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

Nature volume 313, pages 152–154 (1985)Cite this article

Abstract

Phenylalanine 87 of yeast iso-1-cytochromc c (Phe 82 in horse heart and bonito) is phylogenetically conserved1 and occurs near the surface of the protein2. It has been suggested that this residue is directly involved in electron transfer between cytochrome c and cytochrome c peroxidase (CCP)3 and may also control the polarity of the haem environment4. 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 mutagenesis5 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 introduced6 into a yeast strain7 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 oxidase7. The purified mutant proteins are similar to wild type with respect to their visible spectra8, 20–70% as active as wild-type protein in the CCP assay9, 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.

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Authors and Affiliations

  1. Department of Biochemistry, Faculty of Medicine, University of British Columbia, Vancouver, British Columbia, Canada, V6T 1W5

    Gary J. Pielak, A. Grant Mauk & Michael Smith

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Pielak, G., Mauk, A. & Smith, M. Site-directed mutagenesis of cytochrome c shows that an invariant Phe is not essential for function. Nature 313, 152–154 (1985). https://doi.org/10.1038/313152a0

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