Site-directed mutagenesis of cytochrome c shows that an invariant Phe is not essential for function

Abstract

Phenylalanine 87 of yeast iso-1-cytochrome c (Phe 82 in horse heart and bonito) is phylogenetically conserved and occurs near the surface of the protein. It has been suggested that this residue is directly involved in electron transfer between cytochrome c and cytochrome c peroxidase (CCP) and may also control the polarity of the haem environment. Because Phe residues are not susceptible to chemical modification, no direct means of studying the functional role of Phe 87 has been available, so we have chosen Phe 87 as our initial target here to test the feasibility of using site-directed mutagenesis as a means of studying structure-function relationships in cytochrome c. We have changed the codon for Phe 87 to that of either a Ser, a Tyr or a Gly. The mutated genes have been introduced into a yeast strain lacking both isozymes of cytochrome c. Unlike the recipient strain, transformants grow on a non-fermentable carbon source, indicating that the mutant proteins can reduce cytochrome oxidase. The purified mutant proteins are similar to wild type with respect to their visible spectra, 20-70% as active as wild-type protein in the CCP assay, and their reduction potentials are lowered by as much as 50 mV. Thus Phe 87 is not essential for cytochrome c to transfer electrons but is involved in determining the reduction potential.

The effects of multiple amino acid substitutions on the polypeptide backbone of tuna and horse cytochromes c

“…In such cases NMR assignments must rely on the comparison of entire spin systems rather than individual resonances [15] or on the use of sequential assignment procedures [16,171. Changes in a semi-surface residue, Phe 82, produced by single-site-directed mutagenesis, have also been shown to produce small structural changes at sites well-removed from the site ofmodification [18,19]. These studies have also shown how sensitive the redox potential may be to substitution.…”

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

“…Furthermore, recent site-directed mutagenesis experiments of the CYCZ gene indicate that additional highly conserved residues can be functionally replaced. Serine, tyrosine, and glycine were shown to functionally replace the invariant Phe87 in vitro [22], and arginine can functionally replace the conserved Lys77 residue both in vitro and in vivo [23]. The Arg77 replacement is especially interesting since Lys77 is trimethylated in fungal and plant species and has been suggested to be involved in both mitochondrial import and association of iso-1-cytochrome c with the cytochrome c oxidase complex.…”

Two conserved tryptophan residues of tumor necrosis factor and lymphotoxin are not involved in the biological activity

“…Pielak reported that the phylogenetically conserved phenylalanine 87 in yeast iso-1-cytochrome c is not directly involved in electron transfer, but performs a structural function by controlling the polarity of the heme environment [16]. Also, cyglobin, mutated at the strongly conserved positions 127 and 141, seems to be functionally active, and persons carrying these mutations are asymptomatic [17,18].…”