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Charge Transfer Reactions of the Cytochrome bc1 Complex

细胞色素 bc1 复合物的电荷转移反应

基本信息

批准号：
8318350
负责人：
COLIN A WRAIGHT
金额：
$ 7.93万
依托单位：
UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
依托单位国家：
美国
项目类别：
财政年份：
1996
资助国家：
美国
起止时间：
1996-07-01 至 2012-05-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8318350
关键词：
Accounting Affect Affinity Aging Aging-Related Process Amino Acids Basic Science Behavior Binding Binding Sites Biochemical Biological Assay Carotenoids Catalytic Domain Charge Chronic Complex Coupled Coupling Cytochrome b Group Cytochrome bc1 Complex Cytochromes Cytochromes c2 DNA Diffusion Disease Electron Transport Electron Transport Complex III Electrons Electrostatics Engineering Environment Equilibrium Free Radicals Generations Genetic Crossing Over Goals Health Heme Histidine Human Hydroquinones In Situ Ionic Strengths Iron Kinetics Knowledge Light Measurement Medical Research Membrane Membrane Potentials Methods Mitochondria Modification Molecular Molecular Biology Mutation Myopathy Naphthoquinones Nature Optics Oxidation-Reduction Pathogenesis Pharmaceutical Preparations Physiological Play Preparation Procedures Process Production Property Protocols documentation Protons Quinones Reaction Reactive Oxygen Species Relative (related person)Research Research Personnel Rhodobacter sphaeroides Roentgen Rays Role Series Side Site Source Species Specificity Spectroscopy, Fourier Transform Infrared Spectrum Analysis Structure Sulfur Superoxides System Testing Therapeutic Agents Thermodynamics Time Ubiquinone Ursidae Family Variant Work absorption analog base cofactor dimer driving force fungus infrared spectroscopy inhibitor/antagonist ionization monomer mutant novel pH gradient polypeptide programs research study respiratory response semiquinone stigmatellin uptake

项目摘要

DESCRIPTION (provided by applicant): The cytochrome (cyt) bc1 complex plays a central role in respiratory and photosynthetic electron transport chains, but is also implicated in the production of damaging free radicals and reactive oxygen species relevant to pathogenesis and aging. In addition, the bc1 complex is the target of many biocidal drugs, with significant species specificity and range. Understanding the molecular mechanisms of the bc1 complex is therefore of major importance for both medical and basic research. Biochemical, biophysical and molecular biology studies have driven significant progress in the characterization of the structure and function of bc1 complexes, culminating in the X-ray structure of bc1 crystals from different sources, demonstrating that it is a homodimer. The proposed research focuses on the molecular mechanisms of bc1 activity, utilizing the kinetic and spectroscopic advantages of light activation of the complex from Rhodobacter sphaeroides, as well as its excellent molecular engineering attributes. The proposed research combines methods of high-throughput kinetic (single and multichannel) optical spectroscopy, chemometrics, infrared spectroscopy (FTIR), EPR and direct electric potential measurements, with the selective use of mutants in a histidine-tagged background. A novel dual affinity tag procedure is proposed for the generation of heterodimers with different mutations in each monomer. Specific aims include: (1) characterization of molecular mechanisms of coupling between electron transfer and protolytic reactions in the bc1 complex; (2) determination of the kinetics and equilibria between components as a function of transmembrane proton gradient, Ap, thereby identifying membrane potential (??) and pH gradient (?pH) sensitive steps; (3) characterization of changes in behavior of the bc1 complex as ??; builds up, such as cross-over ET between monomers, superoxide production, etc., using a calibrated carotenoid bandshift as voltmeter; (4) using a multi-pronged approach to demonstrate that electron transfer occurs between monomers; (5) testing our hypothesis that monomer-monomer electron transfer is efficient only under coupled conditions, and characterization the response of the monomer-monomer electron transfer to different factors, in particular ?pH, ??, and T; (6) determination of the energetics of the b-hemes and the Qj site quinone states. To achieve these goals, protocols have been devised for the specific isolation of many steps in the turnover of the bc1 complex, for study by multichannel spectroscopy, FTIR, and electrometric methods. These include: (i) Preparation of the system - reaction centers (RCs) and bc1 - in defined states by changing redox potential, pH and ionic strength, (ii) Separation of the RC and bc1 reactions on the basis of two-flash experiment (distinguishing donor and acceptor sides of the bc1 complex), (iii) Separation of reactions by removing electron-transport components (cyt c2, QB and other quinones) by traditional biochemical (extraction) methods, (iv) Isolation of different reactions by specific inhibitors of the bc1 complex and RC. (v) Deconvolution of spectral, kinetic and thermodynamic parameters, (vi) Use of selected mutants in a histidine-tagged environment to limit or eliminate specific reactions or states of the bc1 complex; (vii) Creation of a heterodimer of the bc1 complex by means of dual affinity tags. The relevance of this work to human health is significant. The cyt bc1 complex, or Complex III, is a major site of production of damaging oxidizing species that cause slow accumulation of damage to mitochondria, especially the DNA. This is thought to be a significant contributor to the aging process. Several chronic and congenital diseases (myopathies) also arise from malfunctioning bc1 complex. Finally, this activity is a target for new therapeutic agents, especially for fungi and parasitic protists.

