Reconstitution of horse heart cytochrome c: interaction of the components obtained upon cleavage of the peptide bond following methionine residue 65.
Horse heart cytochrome c can be divided into a heme peptide of 65 residues (H65CNBr) and a nonheme peptide of 39 residues (N39CNBr) by treatment of the molecule with cyanogen bromide. Upon mixture of the two peptides in aqueous solution, a 1: 1 complex with properties closely resembling those of the parent heme protein can be formed. The reaction can conveniently be effected in sodium acetate buffer of pH 4.7, with the H65CNBr in the reduced form. The heme peptide is predominantly in the high-spin state under these conditions, and, upon the addition of N39CNBr, is converted to a complex with absorption and circular dichroism spectra which correspond closely to those of ferrocytochrome c. If N39CNBr is added to the oxidized form of H65CNBr, the spectral properties of the product differ appreciably from those of the parent protein. A complex with absorption and circular dichroism spectra comparable to those of ferricytochrome c can readily be obtained, however, through oxidation of the product of the reaction of N39CNBr with H65CNBr in the reduced state. The complex formed in this manner has an absorption band at 695 nm, exhibits no tryptophan or tyrosine fluorescence, and is active in the succinate oxidase system.
D000818:Animals; D002942:Circular Dichroism; D003488:Cyanogen Bromide; D003580:Cytochromes / Q000032:analysis* / Q000494:pharmacology; D005453:Fluorescence; D006418:Heme / Q000032:analysis; D006736:Horses; D008099:Liver / Q000187:drug effects / Q000201:enzymology; D008715:Methionine; D009206:Myocardium / Q000032:analysis*; D010084:Oxidation-Reduction; D010101:Oxygen Consumption; D010455:Peptides / Q000032:analysis / Q000494:pharmacology; D051381:Rats; D013057:Spectrum Analysis; D013385:Succinate Dehydrogenase / Q000378:metabolism; D014466:Ultraviolet Rays
D003580:Cytochromes; D010455:Peptides; D006418:Heme; D008715:Methionine; D013385:Succinate Dehydrogenase; D003488:Cyanogen Bromide