A study of the cytochrome c haemochromogen

Abstract

The amino acid sequence of that part of the horse-heart cytochrome c protein remaining attached to the heme after digestion with pepsin ("core") has been shown to be identical with that demonstrated by Tuppy and Paleus (1955) for ox-and chicken-heart cytochrome c. It was possible to separate by cation-exchange column chromatography a series of "denatured" cytochromes c having properties ranging from those characteristic of native cytochrome c to those of the pepsin-digested cytochrome c "core"; the properties studied included enzymic activity in the cytochrome oxidase and succinic oxidase systems, rate of ascorbic acid oxidation, pH range of stability of the reduced spectrum and percentage combination with CO at various pH values. The formation of an intramolecular hemochromogen in the pepsin-digested cytochrome c "core" probably occurred at alkaline pH values (pH 11-12). That this was sterically possible was shown by an atomic model, and required an unfolded peptide chain of 4 amino acids between the 2 presumed hemochromogen-forming groups. The visible spectrum of the "core", in the reduced state, approached that of native reduced cytochrome c at alkaline pH values, but did not coincide with it. The addition of a large excess of hemochromogen-forming bases, containing a- or b -amino groups, did not affect the extinction of the a-band maximum of the "core" at this alkaline pH, whereas the addition of imidazole-containing bases decreased it by 5-8%. This was interpreted as evidence that the hemochromogen-forming residues in cytochrome c are probably not both histidines, at least at pH 11. The hypothesis that the imidazole group of the histidine next to the cysteine and the c -amino group of the lysine next to the other cysteine, in the pepsin-digested cytochrome c "core", are the hemochromogen-forming groups in native cytochrome c is discussed.