Cytochrome c is the only macromolecular component of the mitochondrial respiratory chain that is not an integral protein of the inner mitochondrial membrane, but rather can readily dissociate from the outer surface of that membrane into the intermembrane space. A wealth of information is available concerning many facets of its operation, but there still exists a lack of knowledge about fundamental aspects of the mechanics of its function under normal intracellular physiological conditions, and whether these are correlated with the various states of mitochondrial respiratory chain function. Thus, the transfer of electrons between reductase and oxidase, the major but not the only function of cytochrome c, could take place with very little movement of the protein (solid state model), by two dimensional diffusion along the outer surface of the inner mitochondrial membrane, by free diffusion in the intermembrane space, or by combinations of these processes. Such a concept is provided by the dynamic aggregate model which postulates both freely diffusing and associated forms of all electron transfer components, so that the degree of mobility required for cytochrome c function will depend on the proportional equilibrium of such dissociated and associated forms. To describe its physiological function is it proposed to: (1) Employ a newly developed set of isomeric cytochrome c derivatives to determine, by covalent cross-linking, the number of electron-accepting sites on cytochrome c oxidase, cytochrome c peroxidase and other electron exchange partners, establishing the relation between the site of cytochrome c binding and its effect on the kinetics of reaction; (2) employ difference resonance Raman spectroscopy to study the interaction between cytochrome c and cytochrome oxidase as well as cytochrome c peroxidase, and determine accurate binding affinities in solution; (3) study carboxyl-modified yeast cytochrome c peroxidase and cytochrome b5 to verify the localization of their interaction domains for cytochrome c; (4) obtain cytochromes c with any desired amino acid residue exchange by recombinant DNA procedures, and employ them to examine the structures that specify the remarkable functional adaptation of yeast cytochrome c peroxidase, the functional significance of the constant angle between dipole moment and heme plane, and the functional difference between primate and non-primate cytochromes c; (5) study the biosynthesis of cytochrome c to develop procedures for introducing labelled cytochrome c into intact mitochondria.
