The succinic oxidase segment of heart mitochondria involves succinate dehydrogenase, Complex III (which is an aggregate of eight different polypeptides including cytochromes b and c1 and a non heme iron protein), cytochrome c and cytochrome c oxidase (a complex of seven different polypeptides, with hemes a and a3 and two copper atoms). We have purified and characterized the component polypeptides of both complex III and cytochrome c oxidase and our recent studies have been directed at understanding the arrangement of the various polypeptides and their associated electron acceptor groups in each complex. In particular we have been conducting cross-linking studies with cleavable bifunctional reagents in order to determine the proximity of components one to another in each complex. Our main objective now is to determine the orientation of both Complex III and cytochrome c oxidase in the mitochondrial inner membrane. Labeling studies with the membrane impermeable protein modifying agent (35S) diazobenzenesulfonate are planned. Intact mitochondria and inverted inner membrane preparations will be labeled, the membrane will be solubilized with detergents and Complex III and cytochrome c oxidase will be isolated by immunoprecipitating with antibodies specific to each. The labeling of different components from the intracristal and matrix sides of the membrane will be examined after separating polypeptides by SDS polyacrylamide gel electrophoresis. A combination of cross-linking studies and the labeling experiments should provide a picture of the arrangement of polypeptides in both electron transfer complexes. The studies on cytochrome c oxidase will be complimented by ongoing electron diffraction studies of this complex in reconstituted membranes. Cross-linking experiments will be performed to determine the site of binding of cytochrome c to both cytochrome c oxidase and Complex III. Cross-linking and immunological studies are planned to examine the association of succinate dehydrogenase with the inner membrane. All the studies described are part of our long term project to understand the structure of succinic oxidase. This structural information will we believe help ellucidate the mechanism of electron transfer and its coupling to ATP synthesis.