PROTON AND ELECTRON TRANSFER AND ENERGY COUPLING

我们提出了一种多方面的方法来研究详细的结构-功能 产生能量的电子和质子转移过程的分析 呼吸链部位I和部位III的转导。 对于工地I，我们将：(1)继续努力确定和表征 使用两者的亚基多肽的基本氧化还原组分 哺乳动物复合体I(In)的亲水和疏水组分 与哈特菲和拉根的实验室合作)；(2)关键 比较质子泵和局域化学渗透回路模型的I位能量 耦合基于对所有相关氧化还原反应的详细分析 外加膜电位的组成(Delta X)和pH梯度 (Delta PH)；(3)检测自旋和/或氧化还原 络合物I和I的相邻本征氧化还原组分的相互作用 完整的线粒体膜制剂；(4)进行比较研究 使用细菌系统的I位能量转导机制 (脱氮副球藻和大肠杆菌)。前者含有氧化还原 与哺乳动物非常相似的组件和能量耦合装置 系统，但亚基结构更简单，而大肠杆菌似乎有 在站点I有或没有能量耦合的更简单的氧化还原组分。 第三，我们将：(1)揭开分子机制的复杂性 利用功能活性但结构上的细胞色素C氧化酶 亚基I、II或III(In)的扰动双突变体(回复突变体) 与Tzagoloff和SLonimski的实验室合作)，并确定 单个氧化还原的EPR、光学、热力学和动力学参数 细胞色素c氧化酶的中心；(2)检查自旋和/或氧化还原 电子载流子之间的相互作用，并将它们与它们的 相对于邻近氧化还原分量的空间组织和 连续波饱和脉冲法测定线粒体内膜 电子病历技术。

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.