Yeast iso-l-cytochrome c: Genetic analysis of structural requirements

We describe the use of classical and molecular genetic techniques to investigate the folding, stability, and enzymatic requirements of iso-l-cytochrome cfrom the yeast Saccharomyces cerevisiae. Interpretation of the defects associated with an extensive series of altered forms of iso-l-cytochrome cwas facilitated by the recently resolved three dimensional structure of iso-l-cytochrome c[(1987) J. Mol. Biol. 199, 295–314], and by comparison with the phylogenetic series of eukaryotic cytochromes c. Residue replacements that abolish iso-l-cytochrome cfunction appear to do so by affecting either heme attachment or protein stability; no replacements that abolish electron transfer function without affecting protein structure were uncovered. Most nonfunctional forms retained at least partial covalent attachment to the heme moiety; heme attachment was abolished only by replacements of Cys19 and Cys22, which are required for thioether linkage, and His23, a heme ligand. Replacements were uncovered that retain function at varying levels, including replacements at evolutionarily conserved positions, some of which were structurally and functionally indistinguishable from wild type iso-l-cytochrome c.