Charge Transfer Reactions of the Cytochrome bc1 Complex

描述（由申请人提供）：细胞色素（cyt）bc 1复合物在呼吸和光合电子传递链中起核心作用，但也参与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，bc 1复合物是许多杀生物药物的靶标，具有显著的物种特异性和范围。因此，了解bc 1复合物的分子机制对医学和基础研究都具有重要意义。生物化学、生物物理学和分子生物学研究推动了bc 1复合物结构和功能表征的重大进展，最终获得了不同来源的bc 1晶体的X射线结构，证明它是一种同源二聚体。拟议的研究重点是bc 1活性的分子机制，利用动力学和光谱的优势，光激活的复杂的球形红细菌，以及其优良的分子工程属性。拟议的研究结合了高通量动力学（单通道和多通道）光学光谱，化学计量学，红外光谱（FTIR），EPR和直接电位测量的方法，选择性地使用组氨酸标记的背景中的突变体。提出了一种新的双亲和标签程序，用于产生在每个单体中具有不同突变的异源二聚体。具体目标包括：（1）表征bc 1复合物中电子转移和质子水解反应之间耦合的分子机制;（2）测定作为跨膜质子梯度Ap的函数的组分之间的动力学和平衡，从而确定膜电位（？？）pH梯度（？pH）敏感步骤;（3）表征bc_1络合物的行为变化;积累，如单体之间的交换ET，超氧化物的产生等，使用校准的类胡萝卜素带移电压表;（4）使用多管齐下的方法来证明单体之间发生电子转移;（5）测试我们的假设，单体单体电子转移是有效的，只有在耦合条件下，和表征的单体电子转移的响应不同的因素，特别是？pH值，？？，（6）b-血红素和Qj位醌态的能量学测定。为了实现这些目标，协议已被设计为特定的隔离的许多步骤，在营业额的bc 1复杂的，研究多通道光谱，FTIR和电测量方法。其中包括：（i）通过改变氧化还原电位、pH值和离子强度，制备处于确定状态的系统-反应中心（RC）和bc 1-。（ii）基于两闪实验分离RC和bc 1反应（区分bc 1复合物的供体和受体侧），（iii）通过去除电子传输组分分离反应（cyt c2、QB和其他醌类）的分离;（iv）通过bc 1复合物和RC的特异性抑制剂分离不同的反应。(v)解卷积的光谱，动力学和热力学参数，（六）使用选定的突变体在组氨酸标记的环境中，以限制或消除特定的反应或状态的bc 1复合物;（七）创建一个异源二聚体的bc 1复合物通过双亲和标签。这项工作对人类健康的意义重大。cyt bc 1复合物，或复合物III，是产生破坏性氧化物质的主要场所，其导致线粒体，特别是DNA损伤的缓慢积累。这被认为是衰老过程的重要因素。一些慢性和先天性疾病（肌病）也是由bc 1复合体功能障碍引起的。最后，这种活性是新治疗剂的靶标，特别是对于真菌和寄生原生生物。

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.