The oxidation of ferrocytochrome c in nonbinding buffer.

The apparent equilibrium constant and rate of oxidation was investigated for the reaction of cytochrome c with iron hexacyanide. It was found that if horse heart ferricytochrome c was exposed to ferricyanide (to oxidize traces of reduced protein) the cytochrome subsequently, even after extensive dialysis, had an apparent equilibrium constant different from that of electrodialyzed protein. The effect of ferricyanide ion apparently cannot be removed by ordinary dialysis. The ionic strength dependence of the apparent equilibrium constant and bimolecular oxidation rate constant was measured in the range 1--200 mM using Tris--cacodylate or potassium phosphate buffers at pH 7.0, and electrodialyzed horse heart cytochrome c. The oxidation reaction proceeded very rapidly. Extrapolated to zero ionic strength, kox (approximately 9 X 10(9) M-1 S-1) was about 7% of that calculated for a diffusion-limited reaction. Since the exposed heme edge occupies only the order of 3% of the surface area, electron transfer apparently results at nearly every collision with the active-site region. An effective charge of + 7.8 units was estimated for the oxidation reaction. The rate of oxidation of Pseudomonas aeruginosa c551 was much slower (kox at mu = 0 was the order of 6 X 10(3)), and was not consistent with diffusion-limited kinetics.