Analysis of the invariant Phe82 residue of yeast iso-1-cytochrome c by site-directed mutagenesis using a phagemid yeast shuttle vector

A phagemid (pING4) carrying the yeast iso-1-cytochrome c genewas constructed which bears all the elements necessary for replicationin yeast and bacteria and may be converted into a single-strandedform of DNA for site-directed mutagenesis and nucleotide sequencing.The recombinant vector was used to create a complete set of19 amino acid changes at position 82, a phylogenetically conservedphenylalanine residue in mitochondrial cytochrome c. All thedifferent forms of cytochrome c were functional in vivo, basedupon their ability to support respiration when the mutant proteinswere expressed in a yeast strain (otherwise devoid of cytochromec) grown on non-fermentable carbon sources, with only the straincontaining the Cys82 variant having a substantially decreasedgrowth rate. These results are interpreted in terms of the availablestructural and functional information previously reported ona subset of cytochrome c proteins with mutations at position82.