CHARGE TRANSFER REACTIONS OF THE BE COMPLEX

复杂的电荷转移反应

• 批准号: 6019108
• 负责人: COLIN A WRAIGHT
• 金额: $ 14.97万
• 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 2001-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6019108
• 关键词: Rhodospirillales; acidity /alkalinity; charge transfer complex; chromatophore; cytochrome b; cytochrome c; electrical potential; electron transport; enzyme complex; enzyme mechanism; membrane potentials; photosynthetic reaction centers; spectrometry; ubiquinone

DESCRIPTION: Respiratory and photosynthetic electron transport chains share common design features for separating charge across membranes, using the free energy of oxidation-reduction reactions. Across diverse phyla and even kingdoms, a small number of highly conserved membrane protein complexes are primarily responsible for these conversions. Among these, the cytochrome bc1 complex plays a central role in most systems. In spite of a broad knowledge-base on the functioning of the bcl complex, our understanding of the mechanism is very crude, and even gross features of its internal organization are subjects of controversy. The area of greatest ignorance is the nature of the charge separating reactions leading to electric potential generation ("electrogenesis"). In addition to electron transfer processes, which have been partially characterized by much prior work, these include proton transfer events that are almost completely obscure. This proposal focuses on the "electrogenic" steps of bc1 turnover, utilizing the kinetic advantages of light activation of the complex from Rhodobacter sphaeroides. The proposed research combines a direct electric potential measurement with optical spectroscopy, and the selective use of mutants, to address both electrogenic electron transfers between cofactors and the potentially electrogenic protonation steps of the quinone reactions in the bc1 complex. The specific aims include: (i) localization of the electrogenic steps and determination of the magnitudes of contributions from the high potential chain (QO-FeSR-c1-c2), bH-Qi electron transfer, and proton uptake to the Qi site; (ii) identification of the charge carrying species - whether electrons or protons, and (iii) characterization of the protolytic reactions resulting in proton uptake and proton release. To this end, protocols are devised for specific isolation of many steps in the turnover of the bcl complex, for study by electrometric and spectroscopic methods. These include: (i) Preparation of the system - reaction centers (RCs) and bcl - 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 bcl complex). (iii) Separation of reactions by removing electron-transport components (cytochrome c2, QB and other quinones) by traditional biochemical (extraction) methods; (iv) Isolation of different reactions by specific inhibitors of the bcl complex and RC. (v) Deconvolution of spectral, kinetic and thermodynamic parameters. (vi) Use of selected mutants to limit or eliminate specific reactions.

描述：呼吸和光合电子传递链共享 跨膜分离电荷的常见设计特征， 氧化还原反应的自由能。 跨越不同的门，甚至 王国，少数高度保守的膜蛋白复合物是 主要负责这些转换。 其中，细胞色素 BC 1复合体在大多数系统中起着中心作用。 尽管有一个广泛的 关于bcl复合体功能的知识基础，我们对 机制是非常粗糙的，甚至是其内部的粗糙特征。 组织是有争议的问题。 最无知的领域是 导致电势的电荷分离反应的性质 发电（electrogenesis）。 除了电子转移过程之外， 这些已经部分地由许多先前的工作表征，这些包括 几乎完全不为人知的质子转移事件。 该建议侧重于bc 1周转的“生电”步骤，利用 光活化红细菌复合物的动力学优势 sphaeroides 这项研究结合了直流电势 测量与光学光谱，并选择性地使用突变体， 解决了辅因子之间的生电电子转移和 潜在的产电质子化步骤的醌反应中， bc 1复合体。 具体目标包括：（一） 产电步骤和确定贡献的大小 高电位链（QO-FeSR-c1-c2），bH-Qi电子转移，和 质子吸收到齐网站;（ii）识别的电荷携带 物种-无论是电子或质子，和（iii）表征的 导致质子吸收和质子释放的质子分解反应。 为此，设计了用于特定分离许多步骤的方案， bcl复合物的周转，用于通过电测量和 光谱方法 这些措施包括：（一）系统的准备工作- 反应中心（RC）和bcl -通过改变氧化还原 电位、pH和离子强度。 (ii)RC和bc 1的分离 反应的基础上，两个闪光实验（区分供体和 Bcl复合物的受体侧）。 (iii)反应分离， 除去电子传递组分（细胞色素c2、QB和其它 醌类）;（iv）分离 bcl复合物和RC的特异性抑制剂的不同反应。 （五） 光谱、动力学和热力学参数的解卷积。 (vi)使用 以限制或消除特定的反应。