Complexation which facilitates rejoining of horse cytochrome c apofragment [Homoser-lactone65](1-65) or [Homoser-lactone65] (23-65) to apofragment (66-104)

We have shown that two CNBr fragments of horse apocytochrome c, [Homoser-lactone⁶⁵](1–65) and (66–104), bind to the ferric heme fragment (1–25)H to form a non-productive three-fragment complex, and that when the heme of this complex has been kept reduced for 48 h at 25°, the peptide bond between residues 65 and 66 is restored with a yield of 24% or more. We have also shown that another CNBr fragment [Homoser-lactone⁶⁵](23–65), but not [Homoser-lactone⁶⁵](39–65), similarly rejoins to fragment (66–104) in the presence of the ferrous heme fragment with 25% or more yield. For complex of ferro-heme fragment [Hse-lacton⁶⁵](1–65)H and apofragment (66–104) of horse cytochrome c, which restores the peptide bond between residues 65 and 66 (located on the left side of the heme) (cf. Harbury, H.A. (1978) in Semisynthetic Peptides and Proteins (Offord, R.E. & DiBello, C., eds.), pp. 73–89, Academic Press, New York), Corradin & Harbury have suggested that axial ligation of methionine 80 to the heme (on the left side) is important. Consistent with their idea, fragment [Hse⁸⁰](66–104) was found not to link to [Hse-lactone⁶⁵](1–65) in the presence of ferro(1–25)H. Furthermore, the present studies indicate that the interaction involving residues 26 to 38 (on the right side) is also important for such a conformation which assists in the rejoining of the two apofragments. Combining these two ideas, we suggest that restoration of the peptide bond between residues 65 and 66 reflects the structural integrity of these complexes in the reduced form. Thus, the present reaction system can be used not only for chemical synthesis of [Homoser⁶⁵] apocytochrome c but also to extend amino acid substitution studies of cytochrome c to residues 1 to 64.