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 包含由FE和无机组成的假体群的多种蛋白质 各种结构的硫，称为Fe-S簇。除了 电子传输的良好作用，Fe-S蛋白参与了A 多种非还原过程，包括感应和调节 功能。在此应用程序中，我们建议采用合并 光谱/快速运动方法研究Fe-S的生物合成 群集并研究Fe-S群集新发现的功能作用 稳定自由基中间体。已经确定了一对 氮固定基因产物NIFU和NIFS对于组装至关重要 用于氮酶系统的Fe-S簇。 NIFS和 NIFU分别称为ISC和ISCU，在广泛的范围内发现 已经提出了从细菌到人类的生物，因此已提出 参与生物学中Fe-S簇的大会/修复。 在这里，提出了实验来研究Fe-S的机制 生物合成并确定NIFU/NIFS和ISCU/ISC的角色 重要的生物过程。为了增强我们对 Fe-S群集功能，选择了三种功能多样的蛋白 拟议的研究：丙酮酸甲酸酯酶激活酶（PFL-AE）， 铁蛋白：硫氧还蛋白还原酶（FTR）和生物素合酶。 PFL-AE激活 通过催化糖基自由基的产生 在PFL中。 FTR催化二硫基团的还原性切割 用于酶活性的硫氧还蛋白，生物素合酶转化了二乙基蛋白酶 生物素。到目前为止积累的证据表明，这三种酶都采用 基于4FE-4S群集介导的位点特异性U（3）-s（2-）的化学 各自的功能。拟议的研究旨在评估有效性 该建议并确定涉及的详细机械步骤 催化循环。拟议研究的首选方法是 Mossbauer和EPR光谱镜，特别适合该研究 含有铁的蛋白质。将使用快速冻结动力学技术 捕获中间体以进行光谱表征和 动力学研究。只要有可能，其他互补技术就这样 作为共振拉曼，Endor和ExAFS将用于获得进一步的结构 有关反应中间体的信息。特定于网站的变体将是 经过工程，生产和进行类似的动力学/光谱学 为了定义特定的功能作用的研究 残留物。预计分子水平上的详细机械见解将 从拟议的调查中得出。