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 lesion. 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 stability, biosynthesis, as well as electron transport and binding affinities with the reductase and oxidase complexes of the mitochondrial respiratory chain. Secondary objectives are the continued improvement of th methodologies employed to obtain mutant holocytochromes c in yeast, and the development of more efficient techniques to produce large quantities of wild-type and mutant apocytochromes c in Escherichia coli. 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. Thes include the recognition of the apoprotein by the outer mitochondrial membrane, its transport through the membrane, the enzymecatalyzed 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, the nucleotide sequences of cloned eukaryotic cytochrome c genes will be used to calculate statistical phylogenetic trees that define the mutations which occurred in the different lines of evolutionary descent considered. This will allow the production of extinct evolutionary forms of the protein by expressing genes artificially changed through site-directed mutagenesis, and thus retrace the functional concomitants of past evolutionary transitions.
