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 metabolis m, and substantial amounts of protein could be extracted from permeabi lized cells. The purified analogs, Cysl4Ala, Gly37Arg, Arg38Lys, Arg38 Gly, Gly84Ser, and Thr28Ile,Gly84Ser, were examined for changes in fun ctional properties, since the majority of these residues are strongly or absolutely conserved. Indeed, although growth rates of the host yea st strains were very similar, there was great divergence in both physi cochemical and biological properties, which have been rationalized in terms of changes to the stability of the cytochrome fold, and to the d ipole moment of the protein. Interestingly, although modification of e lectrostatic properties in some mutants can apparently produce a twofo ld increase in electron transfer efficiency, such changes are not evol utionarily acceptable. The ''improvement'' is illusory. We suggest tha t an associated decrease in the stability of the heme crevice offsets any advantage of increased transfer rates.