The effect of complex-formation with polyanions on the redox properties of cytochrome c

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Abstract
The stable complex formed between mammalian [horse heart] cytochrome c and phosvitin at low ionic strength was studied by partition in an aqueous 2-phase system. Oxidized cytochrome c binds to phosvitin with a higher affinity than reduced cytochrome c. The difference was equivalent to a decrease of the redox potential by 22 mV on binding. Complex formation with phosvitin strongly inhibited the reaction of cytochrome c with reagents that react as negatively charged species, e.g., ascorbate, dithionite, ferricyanide and tetrachlorobenzoquinol. Reaction with uncharged reagents such as NNN''N''-tetramethylphenylenediamine and the reduced form of the N-methylphenazonium ion (present as the methylsulfate) was little affected by complex formation, whereas oxidation of the reduced cytochrome by the positively charged tris(phenanthroline)cobalt(III) ion was greatly stimulated. A similar pattern of inhibition and stimulation of reaction rates was observed when phosvitin was replaced by other macromolecular polyanions such as dextran sulfate and heparin, indicating that the results were a general property of complex formation with polyanions. A weaker but qualitatively similar effect was observed on addition of inositol hexaphosphate and ATP. The effects of complex formation with polyanions on the reactivity of cytochrome c with redox reagents may be mainly the result of replacing the positive charge on the free cytochrome by a net negative charge. Any steric effects on polyanion binding are small in comparison with such electrostatic effects.