Biosynthesis and Novel Function of Fe-S clusters

Fe-S团簇的生物合成和新功能

• 批准号: 7393155
• 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 2010-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7393155
• 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）： 铁硫蛋白（Fe-S）是一类功能多样的蛋白质，含有由不同结构的铁和硫组成的辅基，称为Fe-S簇。它们在呼吸和光合电子传递链中具有介导生物电子传递的功能作用，并参与必需有机元素的代谢。它们还参与各种各样的非氧化还原过程，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Mossbauer和EPR光谱法结合快速冷冻淬灭技术来研究（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还原酶和S-腺苷甲硫氨酸（SAM）依赖性Fe-S酶。目前已知的铁硫团簇生物合成机制有三种：固氮特异性NIF系统、普遍存在的“管家”铁硫团簇组装ISC系统和新发现的“硫活化”SUF系统。本研究项目的重点是NIF和ISC系统。实验的目的是研究机制，NIF和ISC系统用于集群组装和运输。重点是从支架蛋白到靶蛋白的组装簇的运输。此外，六个isc基因产物的体内功能作用将通过使用全细胞穆斯堡尔谱和受控的细菌表达系统，允许实时耗尽的六种蛋白质。为了研究Fe-S簇合物的新功能，首先选择了三种功能不同的酶。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化物基团的还原裂解以用于酶活化。PFL-AE通过催化PFL中甘氨酰自由基的产生来激活丙酮酸甲酸裂解酶（PFL），BioB将脱硫生物素转化为生物素。在本预算期间，在了解这些酶中Fe-S簇的功能方面取得了重大进展。结果已经确定，所有三种酶都采用独特的位点特异性Fe基Fe 4S 4簇化学用于其各自的功能。为了进一步确定这些酶催化循环中涉及的详细机械步骤，将使用快速冷冻猝灭和冷冻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，我们建议扩展我们的研究，包括另一个重要的SAM依赖性酶，人类MOCS 1A，催化钼辅因子，MoCo的生物合成的初始步骤。正是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。

项目成果

Characterization of a peroxodiiron(III) intermediate in the T201S variant of toluene/o-xylene monooxygenase hydroxylase from Pseudomonas sp. OX1.

假单胞菌甲苯/邻二甲苯单加氧酶羟化酶 T201S 变体中过氧二铁 (III) 中间体的表征。

• DOI: 10.1021/ja9011782
• 发表时间: 2009
• 期刊: Journal of the American Chemical Society
• 影响因子: 15
• 作者: Song,WoonJu;Behan,RachelK;Naik,SunilG;Huynh,BoiHanh;Lippard,StephenJ
• 通讯作者: Lippard,StephenJ

Spectroscopic properties of desulfoferrodoxin from Desulfovibrio desulfuricans (ATCC 27774).

来自脱硫弧菌 (ATCC 27774) 的脱硫铁还蛋白的光谱特性。

• 发表时间: 1994
• 期刊: The Journal of biological chemistry
• 作者: Tavares,P;Ravi,N;Moura,JJ;LeGall,J;Huang,YH;Crouse,BR;Johnson,MK;Huynh,BH;Moura,I
• 通讯作者: Moura,I

Role of histidine-86 in the catalytic mechanism of ferredoxin:thioredoxin reductase.

组氨酸 86 在铁氧还蛋白：硫氧还蛋白还原酶催化机制中的作用。

• DOI: 10.1021/bi802074p
• 发表时间: 2009
• 期刊: Biochemistry
• 影响因子: 2.9
• 作者: Walters,ElizabethM;Garcia-Serres,Ricardo;Naik,SunilG;Bourquin,Florence;Glauser,DominiqueA;Schürmann,Peter;Huynh,BoiHanh;Johnson,MichaelK
• 通讯作者: Johnson,MichaelK

Characterization of the iron-binding site in mammalian ferrochelatase by kinetic and Mössbauer methods.

通过动力学和穆斯堡尔方法表征哺乳动物亚铁螯合酶中的铁结合位点。

• DOI: 10.1074/jbc.270.44.26352
• 发表时间: 1995
• 期刊: The Journal of biological chemistry
• 作者: Franco,R;Moura,JJ;Moura,I;Lloyd,SG;Huynh,BH;Forbes,WS;Ferreira,GC
• 通讯作者: Ferreira,GC

Spectroscopic and functional characterization of iron-bound forms of Azotobacter vinelandii (Nif)IscA.

• DOI: 10.1021/bi300664j
• 发表时间: 2012-10-16
• 期刊: Biochemistry
• 影响因子: 2.9
• 作者: Mapolelo DT;Zhang B;Naik SG;Huynh BH;Johnson MK
• 通讯作者: Johnson MK