Charge Transfer Reactions of the Cytochrome bc1 Complex

超出提供的空间。 在呼吸和光合作用电子传递链中起核心作用的细胞色素BC[复合体也是 与与发病和衰老有关的破坏性自由基和活性氧的产生有关。 因此，了解BCI复合体的分子机制对医学和基础研究具有重要意义。 生化、生物物理和分子生物学研究推动了结构和分子生物学表征方面的重大进展 B^络合物的功能，最终形成来自不同来源的BC[晶体]的X射线结构。尽管有广博的知识- 基于BC]的复杂功能，我们对其功能的分子机制的理解是粗略的。有了分子 Bc[络合物的结构我们现在可以研究b^络合物的分子机制，即 辅因子和蛋白质在分子和原子水平上的时间依赖和同步相互作用的序列。 该方案利用光的动力学和光谱优势，重点研究了BCI周转的分子机制。 球形红细菌复合体的活化。建议的研究结合了强大的红外方法 光谱(FTIR)、多通道光学光谱、化学计量学和直接电位测量，具有选择性 在组氨酸标记的背景中使用突变体。具体目标包括：(1)质子化和电生化步骤的定位 细胞色素b的周转及其对净发电的相对贡献；(2)细胞色素b的分子特征 质子分解反应导致质子吸收和质子释放；(3)应用FTIR鉴定辅因子的红外光谱谱带，以及 蛋白质对Be]复合体b‘和V中心的特定抑制物结合的反应；(4)FTIR和 质子传导途径的主要成分，特别是氨基酸残基的诱变；以及(5)表征 BCJ络合物中泛醌的电子转移和质解反应之间的耦合的分子机制。 为了实现这些目标，已经为BCJ综合体的周转中的许多步骤的具体隔离设计了协议， 用于FTIR、多通道光谱和电测方法的研究。这些步骤包括：(一)制备体系--反应 中心(RCS)和Be，-通过改变氧化还原电位、pH和离子强度处于定义的状态，(Ii)RC和Be的分离！ 基于双闪实验的反应(区分BCI络合物的给体和受体侧)，(Iii)分离 通过传统的生物化学(提取)去除电子传递成分(细胞色素c2、qb和其他苯二酚)的反应 方法(Iv)用BCI复合体和RC的特异性抑制剂分离不同的反应。(V)光谱、动力学反褶积 和热力学参数，(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特定反应 或公元前[建筑群]的状态。

EXCEED THE SPACE PROVIDED. The cytochrome bc[ complex, which plays a central role in respiratory and photosynthetic electron transport chains, is also implicated in the production of damaging free radicals and reactive oxygen species relevant to pathogenesis and aging. Understanding the molecular mechanisms of the bci complex is therefore of major importance for medical and basic research. Biochemical, biophysical and molecular biology studies have driven significant progress in the characterization of the structure and function of b^ complexes, culminating in the X-ray structure of bC[ crystals from different sources. In spite of a broad knowledge- base on bC]complex function, our understanding of the molecular mechanisms governing its function is crude. With the molecular structure of the bc[ complex in hand we are now in a position to study the molecular mechanisms of the b^ complex, i.e., the sequence of time dependent and synchronous interactions between cofactors and protein at molecular and atomic levels. This proposal focuses on the molecular mechanisms of bci turnover, utilizingthe kinetic and spectroscopic advantages of light activation of the complex from Rhodobacter sphaeroides. The proposed research combines the powerful methods of infrared spectroscopy (FTIR), multichannel optical spectroscopy, chemometrics, and direct electric potential measurements, with the selective use of mutants in a histidine-tagged background. Specific aims include: (1) localization of protonogenic and electrogenic steps in cytochrome b^ turnover and determination of their relativecontributions to net electrogenesis; (2) molecular characterization of the protolytic reactions resulting in proton uptake and proton release; (3) application of FTIR to identify IR bands of the cofactors, and responses of the protein due to binding of specific inhibitors in centers b' and V of the be] complex; (4) identification by FTIR and mutagenesis of the principle components of the proton conduction pathways, especially amino acid residues; and (5) characterization of molecular mechanisms of coupling between electron transfer and protolytic reactions of the ubiquinonesin the bcj complex. To achieve these goals, protocols have been devised for the specific isolation of many steps in the turnover of the bcj complex, for study by FTIR, multichannel spectroscopy, and electrometric methods. These include: (i) Preparation of the system - reaction centers (RCs) and be, - in defined states by changing redox potential, pH and ionic strength, (ii)Separation of the RC and be! reactions on the basis of two-flash experiment (distinguishing donor and acceptor sides of the bci complex), (iii) Separation of reactions by removing electron-transport components (cytochrome c2, QBand other quinones) by traditional biochemical (extraction) methods, (iv) Isolation of different reactions by specific inhibitors of the bci complex and RC. (v) Deconvolution of spectral, kinetic and thermodynamicparameters, (vi) Use of selected mutants in a histidine-tagged environment to limit or eliminate specific reactions or states of the bC[complex.