The Regulation of Surface Charged Residues on the Properties of Cytochrome b5

To understand the roles of negatively surface charged residues, the cytochrome b₅ (Cyt b₅) E48A/D60A mutant was constructed. UV-visible and CD spectra confirmed that the mutation did not cause overall structural changes of the protein. The mutant presents an unexpected high stability toward the thermal and denaturant compared with the wild type Cyt b₅, which shows that these surface charged residues can influence the interactions between the heme b group and the polypeptide chain. Functional properties were clarified through the electron transfer reactions between Cyt b₅ and Cyt c. The driving force of the electron transfer reactions is conservative. Although the association constant of Cyt b₅ E48A/D60A with Cyt c is much lower than that of the wild type Cyt b₅, their electron transfer rate constants do not differ significantly. The results show that these surface charged residues play important roles in regulating both the stability and functional properties of Cyt b₅.