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

Abstract

We describe the use of classical and molecular genetic techniques to investigate the folding, stability, and enzymatic requirements of iso‐l‐cytochrome c from the yeast Saccharomyces cerevisiae. Interpretation of the defects associated with an extensive series of altered forms of iso‐l‐cytochrome c was 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 c function 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.

Role of cytochrome c heme lyase in mitochondrial import and accumulation of cytochrome c in Saccharomyces cerevisiae.

“…Mutant yeast and Neurospora strains have been isolated that lack holocytochrome c in vivo because of a deficiency in heme lyase (9,28) or because they contain altered forms of cytochrome c with amino acid substitutions at the site of covalent heme attachment (17). However, the instability of the apo form of iso-1-cytochrome c, the predominant isozyme in S. cerevisiae (11), or of the only apocytochrome c in N. crassa (28) made it difficult to determine whether the defect in heme attachment also resulted in a defect in import.…”

Effects of the Tyr64 Substitution on the Stability of Cytochrome c553, a low Oxidoreduction‐Potential Cytochrome from Desulfovibrio vulgaris Hildenborough

“…During the last decade, numerous mutations have been introduced into codons for conserved amino acids in cytochrome c sequences (for reviews see Mauk, 1991 ;Hampsey et al, 1988). One of the conclusions of this work is that certain amino acids can functionally replace evolutionarily invariant residues, sometimes at activity levels indistinguishable from wild type, indicating that evolutionary invariance does not necessarily imply functional invariance.…”

Dramatic thermostabilization of yeast iso-1-cytochrome c by an asparagine----isoleucine replacement at position 57.

“…for [Ile57]Cyt-SCH3 (8.0 kcal/mol), given in Table 2, is over (15,25) reported that replacement of Gly-34 in iso-1-cytochrome c with Ser, Asn, or Asp caused instability in vitro, as inferred from lability during extractions, and caused diminished function in vivo, as measured by less growth in lactate medium. The loss of function by the replacements of Gly-34 was attributed to the inability of the protein to accommodate a side chain larger than the glycine proton, thereby altering the bond angles at position 34 (15,25).…”

“…The loss of function by the replacements of Gly-34 was attributed to the inability of the protein to accommodate a side chain larger than the glycine proton, thereby altering the bond angles at position 34 (15,25). Similarly, iso-1-cytochromes c are functional with the The forces which stabilize proteins act simultaneously on every residue, resulting in a small net stabilization.…”