Two membrane proteins with important and well-defined physiological functions in cellular respiration (cytochrome c oxidase from beef heart and yeast mitochondria; HCO3-/Cl- anion exchange protein from human red blood cells) are under study to relate their biological functions to their chemical structures and three-dimensional structures in the membrane. (1) Work toward elucidation of the structure of the metal sites of cytochrome c oxidase, their spatial distribution and functional role in electron transfer, dioxygen reduction and energy conservation will be continued. Particular emphasis will be devoted to the ligands of CuB, the nature of the """"""""pulsed"""""""" enzyme, the electronic structures of the two dioxygen intermediates at the three-electron level of reduction, the sequence of intramolecular electron transfer, redox interactions among the metal sites and their role in the energy conservation process, and the site of redox-linked proton translocation in the protein. A hypothesis of redox-linked proton-translocation based on the concept of electron-gating will be tested and the details extended or refined as necessary. (2) Similarly, the structure of the anion transport sites of the Cl-/HCO3- anion exchange protein is being studied by 35Cl/37Cl nuclear magnetic resonance (NMR) spectroscopy and amino acid-specific modification experiments, followed by chemical analysis by HPLC. The goals are to develop a molecular picture describing the anion translocation event, the structure of the transport site(s), their location(s) in the primary sequence of the protein and relative to the membrane, and the minimal structure containing the intact anion transport machinery. (3) To investigate the interactions of these proteins with the lipids in the membranes in which they are embedded, purified cytochrome c oxidase as well as the HCO3-/Cl- exchange protein are being reconstituted into lipid membranes of well-defined chemical compositions. The state of aggregation of the intramembraneous particles in the reconstituted membranes is being visualized by freeze-fracture electron microscopy, and the protein distributions thus derived are correlated with the motional state of the bilayer inferred from magnetic resonance experiments to provide insights on the nature of the lipid deformations induced by the membrane proteins in their immediate surroundings. These studies on lipid-mediated protein-protein forces are designed to develop an understanding of the significance of protein-lipid interactions in biological function.
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