Absorption spectra and some other properties of cytochrome c and of its compounds with ligands

Abstract

A brief account is given of the development of our knowledge of certain aspects of cytochrome c since its early preparations from baker’s yeast, on which the fundamental properties of this haemoprotein were established. In view of the extensive work which is now being carried out on cytochromes of group c isolated from different organisms, it was found impor­tant to re-investigate certain properties of cytochrome c which can easily be prepared from mammalian heart muscle and purified to 0.45 % Fe. The absorption spectra of oxidized (Fe³⁺) and reduced (Fe²⁺) cytochrome c (at pH 6.8, 13 and 14) and of their compounds with ligands such as CO (Fe²⁺), CN' (Fe²⁺ and Fe³⁺), N'₃ (Fe³⁺), NO (Fe²⁺ and Fe³⁺) and F'(Fe³⁺) were re-determined and uniformly presented. Marked intensification of the Soret- and α-bands of ferro-cytochrome c was observed at pH14. Although CN'- and N'₃-ferri-cytochrome c are formed within the physiological range of pH, they play no part in cyanide or azide inhibition of cell respiration or of the catalytic activity of the respiratory chain in the heart muscle preparation. Nitric oxide reacts with both ferri- and ferro-cytochrome c forming two spectroscopically distinct compounds: I and II analogous to compounds of NO with methaemoglobin and haemoglobin respectively. The nature of these compounds is discussed in relation to the view recently put forward that compound I, which was found to be diamagnetic, has a ferro-cytochrome c nitrosyl structure. The inability of ferro-cytochrome c to react with O₂ and ligands (CO, CN ' and NO) within the physiological range of pH, is due not only to the embedded position of the haem within the fold of the protein, to which it is linked both by its iron and porphyrin side chains (2 and 4), but also to the strong affinity of the divalent iron for the nitrogenous groups of the protein, as well as to the nature of the protein fold which exerts greater steric hindrance to ligands in ferro- than in ferri-cytochrome c. This is in agreement with the view that the tertiary protein structure is more open in ferri- than in ferro-cytochrome c. Criteria of the purity of cytochrome c were re-examined in the light of recent work on its purification. The thermostability of cytochrome crecognized during the early study of this compound was further investigated. It was found that ferro-cytochrome cin solutions at pH 9.1 to 10.2, containing carbon monoxide or cyanide, when heated to 100 °C becomes completely con­verted into the corresponding CO- or CN'-derivative, which, on cooling, immediately dissociate; up to 80% of heat-denatured cytochrome c was found to revert to the original native state. The striking similarities of yeast and mammalian cytochrome c in all their properties, including their equally successful incorporation within the cytochrome cdeficient heart muscle succinic oxidase system, contrasts with the marked dissimilarities in the structures of their haemopeptide cores, which were found to differ in the contents and sequences of five of their eleven amino acids. Similar discrepancies between the primary protein structures and the biological properties have recently been recorded for the α and β peptide chains of human haemoglobin and the single chain of sperm whale myoglobin. The effect of the valency of iron on the general properties of protein of cytochrome cand of other haemoproteins is discussed.