Mechanism of oxidation of oxymyoglobin by copper ions: comparison of sperm whale, horse, and pig myoglobins

The influence of Cu²⁺ concentration, pH, and ionic strength of the solution as well as redox-inactive zinc ions on the rate of oxidation of sperm whale, horse, and pig oxymyoglobins (oxy-Mb) by copper ions has been studied. These myoglobins have homologous spatial structures and equal redox potentials but differ in the number of histidines located on the surface of the proteins. It was shown that oxy-Mb can be oxidized in the presence of Cu²⁺ through two distinct pathways depending on which histidine binds the reagent and how stable the complex is. A slow pH-dependent catalytic process is observed in the presence of equimolar Cu²⁺ concentration for sperm whale and horse oxymyoglobins. The curves of pH dependence in both cases are sigmoid with pK _eff corresponding to the ionization. The process is caused by the strong binding of Cu²⁺ to His113 and His116, an analogous His residue being absent in pig Mb. In contrast, rapid oxidation of 10-15% of pig oxy-Mb is observed under the same conditions (fast phase), which is not accompanied by catalysis because the reduced copper is apparently not reoxidized. The complexing of Cu²⁺ with His97 situated near the heme is probably responsible for the fast phase of the reaction. The affinity of His97 for Cu²⁺ must be significantly lower than those of the catalytic His residues since the fast phase does not contribute markedly to the rate of sperm whale and horse oxy-Mb oxidation. Increasing copper concentration does not produce a proportional growth in the oxidation rate of sperm whale and horse oxy-Mbs. Which Cu²⁺ binding sites of Mb make main contributions to the His reaction rate at different Cu²⁺/Mb ratios from 0.25 to 10 is discussed.