ABSTRACT
A comparative study of the rates of ferrocyanide-catalyzed oxidation of several oxymyoglobins by molecular oxygen is reported. Oxidation of the native oxymyoglobins from sperm whale, horse and pig, as well as the chemically modified (MbO(2)) sperm whale oxymyoglobin, with all accessible His residues alkylated by sodium bromoacetate (CM-MbO(2)), and the mutant sperm whale oxymyoglobin [MbO(2)(His119-->Asp)], was studied. The effect of pH, ionic strength and the concentration of anionic catalyst ferrocyanide, [Fe(CN)(6)](4-), on the oxidation rate is investigated, as well as the effect of MbO(2) complexing with redox-inactive Zn(2+), which forms the stable chelate complex with functional groups of His119, Lys16 and Asp122, all located nearby. The catalytic mechanism was demonstrated to involve specific [Fe(CN)(6)](4-) binding to the protein in the His119 region, which agrees with a high local positive electrostatic potential and the presence of a cavity large enough to accommodate [Fe(CN)(6)](4-) in that region. The protonation of the nearby His113 and especially His116 plays a very important role in the catalysis, accelerating the oxidation rate of bound [Fe(CN)(6)](4-) by dissolved oxygen. The simultaneous occurrence of both these factors (i.e. specific binding of [Fe(CN)(6)](4-) to the protein and its fast reoxidation by oxygen) is necessary for the efficient ferrocyanide-catalyzed oxidation of oxymyoglobin.
Subject(s)
Ferrocyanides/chemistry , Myoglobin/chemistry , Oxygen/chemistry , Point Mutation , Amino Acid Substitution , Animals , Catalysis , Histidine , Horses , Isoelectric Focusing , Kinetics , Metmyoglobin/chemistry , Metmyoglobin/genetics , Muscle, Skeletal/metabolism , Myoglobin/genetics , Oxidation-Reduction , Sperm Whale , Swine , Zinc/metabolismABSTRACT
A comparative study of the rate of ferrocyanide-catalyzed oxidation of native sperm whale MbO2, its chemically modified derivative in which all accessible His residues are alkylated by sodium bromoacetate, (CM-MbO2), and mutant sperm whale MbO2 with His119 replaced by Asp residiue, [MbO2(His119-->Asp)] was carried out. The influence of pH, ionic strength, and [Fe(CN)6]4- concentration on the oxidation rate was investigated, as well as the effect of complexing MbO2 with redox-inactive Zn2+ ion, which, at the equimolar Zn2+ concentration, forms a stable complex with His119(GH1) on the protein surface. It was shown that the mechanism of the catalysis involves specific binding of [Fe(CN)6]4- to the protein at the His119(GH1) region, which is in agreement with a large positive electrostatic potential and the presence at this site of Mb of a cavity large enough to accommodate [Fe(CN)6]4- anion. The protonation of nearby His113 and His116 residiues (especially of the latter) plays a very important role in the catalysis, promoting the fast oxidation of bound [Fe(CN)6]4- by dissolved oxygen. Only the presence of these both necessary conditions in MbO2 structure provides its effective oxydation catalyzed by ferrocyanide.
Subject(s)
Amino Acid Substitution/genetics , Ferrocyanides/chemistry , Metmyoglobin/chemistry , Myoglobin/chemistry , Oxygen/chemistry , Point Mutation , Whales , Animals , Catalysis , Metmyoglobin/genetics , Myoglobin/genetics , Oxidation-Reduction , Whales/geneticsABSTRACT
The influence of pH, ionic strength of the solution, and [Fe(CN)6]4- concentration on the rate of oxidation of sperm whale, horse, and pig oxymyoglobins, which is catalyzed by ferrocyanide ions, was studied. These myoglobins have homologous spatial structures and identical redox potentials but differ by the amount of His residues located on the protein surface. The effect of the MbO2 complexing with redox-inactive Zn2+ ion on the reaction rate was also examined. At the equimolar Zn2+ concentration, zinc ions form a stable complex with His119(GH1). It was found that the kinetic behavior of horse MbO2, which lacks His12(A10) substituted for by Gln, is fully analogous to one of sperm whale MbO2, while the oxidation of pig MbO2, three histidines of which, His12, His113(G14), and His116(G17), are replaced by Gln, is strongly inhibited. The mechanism of the catalysis was shown to involve specific binding of [Fe(CN)6]4- to the protein at the His119(GH1) site, which is in accord with the large positive electrostatic potential of this site and the presence here of a cavity large enough to accommodate [Fe(CN)6]4-. The nearby His113 and His116 residiues, which are absent in pig Mb, also play a very important role in the catalysis, because their protonation (especially of the last residue) is most likely responsible for the week oxidation of bound [Fe(CN)6]4- by dissolved oxygen.
