Increasing the redox potential of isoform 1 of yeast cytochrome c through the modification of select haem interactions.
The oxidation-reduction potential of eukaryotic cytochromes c varies very little from species to species. We have introduced point mutations into isoform 1 of yeast cytochrome c (iso-1-cytochrome c) to selectively engineer a protein with a higher redox potential. Of the ten different mutant proteins generated for the present investigation Y67R, Y67K and W59H were found to be non-functional. Three other mutant proteins, L32M, L32T and T49K, were functional, but too unstable for biophysical studies. Mutant cytochromes c K79S, K79T, Y48H and Y48K were purified and characterized. The Y48K mutant is the only one that exhibits a significant increase of +117 mV in redox potential compared with the wild-type protein while still supporting oxidative phosphorylation in vivo. Low temperature difference spectroscopy confirmed the formation of the holoprotein, while adsorption and CD spectroscopy reveal perturbations in the structure of Y48K iso-1-cytochrome c.
Lett
C Marc
CM
Department of Chemistry, University of Waterloo, 200 University Avenue West, Waterloo, Ontario, Canada, N2L 3G1.
Guillemette
J Guy
JG
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001665:Binding Sites; D002942:Circular Dichroism; D003574:Cytochrome c Group / Q000737:chemistry / Q000378:metabolism*; D045304:Cytochromes c; D004563:Electrochemistry; D006418:Heme / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D011487:Protein Conformation; D020033:Protein Isoforms / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology; D029701:Saccharomyces cerevisiae Proteins; D013053:Spectrophotometry
C014059:CYC1 protein, S cerevisiae; D003574:Cytochrome c Group; D020033:Protein Isoforms; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D006418:Heme; D045304:Cytochromes c