THE DIRECT ELECTROCHEMISTRY OF CYTOCHROME B(5) AND ITS MUTANT PROTEINS

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 neg atively charged protein and negatively charged electrode b was promote d by the multivalent cations such as Mg2+ or Cr(NH3)(6)(3+) ions. When the protein solution was titrated by Mg2+ ion, the conditional reduct ion potential E-0' shifted to the positive direction and reached a pla teau 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 o f cytochrome b(5) and its mutants obtained by spectroelectrochemical t itration in the presence of 0.01 mM Ru(NH3)(6)(3+), pH 7.0, I=0.1 M ph osphate 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. Th e results demonstrate that the mutagenesis at surface residues Glu44 a nd Glu56 does not alter the reduction potential of cytochrome b(5) sig nificantly. However, the studies on binding between cytochrome b(5) an d Mg2+ ion by the electrochemistry and NMR show that although the Mg2 ion has the same interaction with these negatively charged residues a nd 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. (C) 1997 Elsevier Science S.A.