Spectral and electrochemical studies of cytochrome c peptide complexes.

Hemepeptides 14-21, 11-21, 11-26, l-38, 1-65, and I-80 and non-hemepeptides 66-104,67-104 (guanidinated), and l104 (apoprotein) have been purified from horse heart cytochrome c. Addition of non-hemepeptide l-104 to any of the ferrohemepeptides or addition of non-hemepeptides 66-104 or 67-104 to ferrohemepeptides l-6.5 or l-80 produces changes in the visible absorption spectrum of the ferrohemepeptides, indicating ligation of the sulfur of methionine 80 in the non-hemepeptide with the heme iron of the hemepeptide. The absorption spectra, circular dichroic spectra, formal potential, and electron transfer capability of these peptide complexes have been measured. Noncovalent peptide complexes l-38:1-104, l-65:1-104, and l-80:1-104 and the covalent complex l-65-66-104 exhibit the structural parameters and biological function characteristic of the native protein. These results suggest that the native tertiary structure can be generated from overlapping peptides. By contrast, noncovalent complexes 14-21:1-104, ll-21:1-104, ll-26:1-104, l-65:67-104, and l-80:66-104, while containing substantial secondary structure as judged by optical measurements, appear to have an exposed heme moiety in both oxidation states, resulting in a low formal potential and no biological function. Combination of ferrohemepeptide l-65, having a homoserine lactone at position 65, with peptide 66-104, but not with peptide 67-104, produces a peptide linkage between the COOH terminus of the hemepeptide and the NH, terminus of the non-hemepeptide. This comparison suggests that the peptides are precisely oriented in a noncovalent complex such that a peptide bond cannot be formed across the gap resulting from the absence of residue 66. Comparison of the properties of the noncovalent complex l-65:66-104 with the covalent complex l-65-66-104 indicates that, while both complexes in the ferro form have a native tertiary structure,