The mitochondrial cytochromes c of the present species are presumed to collectively reflect evolutionary events occurring for the last 1.5 billion years. These evolutionary secrets, if uncovered through finding the roles of the invariant residues, may help understanding the principles underlying structure-function of proteins. In this context, it would be worthwhile to chemically synthesize the ancestral form of cytochrome c predicted by W.M. Fitch and E. Martgoliash and test whether it is active. The feasibility of this project is supported by our finding that the complementing fragments are completely exchangeable between horse and Candida krusei cytochromes c for formation of hybrid complexes (see the other report). As a first step, we plan to synthesize horse cytochrome c via synthesis of apocytochrome c, using the Merrifield solid phase method and joining two apofragments (complexation assisted rejoining, see the previous report), followed enzymatic attachment of heme, using cytochrome c synthase found in this Section. In extending the joining procedure we have shown that apofragment (Hse-lactone-65)(23-65) (homoserine-lactone), but not (Hse-lactone-65)(39-65), of horse cytochrome c rejoins to apofragment (66-104) in the presence of reduced heme fragment (1-25)H and that intact methionine 80 is essential for this rejoining reaction. On the basis of these results, to make semi-synthetic (Hse-65)(28-104), we carried out synthesis of fragment (Gly-66)(28-66) of horse cytochrome c, containing tryptophan at position 59. In the effort to develop a second procedure for joining of fragments, we found a novel carboxypeptidase A catalyzed transpeptidation as follows. Synthesized radiolabeled tripeptide Leu-Met-His-amide or Leu-Arg-Met-amide and excess of heme fragment (Hse-69)(1-69)H of yeast cytochrome c or (Hse-65)(1-65)H of horse cytochrome c were incubated with carboxypeptidase A in the presence of 50 to 70% glycerol at pH 10.0 at 20 to 30 degrees C. Analyses of the product indicated that both labeled tripeptides replaced the carboxyterminal homoserine with the efficiency up to 50% after 7 days.
