The main role of inner histidines in the molecular mechanism of myoglobin oxidation catalyzed by copper compounds.

In the presence of Cu(2+) and Cu(Gly)(2), the oxidation of two native MbO(2)'s (Mb = myoglobin), from the sperm whale and horse, and also two chemically modified sperm whale MbO(2)'s alkylated at solvent-accessible histidines by sodium bromoacetate (CM-MbO(2)) and by iodoacetamide (CA-MbO(2)) have been studied at different pH's, ionic strengths, and concentrations of the copper reagent. The influence of competitive redox-inactive zinc ions on the reaction rate is investigated as well. Localization of Cu(Gly)(2) in sperm whale met-Mb and CM-met-Mb has been examined using the high-resolution NMR method. The obtained data suggest that binding of copper compounds to the surface histidines (all of them are 1.8-2.7 nm apart from the heme) has only a minor, no more than 35%, contribution to the overall reaction rate, in particular under a large excess of the reagent (more than 8-10-fold). The noticeable contribution of His113(116), His48, and His81, which have the greatest affinity to copper according to NMR data, is revealed only at small concentrations of copper, less than a 5-fold excess relative to the protein. The main contribution to the reaction rate must be from the binding of copper to the inner histidines, His97 (0.62 nm from the heme), and possibly to the distal His64. Both are inaccessible to the modification by alkylating reagents and have much lower affinity to copper than all surface histidines, because they are hydrogen-bonded, the former with the carboxyl group of the heme propionate and the second with the liganded O(2).