Biosynthesis and Novel Function of Fe-S clusters

Fe-S团簇的生物合成和新功能

• 批准号: 7217335
• 负责人: Boi-Hanh V. Huynh
• 金额: $ 23.79万
• 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 2009-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7217335
• 关键词: Aconitate Hydratase; Address; Anabolism; Azotobacter vinelandii; Biochemical; Biological; Biological Assay; Biology; Biotin; Budgets; Carbon; Cells; Chemistry; Chromosomes; Class; Collaborations; Complement; Complex; Coupled; Development; Diploidy; Disulfides; Electron Spin Resonance Spectroscopy; Electron Transport; Electronics; Elements; Environment; Enzyme Activation; Enzymes; Ferredoxin; Freezing; Future; Gene Cluster; Generations; Genetic; Goals; Heme; Housekeeping; Human; Hydrogen; In Vitro; Individual; Investigation; Iron; IscS protein; Kinetics; Laboratories; Laboratory Research; Measurement; Mediating; Metabolism; Metals; Molecular; Molecular Biology Techniques; Molecular Chaperones; Mossbauer Spectroscopy; Nitrogen; Nitrogen Fixation; Oxidation-Reduction; Oxidoreductase; Oxygen; Participant; Process; Progress Reports; Prosthesis; Proteins; Purpose; Range; Reaction; Research; Research Personnel; Research Project Grants; Role; Role playing therapy; Scaffolding Protein; Site; Staging; Structure; Sulfur; System; Techniques; Temperature; Thioredoxin; Time; Work; absorption; base; biotin synthase; design; desthiobiotin; disulfide bond; ferredoxin-thioredoxin reductase; formate C-acetyltransferase; formate acetyltransferase activating enzyme; in vivo; insight; interest; molybdenum cofactor; nitrogenase reductase; novel; programs; research study; respiratory; success

DESCRIPTION (provided by applicant): Iron-sulfur (Fe-S) proteins are a group of functionally diverse proteins that contain prosthetic groups composed of Fe and sulfur of various structures, termed Fe-S clusters. They have a well established functional role of mediating biological electron transfer in the respiratory and photosynthetic electron transfer chains and are involved in the metabolism of essential organic elements. They are also involved in a diverse range of non-redox processes including sensing and regulatory function. This proposal seeks support to continue the PI's research project of employing a combined Mossbauer and EPR spectroscopic approach together with the rapid freeze-quench technique to investigate (1) the biosynthesis of Fe-S clusters and (2) the newly emerged functions of Fe-S clusters found in two classes of Fe-S enzymes: ferredoxin-dependent disulfide reductases and S-adenosylmethionine (SAM)-dependent Fe-S enzymes. At present, there are three known Fe-S cluster biosynthesis machineries: the nitrogen fixation specific NIF system, the ubiquitous "housekeeping" iron-sulfur cluster assembly ISC system, and the newly discovered "sulfur mobilization" SUF system. This research project focuses on the NIF and ISC systems. Experiments are designed to investigate the mechanism that the NIF and ISC systems use for cluster assembly and transport. The emphasis is on the transport of the assembled clusters from the scaffold proteins to the targeted proteins. In addition, the in vivo functional roles of the six isc gene products will be investigated by using whole cell Mossbauer spectroscopy and a controlled bacterial expression system that permits real-time depletion of each of the six proteins. For the studies of the novel functions of Fe-S clusters, three functionally diverse enzymes were chosen initially. They are, ferredoxin:thioredoxin reductase (FTR), pyruvate formate-lyase-activating enzyme (PFL-AE), and biotin synthase (BioB). FTR catalyzes the reductive cleavage of disulfide groups in thioredoxins for enzyme activation. PFL-AE activates pyruvate formate lyase (PFL) by catalyzing the generation of a glycyl radical in PFL, and BioB converts dethiobiotin to biotin. Significant progress has been made during the current budget period in understanding the functions of the Fe-S clusters in these enzymes. The results have established that all three enzymes employ a unique site-specific Fe-based Fe4S4 cluster chemistry for their respective functions. In an effort to further determine the detailed mechanistic steps involved in the catalytic cycles of these enzymes, rapid freeze-quench and cryoreduction techniques will be used to trap reaction intermediates for spectroscopic characterization and kinetic investigations. In addition, we propose to extend our study to include another important SAM-dependent enzyme, the human MOCS1A, which catalyzes the initial steps in the biosynthesis of molybdenum cofactor, MoCo. It is by studying these functionally diverse enzymes that we hope to identify factors that are essential for controlling the reactivity of Fe-S clusters.

描述（由申请人提供）： 铁硫 (Fe-S) 蛋白是一组功能多样的蛋白质，含有由不同结构的铁和硫组成的辅基，称为 Fe-S 簇。它们在介导呼吸和光合电子传递链中的生物电子传递方面具有明确的功能作用，并参与必需有机元素的代谢。它们还参与各种非氧化还原过程，包括传感和调节功能。该提案寻求支持继续 PI 的研究项目，即采用穆斯堡尔和 EPR 光谱相结合的方法以及快速冷冻猝灭技术来研究 (1) Fe-S 簇的生物合成和 (2) 在两类 Fe-S 酶中发现的 Fe-S 簇的新功能：铁氧还蛋白依赖性二硫键还原酶和铁氧还蛋白依赖性二硫键还原酶。 S-腺苷甲硫氨酸 (SAM) 依赖性 Fe-S 酶。目前，已知的铁硫簇生物合成机制有三种：固氮特异性NIF系统、无处不在的“看家”铁硫簇组装ISC系统和新发现的“硫动员”SUF系统。该研究项目重点关注 NIF 和 ISC 系统。实验旨在研究 NIF 和 ISC 系统用于簇组装和运输的机制。重点是组装的簇从支架蛋白到目标蛋白的运输。此外，将通过使用全细胞穆斯堡尔光谱和允许实时消耗六种蛋白质中每一种的受控细菌表达系统来研究六种 isc 基因产物的体内功能作用。为了研究 Fe-S 簇的新功能，最初选择了三种功能多样的酶。它们是铁氧还蛋白：硫氧还蛋白还原酶（FTR）、丙酮酸甲酸裂解酶激活酶（PFL-AE）和生物素合酶（BioB）。 FTR 催化硫氧还蛋白中二硫基的还原裂解，从而激活酶。 PFL-AE 通过催化 PFL 中生成甘氨酰自由基来激活丙酮酸甲酸裂解酶 (PFL)，而 BioB 将脱硫生物素转化为生物素。在当前预算期间，在了解这些酶中 Fe-S 簇的功能方面已经取得了重大进展。结果表明，所有三种酶均采用独特的位点特异性 Fe 基 Fe4S4 簇化学来实现各自的功能。为了进一步确定这些酶催化循环中涉及的详细机制步骤，将使用快速冷冻淬灭和低温还原技术来捕获反应中间体，以进行光谱表征和动力学研究。此外，我们建议扩大我们的研究范围，纳入另一种重要的 SAM 依赖性酶，即人类 MOCS1A，它催化钼辅因子 MoCo 生物合成的初始步骤。通过研究这些功能多样的酶，我们希望确定控制 Fe-S 簇反应性所必需的因素。