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.

说明：(申请人提供)铁-S蛋白是一组具有功能的 含有由铁和无机组成的假体基团的各种蛋白质 硫的各种结构，称为铁-S团簇。除 电子传递的作用已经确立，铁-S蛋白参与了一种 各种非氧化还原过程，包括传感和监管 功能。在此应用程序中，我们建议使用组合的 光谱/快速动力学方法研究铁-S的生物合成 并研究了新发现的Fe-S团簇的功能作用。 稳定激进中间体。已经确定有一对 固氮基因产物NifU和NIFS是组装所必需的 铁-S簇为固氮酶系统。NIFS和NIFS的同系物 NifU，分别称为ISCS和ISCU，存在于广泛的 因此，已经提出了从细菌到人类的各种生命有机体 参与生物中铁-S团簇的组装/修复。 在此，提出了用实验来研究铁-S的作用机理 NifU/NIFS和IscU/ISCS在生物合成中的作用 重要的生物过程。为了增进我们对 Fe-S簇功能，选择了三个功能不同的蛋白质用于 建议研究：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)和生物素合成酶。PFL-AE激活 丙酮酸甲酸裂解酶(PFL)催化产生甘氨酸根 在PFL。FTR催化二硫键的还原裂解 硫氧还蛋白用于酶激活，生物素合成酶转化去硫代胆碱 为了生物素。到目前为止积累的证据表明，这三种酶都使用了一种 4Fe-4S团簇介导的U(3)-S(2-)基定点化学 各自的职能。这项拟议的研究旨在评估有效性 并确定所涉及的详细机械性步骤 催化循环。建议研究的选择方法是 穆斯堡尔谱和EPR谱，特别适合于这项研究 含有铁的蛋白质。将使用快速冻结-淬火动力学技术 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，如 作为共振拉曼，Endor和EXAFS将被用来获得进一步的结构 关于反应中间体的信息。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 调查的目的是确定特定的职能角色 残留物。在分子水平上的详细的机械洞察预计将 从拟议的调查中浮现出来。