Effects of Surface Negatively Charged Amino Acid Mutation on Binding between Cytochrome b 5 and Cytochrome c

Abstract

The binding between different Cytochrome b 5 mutants and Cytochrome c has been studied by using of NMR method. The result shows that electrostatic effect plays an important role in maintaining the stability and specificity of the complex formed. The differences in association constants demonstrate the electrostatic contributions of Cytochrome b 5 surface negatively charged residues, which are suggested to be involved in the complex formation in the Brownian dynamics simulations and Salemme models, and to have a significant degree of additivity to the stability of Cyt c-Cyt b 5 complex, and the contribution of Glu48 is a little higher than that of Glu44. Moreover, our result suggests that the docking geometry obtained from Brownian dynamics simulations by Northrup et al, which was involved in the participation of Glu48, Glu56, Asp60 and heme propionate of Cytochrome b 5, do occur in the association between Cytochrome b 5 and Cytochrome c. The competition between the ferricytochrome b 5 mutant I (E48, E56/A, D60/A) and [Cr(oxalate) 3] 3- for ferricytochrome c shows that site III of Cytochrome c, which is a strong binding site to wild-type Cytochrome b 5, still binds to the mutant with relatively weaker strength. Our results indicate that there are other docking domains between Cyt b 5 and Cyt c different from the above mentioned one from Brownian dynamics simulations, at the same time, it also implies that electrostatic interaction on the wild-type Cyt b 5 and Cyt c interface results in flexible association complexes.