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：（由申请人提供）Fe-S蛋白是一组功能性 不同的蛋白质含有辅基组成的铁和无机 各种结构的硫，称为Fe-S簇。除了有 电子传递的作用，铁硫蛋白参与了一个 各种各样的非氧化还原过程，包括传感和调节 功能协调发展的在本申请中，我们提出采用组合的 光谱/快速动力学方法研究Fe-S的生物合成 团簇，并研究新发现的功能作用的Fe-S团簇， 稳定自由基中间体。已经确定，一对 固氮基因产物NifU和NifS是组装所必需的 固氮酶体系的Fe-S簇。NifS的同源物和 NifU，分别称为IscS和IscU，在广泛的 从细菌到人类的生物体，因此， 参与生物学中Fe-S簇的一般组装/修复。 本文通过实验研究了Fe-S的作用机理 NifU/NifS和IscU/IscS在这一过程中所起的作用。 重要的生物学过程。为了加强我们对 Fe-S簇功能，三种功能不同的蛋白质被选择用于 建议的研究：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。FTR催化二硫化物基团的还原裂解， 硫氧还蛋白用于酶激活，生物素合酶转化脱硫生物素 生物素。到目前为止积累的证据表明，所有三种酶都利用了一种 4Fe-4S簇介导的基于位点特异性u（3）-S（2-）的化学， 各自的功能。该研究旨在评估有效性 并确定所涉及的详细机械步骤， 催化循环。拟议研究的选择方法是 穆斯堡尔和EPR光谱，特别适合研究 含铁蛋白质将使用快速冷冻-淬火动力学技术 捕获反应中间体，用于光谱表征， 动力学研究在可能的情况下，其他补充技术，如 由于共振拉曼、ENDOR和EXAFS将用于获得进一步的结构 关于反应中间体的信息。位点特异性变体将 设计、生产和经受类似的动力学/光谱 为确定具体机构的职能作用而进行的调查 残基在分子水平上的详细机制的见解，预计 从拟议的调查中产生。