Multiple molecular forms of bovine heart cytochrome c. V. A comparative study of their physicochemical properties and their reactions in biological systems.

The differences in the physicochemical and electron transfer properties of the multiple forms of bovine heart cytochrome c, Cy I to Cy IV, provide a means of correlating substitutions of aspartyl or glutamyl residues for asparaginyl or glutaminyl residues in the polypeptide chain with the conformation of this heme protein, as well as a new aspect in the study of the relation between its structure and function. The differences in light absorption spectra (at 25” and -190’) and optical rotatory dispersion, as well as in circular dichroic absorption spectra, of the multiple forms indicate that they differ in conformation, and that Cy I represents the native form. This, coupled with the fact that the oxidationreduction potentials of the various forms are ditIerent, indicates that the environment of the heme group, i.e. the hemeprotein interaction, has been affected in Cy II and Cy III. Evidence is presented in support of a difference in conformation in the ferrous as well as in the ferric form, although the difIerence is clearer in the latter form. The different forms are slowly oxidized by molecular oxygen (278 pM) at neutral pH; the fist order rate constant increases in the order Cy I < Cy II < Cy III, and numerically by about 3 times (from 3.0 X lop5 to 8.3 x 10M5 se@). The different forms are reduced by ascorbate at pH 7.0 in a second order reaction. The rate constants decrease in the order Cy I > Cy II > Cy III and numerically by about 3 times (from 7.9 to 2.8 M-~ se@). The different forms are all able to restore succinate oxidation in mitochondria depleted of endogeneous cytochrome c. However, they are not isokinetic. Athough the V’,,,, values are essentially the same, the K’, values for cytochrome c increase in the order Cy I < Cy II < Cy III and numerically by about 5.5 times (from 2.9 to 15.9 PM). The results put into perspective earlier studies on various chemically and enzymically modified forms of cytochrome c, and support the view that certain asparagine and glutamine residues are important to achieve the conformation and the