LOOP REPLACEMENT AND RANDOM MUTAGENESIS OF OMEGA-LOOP-D, RESIDUES-70-84, IN ISO-1-CYTOCHROME-C

To study the role of omega loop D, residues 70-84, in the structure an d function of yeast iso-l-cytochrome c, this loop was replaced with ho mologous and heterologous loops, A novel method was developed for rapi d insertion of these mutations into the yeast chromosome at the CYC1 l ocus. The strains containing these loop replacement cytochromes cannot grow on nonfermentable carbon sources, indicating that the proteins a re nonfunctional. Whole cell difference spectroscopy shows that no hol ocytochrome c is present; however, apoprotein is found by immunoblot a nalysis. Thus, apoprotein is present in these mutant strains, but it c annot bind heme and cannot compete with wild type apoprotein conversio n to holoprotein. This is a unique example of a set of loop replacemen ts that do not produce folded protein, and these results suggest that the loop D amino acid sequence in iso-1-cytochrome c plays a significa nt role in cytochrome c biosynthesis in vivo. To identify the signific ant amino acids in loop D, random mutagenesis of six highly conserved loop residues, Tyr-74, Ile-75, Pro-76, Gly-77, Thr-78, and Lys-79, was accomplished, Sequencing of the random mutants shows that strict cons ervation of none of these residues is required to produce a minimally functional cytochrome c, Preferences are found for small, hydrophilic or aromatic residues at position 74, hydrophobic residues at position 75, glycine and arginine at positions 76 and 77, and beta-branched ami no acids at position 78. Implications for the role of loop D in the st ructure and function of iso-1-cytochrome c are discussed.