Functional correlation in amino acid residue mutations of yeast iso-2-cytochrome c that is consistent with the prediction of the concomitantly variable codon theory in cytochrome c evolution

Fitch and Markowitz' theory of concomitantly variable codons (covarions) in evolution predicted the existence of functional correlation in amino acid residue mutations among present-day cytochromes c. Mutational analysis was carried out on yeast iso-2-cytochrome c, where hydrophobic core residues 12 0, M64, L85, and M98 and surface residue L9 were mutated in selected combin ations, to those found in mammalian and bird cytochromes c, The functionali ty assay is based upon the ability of yeast cells to grow in YPGE medium. F urthermore, experiments on the single M64L and M98L mutations as well as th e double M64L/M98L mutation using NMR showed that the effects of these muta tions are to perturb the structural integrity of the protein, We identified functional correlation in two cases of a pair of residue mutations, the 12 0 --> V and M98 --> L pair and the L9 --> I and L85 --> I pair. In both cas es, only one of the two alternative, putative evolutionary pathways leads t o a functional protein and the corresponding pairs of residue mutations are among those found in present-day cytochromes c. Since valine is predicted to be at position 20 in the ancestral form of cytochrome c, the present dat a provide an explanation for the ancient requirement of leucine rather than methionine in position 98. The present data provide further evidence for t he role of those specific atom-atom interactions in directing a pathway in the evolutionary changes of the amino acid sequence that have taken place i n cytochrome c, in accordance with Fitch and Markowitz.