Amino acid replacements of the evolutionarily invariant tryptophan at position 64 in mutant forms of iso-1-cytochrome c from Saccharomyces cerevisiae

Tryptophan located at position 59 in vertebrate cytochromes c and at position 64 in yeast iso-1-cytochrome c is an evolutionarily invariant residue that is believed to be essential to the operation of the cytochrome c molecule. We show that this residue is replaced in at least partially functional iso-1-cytochromes c from cyc1 revertants of the yeast Saccharomyces cerevisiae. Tryptophan, tyrosine and leucine are found at position 64 in the revertants from the cyc1-84 mutant, confirming the genetic evidence (Sherman et al., 1974) that the mutant contains an UAG nonsense codon and establishing that the site of the mutation corresponds to the normal tryptophan 64. In a revertant from the cyc1.189 mutant, position 64 is occupied by a residue of phenylalanine. All three altered proteins are unstable, implying that tryptophan 64 has an essential and unique role for maintaining the normal structure of the cytochrome c molecule. In addition the iso-1-cytochrome c with leucine 64 and tyrosine 64 have greatly reduced biological activities, while iso-1-cytochrome c with the phenylalanine replacement has at least 20% of the wild-type activity or more. It remains uncertain whether the reduced specific activities are due to distorted tertiary structures or due to the specific lack of the tryptophan residue that may also have a direct functional role.