The effect of pH and ionic strength on the pre-steady-state reaction of cytochrome c and cytochrome aa3.

(1) In the pH range between 5.0 and 8.0, the rate constants for the reaction of ferrocytochrome c with both the high- and low-affinity sites on the cytochrome aa3 increased by a factor of approx. 2 per pH unit. (2) The pre-steady-state reaction between ferrocytochrome c and cytochrome aa3 did nt cause a change in the pH of an unbuffered medium. Furthermore, it was found that this reaction and the steady-state reaction are equally fast in H2O and 2H2O. From these results it was concluded that no protons are directly involved in a rate-determining reaction step. (3) Arrhenius plots show that the reaction between ferrocytochrome c and cytochrome aa3 requires a higher enthalpy of activation at temperatures below 20 degrees C (15--16 kcal/mol) as compared to that at higher temperature (9 kcal/mol). We found no effect of ionic strength on the activation enthalpy of the pre-steady-state reaction, nor on that of the steady-state reaction. This suggests that ionic strength does not change the character of these reactions, but merely affects the electrostatic interaction between both cytochromes.