PROTON AND ELECTRON TRANSFER AND ENERGY COUPLING

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The ultimate goal of the project is to elucidate the structure- function relationship of the Site I and Site III energy coupling. We have three major long term objectives, namely, (A) To test the hypothesis that the mechanism of electron transfer in the Site I is of a cyclic type. This hypothesis enables us to explain (a) the extreme complexity of redox components and protein subunit structure, (b) a high (four to five) stoichiometry of H+/2e- (c) the extremely low midpotential of the cluster N-la. For this goal, we will do the following: (i) study the physico- chemical properties of the H+-carriers, (ii) investigate new inhibitors which are different from conventional rotenone-type compounds, (iii) measure the spatial organization of the redox components in the membrane using continuous wave and pulsed EPR techniques at cryogenic temperature, and (iv) study simpler bacterial systems, such as Paracoccus denitrificans and Thermus thermophilus HB-8. (B) To apply our experience in the study of Site I to clinical problems. Certain diseases are caused by a genetic defect in the iron sulfur proteins in the Site I. We have demonstrated that this defect can be clearly identified by EPR measurement on pathological specimens. We will continue this line of study. (C) To elucidate the structure function relationship of Site III respiration. Since we pioneered the measurement of spin-spin interaction between cyt.a3-NO and other paramagnetic species in bovine heart cytochrome oxidase, we will complete the study by conducting the following experiments: (i) definitive measurement of inter-cluster distance in bovine heart cytochrome oxidase, (ii) direct measurement of T1 and T2 by pulsed EPR with careful resolution of redox poise of cyt.a and CuA, (iii) use of yeast strain whose cyt. c oxidase protein and chromophore structure are perturbed by double mutation. Taking full advantage of studying microorganism, we will study spectral and thermodynamic characteristics of individual redox components and analysis of the redox and spin-spin interactions among redox centers. These studies are expected to open a new horizon in our understanding of the electron and proton transfer process in Site I and III on the molecular level.