Interaction between cytochrome c and cytochrome b5.

The reduction of cytochrome c by cytochrome b5 was studied over a wide range of ionic strengths in four different buffer systems. The reaction rate decreased linearly as the I1/2 was increased, suggesting that electrostatic interactions are important in the interaction. The ionic strength dependence of the reaction rate was in quantitative agreement with the theory of Wherland & Gray [Wherland, S., & Gray, H.B. (1976) Proc. Natl. Acad. Sci U.S.A. 73, 2950] only if the effective radius of the interaction was 2 A. This indicates that the interaction between the two proteins is best described as the sum of n complementary charge interactions, each involving a specific lysine on cytochrome c and a specific carboxyl group on cytochrome b5. The number of complementary charge interactions, n, was calculated to be five to seven, in agreement with the results of our specific modification studies. Ultracentrifugation and gel permeation techniques were used to demonstrate that cytochrome b5 and cytochrome c formed a stable complex at low ionic strength.