Circular dichroism studies: I. Cytochrome c

Abstract

The circular dichroism spectra of the ferri and ferro forms of cytochrome c obtained from horse, beef, chicken, turtle, and Pseudomonas aeruginosa have been measured at pH 7.0 over the wavelength region from 690 to 220 mμ. For purposes of analysis the spectra were arbitrarily divided into five regions: region I, 690-580 mμ; region II, 580-450 mμ (region related to heme coordination); region III, 450-350 mμ (Soret region); region IV, 350-240 mμ (region of protein-heme interactions and Cotton effects due to the aromatic side-chain residues); and region V, below 240 mμ (region of peptide bond absorption). The spectra are complex, exhibiting numerous Cotton effects. The molar ellipticities of all five cytochromes are small and tend to increase in magnitude at shorter wavelengths. The circular dichroism spectra of the mammalian-type cytochrome c of horse, beef, chicken, and turtle are grossly similar to each other. The similarities and differences between the spectra of the mammalian-type cytochromes c were utilized to locate the positions of some of the optically active transitions in regions II, III, and IV. The positions of the two Cotton effects at 280–290 mμ associated with the aromatic side-chain residues of the protein appear to be unaffected by change in oxidation. The spectra of all five ferrocytochromes c are alike in regions II and III. The spectra of the ferri forms of the mammalian-type and Pseudomonas cytochrome c in the Soret region appear to be mirror images of each other. This effect must reflect a difference in the environment of the heme group in the ferricytochromes c of Pseudomonas and mammalian type. Since the effect does not occur in the spectra of the ferrocytochromes c examined, it is likely that the environment of the heme group in the reduced form of cytochrome c remains invariant. An analysis of the relationship of the circular dichroism of all five ferri- and ferrocytochromes c in regions II and III to the structure of the central coordination complex of mammalian-type cytochrome c is presented.