The specific aims of this project center on the structure study of cytochrome c oxidase by digital image processing of electron micrographs of 2-dimensional crystals. Cytochrome oxidase is the terminal component of mitochondrial electron transport which is responsible for generating energy in the form of ATP for use by all cells undergoing aerobic respiration. Cytochrome oxidase can be purified in detergent solubilized form as both a monomer and a dimer which are equally active in electron transfer. Likewise, 2-dimensional crystals of monomers and of dimers have been isolated and preliminary structural studies completed. Dr. Frey has recently obtained images of both crystal forms at a resolution of approximately 10A by cryo-electron microscopy, and the principal goal of this project is to determine the 3-dimensional structures of both monomers and dimers at this resolution. Specific sites on the enzyme will be identified by labeling with molecules visible by electron microscopy. Cytochrome c, binds with high affinity to monomer crystals, and the site of binding has been identified in 2-dimensional projection. A maleimide derivative of a water soluble undecagold cluster compound selectively labels Cys-115 of subunit III, and preliminary results show the location of this compound in 2-dimensional projections of dimer crystals. The binding sites of both molecules will be determined in 3-dimensions. The results will be interpreted to provide not only the position of both binding sites but also the orientation of cytochrome c bound to cytochrome oxidase. finally, the form of cytochrome oxidase most active in proton pumping will be determined by electron microscopy of vesicles reconstituted from purified cytochrome oxidase and phospholipids. The proportion of monomers and dimers observed in these vesicles will be compared with their efficiency of proton pumping to determine whether there is a correlation between the two properties.
