Improving cytochrome c function by protein engineering?: studies of site-directed mutants of the human protein.

We have expressed the gene for human cytochrome c, and six mutants of the native sequence, in yeast defective in its own iso-1-cytochrome c gene. All constructs support strong growth in strict aerobic metabolism, and substantial amounts of protein could be extracted from permeabilized cells. The purified analogs, Cys14Ala, Gly37Arg, Arg38Lys, Arg38Gly, Gly84Ser, and Thr28Ile,Gly84Ser, were examined for changes in functional properties, since the majority of these residues are strongly or absolutely conserved. Indeed, although growth rates of the host yeast strains were very similar, there was great divergence in both physicochemical and biological properties, which have been rationalized in terms of changes to the stability of the cytochrome fold, and to the dipole moment of the protein. Interestingly, although modification of electrostatic properties in some mutants can apparently produce a twofold increase in electron transfer efficiency, such changes are not evolutionarily acceptable. The "improvement" is illusory. We suggest that an associated decrease in the stability of the heme crevice offsets any advantage of increased transfer rates.