Redox properties of cytochrome b5: a mutagenesis and DPV study of the pH and ionic strength dependence of redox potentials and interactions with myoglobin by DPV

To understand the determinants of redox potentials in cytochrome b5 (cyt b5), wild type cyt b5 and its E44A, E56A, E44/56A and F35Y mutants have been characterized. The pH and ionic strength dependence of the redox potentials of cyt b5 and its mutants have been investigated by differential pulse voltammetry (DPV). Variation of pH demonstrated that there are ionizable functional groups that are linked to the redox potential of cyt b5. The Glu44 and Glu56 mutants produced the highest pK values. The F35Y mutant had the lowest pK. These results imply that there are different rearrangements of the charge density distributed in the hydrogen bond networks (involving Glu44 and Glu56) as well as in the aromatic network (involving Phe35). Analysis of the diffusion coefficients Do and the heterogeneous electron transfer rate constants ko′, demonstrate that the acidic residues Glu44 and Glu56 participate in interaction not only with their partner proteins, but also with the surface of the electrode. The residue Phe35 plays an important role in regulating the redox potentials of cyt b5. The results of both the direct electrochemistry and the protein–protein pH titration imply that the cyt b5∣myoglobin interface has both electrostatic and hydrophobic interactions.