PROTON AND ELECTRON TRANSFER AND ENERGY COUPLING

我们提出了一个多方面的方法来详细的结构-功能 分析电子和质子转移过程，导致能量 在呼吸链的位点I和位点III区段中的转导。 对于研究中心I，我们将：（1）加大努力， 使用分解的亚单位多肽的基本氧化还原组分 哺乳动物复合物I的亲水和疏水部分（在 与Hatefi和Ragan的实验室合作）;（2）严格地 比较质子泵和局部化学渗透环路模型的站点I能量 耦合的基础上的所有相关的氧化还原反应的详细分析 施加的膜电位（Δ X）和pH梯度 （3）检查自旋-自旋和/或氧化还原 络合物I的相邻固有氧化还原组分的相互作用和 完整的线粒体膜制备;（4）进行比较研究 使用细菌系统的位点I能量转导机制 （副球菌和大肠杆菌）。 前者含有氧化还原 组件和能量耦合装置非常类似于哺乳动物 系统，但亚基结构较简单，而E.大肠杆菌似乎 更简单的氧化还原组分，在位点I处具有或不具有能量耦合。 为 第三阶段，我们将：（1）解开分子机制的复杂性 细胞色素c氧化酶的功能活性，但结构 亚基I、II或III的扰动双突变体（回复突变体）（在 与Tzagoloff和Slonimski的实验室合作），并确定 EPR，光学，热力学和动力学参数的个别氧化还原 细胞色素c氧化酶的中心;（2）检查自旋-自旋和/或氧化还原 电子载体之间的相互作用，并将它们与它们的 空间组织相对于相邻的氧化还原组分和 线粒体内膜连续波饱和脉冲法 EPR技术。

We propose a multifaceted approach to the detailed structure-function analysis of electron and proton transfer processes which lead to energy transduction in the Site I and Site III segments of the respiratory chain. For Site I, we will: (1) Extend our efforts to identify and characterize essential redox components using resolved subunit polypeptides of both hydrophilic and hydrophobic fractions of mammalian Complex I (in collaboration with Hatefi's and Ragan's laboratories); (2) Critically compare proton pump and local chemiosmotic loop models for Site I energy coupling based on a detailed analysis of the response of all relevant redox components to the applied membrane potential (Delta X) and pH gradient (Delta pH) under various conditions; (3) Examine spin-spin and/or redox interactions of neighboring intrinsic redox components of Complex I and intact mitochondrial membrane preparations; (4) Conduct comparative studies of the Site I energy transduction mechanism using bacterial systems (Paracoccus denitrificans and Escherichia coli). The former contains redox components and energy coupling devices very similar to the mammalian system, but simpler in subunit structure, while E. coli seems to have simpler redox components with or without energy coupling at Site I. For Site III, we will: (1) Unravel the complexity of the molecular mechanism of cytochrome c oxidase utilizing functionally active but structurally perturbed double mutants (revertants) of subunit I, II, or III (in collaboration with Tzagoloff's and Slonimski's laboratories), and determine EPR, optical, thermodynamic, and kinetic parameters of individual redox centers of cytochrome c oxidase; (2) Examine spin-spin and/or redox interactions among the electron carriers and correlate them with their spatial organization relative to the neighboring redox components and to the inner mitochondrial membrane using continuous wave saturation and pulse EPR techniques.