PROTON AND ELECTRON TRANSFER AND ENERGY COUPLING

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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.