描述（由申请人提供）：细胞色素（cyt） bc1复合物在呼吸和光合电子传递链中起核心作用，但也涉及与发病和衰老相关的破坏性自由基和活性氧的产生。此外，bc1复合物是许多生物杀灭药物的靶点，具有显著的物种特异性和作用范围。因此，了解bc1复合体的分子机制对医学和基础研究都具有重要意义。生物化学、生物物理和分子生物学的研究推动了bc1配合物结构和功能表征的重大进展，最终在不同来源的bc1晶体的x射线结构中证明了它是一个同型二聚体。本课题拟重点研究bc1活性的分子机制，利用球形红杆菌光活化配合物的动力学和光谱学优势，以及其优良的分子工程特性。该研究结合了高通量动力学（单通道和多通道）光谱学、化学计量学、红外光谱（FTIR）、EPR和直接电位测量方法，并在组氨酸标记的背景下选择性使用突变体。提出了一种新的双亲和标签程序，用于产生每个单体具有不同突变的异源二聚体。具体目标包括：(1)表征bc1配合物中电子转移与原解反应耦合的分子机制；(2)测定组分之间的动力学和平衡作为跨膜质子梯度Ap的函数，从而确定膜电位（??）和pH梯度（??）pH)敏感步骤；(3)表征bc1配合物的行为变化为？？；建立，如单体之间的交叉ET，超氧化物的生产等，使用校准的类胡萝卜素带移作为电压表；(4)使用多管齐下的方法来证明电子在单体之间发生转移；(5)验证了我们的假设，即单体-单体电子转移仅在耦合条件下有效，并表征了单体-单体电子转移对不同因素的响应，特别是？pH值,? ?， T；(6)测定b-血红素和Qj位点醌态的能量学。为了实现这些目标，已经设计了bc1络合物周转率中许多步骤的特定分离方案，以便通过多通道光谱、FTIR和电测定方法进行研究。这些包括:(i)通过改变氧化还原电位、pH值和离子强度制备体系-反应中心（RC）和bc1 -在规定状态下的反应中心（RC）；（ii）基于双闪实验（区分bc1配合物的供体和受体侧）分离RC和bc1反应；（iii）通过传统的生化（萃取）方法去除电子传递组分（cyt c2、QB和其他醌类）分离反应。（iv）通过特定的bc1复合物和RC抑制剂分离不同的反应。(v)光谱、动力学和热力学参数的反卷积；（vi）在组氨酸标记的环境中使用选定的突变体来限制或消除bc1复合物的特定反应或状态；（vii）通过双亲和标签形成bc1复合物的异源二聚体。这项工作与人类健康的相关性是重要的。cyt bc1复合体，或复合体III，是产生有害氧化物质的主要部位，这些氧化物质会对线粒体，尤其是DNA造成缓慢的损伤积累。这被认为是衰老过程的一个重要因素。一些慢性和先天性疾病（肌病）也可由bc1复合体功能障碍引起。最后，该活性是新的治疗药物的靶点，特别是对真菌和寄生原生生物。