Mechanism of oxidation of oxymyoglobin by copper ions: Comparison of spermwhale, 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 whal e, horse, and pig oxymyoglobins (oxy-Mb) by copper ions has been studied. T hese myoglobins have homologous spatial structures and equal redox potentia ls but differ in the number of histidines located on the surface of the pro teins. It was shown that oxy-Mb can be oxidized in the presence of Cu2+ thr ough two distinct pathways depending on which histidine binds the reagent a nd how stable the complex is. A slow pH-dependent catalytic process is obse rved in the presence of equimolar Cu2+ concentration for sperm whale and ho rse oxymyoglobins. The curves of pH dependence in both cases are sigmoid wi th pK(eff) corresponding to the ionization. The process is caused by the st rong binding Of Cu2+ to His113 and His116, an analogous His residue being a bsent in pig Mb. In contrast, rapid oxidation of 10-15% of pig oxy-Mb is ob served under the same conditions (fast phase), which is not accompanied by catalysis because the reduced copper is apparently not reoxidized. The comp lexing Of Cu2+ with His97 situated near the heme is probably responsible fo r the fast phase of the reaction. The affinity of His97 for Cu2+ must be si gnificantly lower than those of the "catalytic" His residues since the fast phase does not contribute markedly to the rate of sperm whale and horse ox y-Mb oxidation. Increasing copper concentration does not produce a proporti onal growth in the oxidation rate of sperm whale and horse oxy-Mbs. Which C U2+ binding sites of Mb make main contributions to the His reaction rate at different Cu2+/Mb ratios from 0.25 to 10 is discussed.