Traditionally, significant progress towards a comprehensive understanding of the mechanisms by which a protein's structure subserves its function, a prerequisite for the practical control of its physiological activities, has come from studies which assay the changes in a particular activity, resulting either from specific amino acid chemical modifications or from amino acid substitutions created by a genetic lesions. However chemical modifications of amino acid side chains are limited to a small proportion of residues, while surviving genetic modifications are random and necessarily limited to non-lethal mutations. These classical approaches are now being dramatically extended through the use of recombinant DNA techniques to obtain cytochromes c with any desired amino acid sequence. Site-directed mutagenesis of cloned eukaryotic cytochrome c genes and the production of the protein in heterologous expression systems, will be performed to study how particular amino acid substitutions affect protein structure and stability its biosynthesis, as well as electron transport and binding affinities with various physiological reaction partner, such as the mitochondrial cytochrome c oxidase cytochrome c reductase and yeast cytochrome c peroxidase. Since our present technique for the production of mutant cytochromes c requires them to be at least partially functional an important secondary objective is the development of procedures for obtaining the expression in yeast of functionless mutants. The use of mutants of the apoprotein, the biosynthetic intermediate, opens the door to the examination of several physiologically relevant processes that characterize the life cycle of the protein. These include the recognition of the apoprotein by the outer mitochondrial membrane, its transport through the membrane, the enzyme-catalyzed covalent binding of the heme prosthetic group to the apoprotein, the release of the holoprotein into the mitochondrial intermembrane space and the mechanism by which the level of cytochrome c in mitochondria is regulated. Finally, our present system in yeast produces both N-terminally acetylated and non-acetylated rat cytochrome c, both carrying a fully trimethylated lysine 72; it also yields Drosophila melanogaster cytochrome c which has that lysine in tri-, di-, mono- and unmethylated forms, which have been separated by HPLC. These proteins provide a so far unique opportunity for studying the structural and the functional effects of these well known secondary modifications of cytochrome c.
