水平上的详细机制见解预计将 从拟议的调查中得出。