Interaction between adrenodoxin and cytochrome c.

The reduction of horse heart ferricytochrome c by reduced adrenodoxin was found by stopped flow spectroscopy to follow second order kinetics with a rate constant of 7.8 X 10(6) M-1 s-1 in Tris/Cl, pH 7.5, at an ionic strength of 0.2 M, 29 degrees C. The temperature dependence of the rate constant was used to determine that the activation parameters were delta H++++ = 7.7 kcal/mol and delta S = -1.5 cal/mol degrees K. The rate constant decreased rapidly with increasing ionic strength, indicating that electrostatic interactions between the two proteins were important to the reaction. The contribution of individual lysine amino groups to the electrostatic interaction was determined by measuring the reaction rate of specifically trifluoroacetylated or trifluoromethylphenylcarbamylated cytochrome c derivatives. Modification of lysines 13, 27, 72, and 79 surrounding the heme crevice decreased the reaction rate by about 2-fold, while modification of lysine amino groups in other regions of cytochrome c had decreasing effects as the distance from the heme crevice was increased. The interaction domain therefore involves specific complementary charge interactions between lysine amino groups immediately surrounding the heme crevice of cytochrome c and carboxylate groups on adrenodoxin. A semi-empirical relationship was developed for the total electrostatic interaction between the two proteins which is in quantitative agreement with both the ionic strength dependence of the reaction rate and the specific lysine modification studies.