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(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修复基，称为铁-S簇。它们在呼吸和光合作用的电子转移链中具有调节生物电子转移的功能，并参与必需有机元素的新陈代谢。它们还参与各种非氧化还原过程，包括传感和调节功能。这项建议寻求支持继续PI的研究项目，使用穆斯堡尔谱和电子顺磁共振结合快速冷冻-淬灭技术来研究(1)铁-S簇的生物合成和(2)在两类铁-S酶中发现的铁-S簇的新功能：依赖铁氧还蛋白的二硫键还原酶和依赖S-腺苷甲硫氨酸的铁-S酶。目前，已知的铁-S簇状生物合成机制有三种：固氮专属的硝酸钾体系、普遍存在的管家式铁硫簇合物组装体系和新发现的硫动员亚硫酸盐体系。本课题研究的重点是NIF和ISC系统。实验旨在研究NIF和ISC系统用于集群组装和传输的机制。重点是组装的簇从支架蛋白到目标蛋白的运输。此外，将使用全细胞穆斯堡尔光谱和允许实时耗尽六种蛋白质的受控细菌表达系统来研究六种ISC基因产物在体内的功能作用。为了研究铁-S簇的新功能，初步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)和生物素合成酶(BioB)。FTR催化硫氧还蛋白中二硫键的还原裂解以激活酶。PFL-AE通过催化丙酮酸甲酸裂解酶(PFL)中甘氨酸基的生成来激活PFL，Biob将脱硫生物素转化为生物素。在本预算期内，在了解铁-S簇在这些酶中的功能方面取得了重大进展。结果表明，这三种酶都使用了一种独特的基于Fe的Fe4S4簇化学来实现它们各自的功能。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-淬火和冷冻还原技术捕获反应中间体，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，以包括另一种重要的SAM依赖酶-人MOCS1A，它催化钼辅因子MoCo生物合成的初始步骤。正是通过对这些功能不同的酶的研究，我们希望找出控制铁-S团簇反应活性的关键因素。

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