7 FE FDI AS A MODEL FE-S AND REGULATORY PROTEIN

7 FE FDI 作为 FE-S 模型和监管蛋白

• 批准号: 6490036
• 负责人: THOMAS L POULOS
• 金额: $ 23.85万
• 依托单位: UNIVERSITY OF CALIFORNIA-IRVINE
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-01-01 至 2003-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6490036
• 关键词: Azotobacter vinelandii; NAD(P)H oxidoreductase; X ray crystallography; active sites; bacterial genetics; bacterial proteins; electrochemistry; electron transport; enzyme induction /repression; enzyme substrate complex; ferredoxin; gene mutation; microorganism culture; oxidative stress; protein structure function

Iron-sulfur [Fe-S] proteins are ubiquitous occurring in all life forms from the most primitive bacteria and archaea to the most advanced eucaryotes. These proteins are not only central to almost every essential biological electron transfer process but they also function in hydration/dehydration reactions, the generation and stabilization of radical intermediates, iron storage, oxidative stress and iron sensing and the regulation of gene expression. The general goal of the work proposed here is to elucidate the relationships between protein structure and [Fe- S] cluster structure, function, redox properties and reactivity. The experiments described are designed to address three fundamental questions in [Fe-S] protein metallobiochemistry. How does the structure of the protein control the reduction potential of a [4FE-4S]/2+/+ cluster? What is the mechanism of proton transfer from the solvent to a buried [3Fe-4S]0 cluster? What protein factors determine whether a protein will assemble a 3 Fe or a 4 Fe cluster and their interconversion? To answer these questions we have developed Azotobacter vinelandii ferredoxin I (FdI) as an [Fe-S] model system. This is an in depth, multi-disciplinary study where spectroscopic, direct electrochemical and computational methods are applied to [Fe-S] protein variants produced by site-directed mutagenesis, where the data are interpreted based on x-ray structures of mutant proteins and where the physiological consequences are studied by expressing proteins in their native background. The results are applicable to the entire class of [Fe-S] proteins and are of fundamental importance to efforts aimed at changing the reactivity of an existing protein or in the design of new [Fe-S] proteins. In addition to using it as a model we are also characterizing the function of AvFdI. This protein is greater than 90% identical to the 7Fe ferredoxins synthesize by a variety of Pseudomonas species including the important human pathogen P. aeruginosa. Our recent studies provide compelling evidence that FdI has a regulatory function related to an oxidative stress system that responds to the superoxide propagater paraquat. We have shown that FdI controls the expression of its redox partner NADPH:ferredoxin reductase indirectly through a regulatory cascade. Experiments described herein are designed to elucidate the mechanism of this regulation by identifying, purifying and characterizing the components involved in the oxidative stress regulatory system and by studying interactions of these components with each other and with possible effectors (e.g. superoxide, NO).

铁硫[Fe-s]蛋白质无处不在，以所有生命形式发生 从最原始的细菌和古细菌到最先进的 桉树。这些蛋白质不仅对几乎所有必需品都是核心 生物电子传输过程，但它们也起作用 水合/脱水反应，生成和稳定 自由基中间体，铁储存，氧化应激和铁感应以及 基因表达的调节。提出的工作的一般目标 这是为了阐明蛋白质结构与[Fe- S]群集结构，功能，氧化还原特性和反应性。这 所描述的实验旨在解决三个基本问题 在[Fe-s]蛋白质金属生物化学中。结构如何 蛋白质控制[4FE-4S]/2+/+簇的还原电位？什么 是质子从溶剂转移到埋藏的[3FE-4S] 0的机制 簇？哪些蛋白质因子决定蛋白质是否会组装 3 Fe或4 Fe群集及其互换？回答这些 我们已经开发了Azotobacter Vinelandii Ferredoxin I（FDI） [Fe-S]模型系统。这是一项深入的多学科研究 光谱，直接电化学和计算方法是 应用于由位置定向诱变产生的[Fe-s]蛋白质变体， 基于突变体的X射线结构解释数据的地方 蛋白质和生理后果的研究 在本地背景中表达蛋白质。结果适用 到整个[Fe-S]蛋白质的整个类别，至关重要 旨在改变现有蛋白质或中的反应性的努力 新的[Fe-S]蛋白的设计。除了将其用作模型我们 还表征了AVFDI的功能。该蛋白更大 比90％与7FE铁毒素相同，由多种多样 假单胞菌种类，包括重要的人类病原体铜绿假单胞菌。 我们最近的研究提供了令人信服的证据，表明外国直接投资具有监管 与氧化应激系统有关的功能 超氧化大型大型paraquat。我们已经表明，外国直接投资控制 其氧化还原伴侣NADPH的表达：铁氧还原还原酶间接表达 通过监管级联。本文所述的实验被设计为 通过识别，净化和 表征氧化应激调节的成分 系统以及研究这些组件的相互作用 并具有可能的效应子（例如超氧化物，否）。

项目成果

The crystal structure of NADPH:ferredoxin reductase from Azotobacter vinelandii.

NADPH 的晶体结构：来自 Azotobacter vinelandii 的铁氧还蛋白还原酶。

• DOI: 10.1002/pro.5560071207
• 发表时间: 1998
• 期刊: Protein science : a publication of the Protein Society
• 作者: SridharPrasad,G;Kresge,N;Muhlberg,AB;Shaw,A;Jung,YS;Burgess,BK;Stout,CD
• 通讯作者: Stout,CD

Purification and characterization of a NADP+/NADPH-specific flavoprotein that is overexpressed in FdI- strains of Azotobacter vinelandii.

NADP /NADPH 特异性黄素蛋白的纯化和表征，该黄素蛋白在维氏固氮菌 FdI 菌株中过表达。

• 发表时间: 1994
• 期刊: The Journal of biological chemistry
• 作者: Isas,JM;Burgess,BK
• 通讯作者: Burgess,BK

Mössbauer and EPR studies of Azotobacter vinelandii ferredoxin I.

穆斯堡尔和 EPR 对维氏固氮菌铁氧还蛋白 I 的研究。

• DOI: 10.1021/bi00252a014
• 发表时间: 1994
• 期刊: Biochemistry
• 影响因子: 2.9
• 作者: Hu,Z;Jollie,D;Burgess,BK;Stephens,PJ;Münck,E
• 通讯作者: Münck,E

Overexpression of ferredoxin I in Azotobacter vinelandii.

铁氧还蛋白 I 在固氮菌中过度表达。

• DOI: 10.1006/prep.1994.1014
• 发表时间: 1994
• 期刊: Protein expression and purification
• 影响因子: 1.6
• 作者: Vazquez,A;Shen,B;Negaard,K;Iismaa,S;Burgess,B
• 通讯作者: Burgess,B

Structure of C42D Azotobacter vinelandii FdI. A Cys-X-X-Asp-X-X-Cys motif ligates an air-stable [4Fe-4S]2+/+ cluster.

C42D 维氏固氮菌 FdI 的结构。

• DOI: 10.1074/jbc.m004947200
• 发表时间: 2000
• 期刊: The Journal of biological chemistry
• 作者: Jung,YS;Bonagura,CA;Tilley,GJ;Gao-Sheridan,HS;Armstrong,FA;Stout,CD;Burgess,BK
• 通讯作者: Burgess,BK