A near-reversible cyclic voltammetric response of cytochrome b(5) and its mutants, E44A, E56A and E44/56A, was observed at a gold electrode modified with thioglycolic acid. The electron transfer between the negatively charged protein and negatively charged electrode b was promoted by the multivalent cations such as Mg2+ or Cr(NH3)(6)(3+) ions. When the protein solution was titrated by Mg2+ ion, the conditional reduction potential E-0’ shifted to the positive direction and reached a plateau after Mg2+ ion was more than 20mM. Under this condition, the E-0’ of wild type cytochrome b(5) and its mutants are -6 mV (vs. SHE, wild type Cyt b(5)), -7 mV (Cyt b(5) E44A), -2 mV (Cyt b(5) E56A), -3 mV (Cyt b(5) E44/56A) respectively. The conditional reduction potentials of cytochrome b(5) and its mutants obtained by spectroelectrochemical titration in the presence of 0.01 mM Ru(NH3)(6)(3+), pH 7.0, I=0.1 M phosphate buffer are +5 mV (vs. 6 SHE, wild type Cyt b(5)), +6mV (Cyt b, E44A), +6mV (Cyt b(5) E56A), +7mV (Cyt b(5) E44/56A) respectively. The results demonstrate that the mutagenesis at surface residues Glu44 and Glu56 does not alter the reduction potential of cytochrome b(5) significantly. However, the studies on binding between cytochrome b(5) and Mg2+ ion by the electrochemistry and NMR show that although the Mg2+ ion has the same interaction with these negatively charged residues and similar structural perturbation, the chelation of Mg2+ ions by the heme propionate appears a stronger influence on the heme, shifting the reduction potential of proteins.