Charge Transfer Reactions of the Cytochrome bc1 Complex

细胞色素 bc1 复合物的电荷转移反应

• 批准号: 7483671
• 负责人: COLIN A WRAIGHT
• 金额: $ 28.19万
• 依托单位: UNIVERSITY OF ILLINOIS AT URBANA-CHAMPAIGN
• 依托单位国家: 美国
• 财政年份: 1996
• 资助国家: 美国
• 起止时间: 1996-07-01 至 2011-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7483671
• 关键词: Accounting; Affect; Affinity; Age; Aging-Related Process; Amino Acids; Basic Science; Behavior; Binding; Binding Sites; Biochemical; Biological Assay; Carotenoids; Catalytic Domain; Charge; Chronic; Complex; Condition; Coupled; Coupling; Cytochrome bc1 Complex; Cytochromes; Cytochromes b; 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; Membrane; Membrane Potentials; Methods; Mitochondria; Modification; Molecular; Molecular Biology; Mutation; Myopathy; Naphthoquinones; Nature; Numbers; Optics; Oxidation-Reduction; Pathogenesis; Pharmaceutical Preparations; Physiological; Play; Preparation; Procedures; Process; Production; Property; Protocols documentation; Protons; Quinones; Range; Reaction; Reactive Oxygen Species; Relative (related person); Research; Research Personnel; Rhodobacter; 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; Thinking; Time; Ubiquinone; Ursidae Family; Variant; Work; absorption; analog; base; benzoquinone; cofactor; dimer; driving force; fungus; infrared spectroscopy; inhibitor/antagonist; ionization; monomer; mutant; novel; novel therapeutics; pH gradient; polypeptide; programs; research study; respiratory; response; semiquinone; stigmatellin; uptake

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发挥核心作用，但也涉及与发病机制和衰老相关的破坏性自由基和活性氧的产生。此外，bc1复合物是许多杀菌药物的靶标，具有显着的物种特异性和范围。因此，了解 bc1 复合物的分子机制对于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研究推动了 bc1 复合物结构和功能表征方面的重大进展，最终获得了不同来源的 bc1 晶体的 X 射线结构，证明它是同型二聚体。该研究重点关注bc1活性的分子机制，利用球形红杆菌复合物光活化的动力学和光谱优势，以及其优异的分子工程属性。拟议的研究结合了高通量动力学（单通道和多通道）光谱、化学计量学、红外光谱（FTIR）、EPR和直接电位测量的方法，并在组氨酸标记的背景下选择性地使用突变体。提出了一种新的双亲和标签程序，用于生成每个单体中具有不同突变的异二聚体。具体目标包括：（1）表征bc1复合物中电子转移和质子分解反应之间耦合的分子机制； (2) 确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)将bc1复合物的行为变化表征为??；使用校准的类胡萝卜素带移作为电压表建立，例如单体之间的交叉 ET、超氧化物产生等； （4）多管齐下证明单体之间发生电子转移； （5）检验我们的假设，即单体-单体电子转移仅在耦合条件下有效，并表征单体-单体电子转移对不同因素（特别是pH、δ和T）的响应； (6) b-血红素和Qj位醌态能量学的测定。为了实现这些目标，我们设计了针对 bc1 复合物周转中许多步骤的特定分离的方案，以便通过多通道光谱、FTIR 和电测量方法进行研究。这些包括：(i) 通过改变氧化还原电位、pH 和离子强度，以规定的状态制备系统 - 反应中心 (RC) 和 bc1，(ii) 基于两次闪蒸实验（区分 bc1 复合物的供体和受体侧）分离 RC 和 bc1 反应，(iii) 通过去除电子传输组分（细胞色素 c2、QB 和其他）来分离反应。 (iv) 通过 bc1 复合物和 RC 的特定抑制剂分离不同的反应。 (v) 光谱、动力学和热力学参数的解卷积， (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)通过双亲和标签创建bc1复合物的异二聚体。这项工作与人类健康具有重要意义。 cyt bc1 复合体（或复合体 III）是产生破坏性氧化物质的主要位点，这些氧化物质会导致线粒体（尤其是 DNA）损伤缓慢累积。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）也由 bc1 复合体功能障碍引起。最后，这种活性是新治疗剂的目标，特别是对于真菌和寄生原生生物。