Glutamic acid 133 or 131 is involved in the electron-transfer reaction from Fe(II)(CN)6 4− to the cupric ions in human- or bovine-copper, zinc-superoxide dismutase

Abstract

The electron-transfer reactions from Fe(II)(CN)64− to the cupric ion in human- or bovine-copper, zinc-superoxide dismutase were followed at various pHs from pH 5.0 to 8.0 by the micro-stopped-flow method. From pH 5.0 to 8.0, Fe(II)(CN)64− first forms an adduct through the charge interaction with Arg 143 or 141 of the active cavity; electron-transfer then occurs from Fe(II)(CN)64− to the cupric ion of the enzyme in human- or bovine-copper, zinc-superoxide dismutase. The formation-constant values of the adducts in human- and bovine-copper, zinc-superoxide dismutase are almost constant between pH 7.0 and 8.0, but increase with decreasing pH at pH values lower than 7.0. In contrast, the electron-transfer rate constants from Fe(II)(CN)64− to the cupric ion of the enzyme after adduct-formation in human- and bovine-copper, zinc-superoxide dismutase are nearly constant from pH 5.0 to 8.0 and give almost the same values in bovine- or human-copper, zinc-superoxide dismutase. The pH dependencies of adduct-formation-constants in human- and bovine superoxide dismutase indicate that the protonation of some residue with pKa value less than 5.0 accelerates the formation of adducts formed through the charge interaction between Arg 143 (human) or Arg 141 (bovine) and Fe(II)(CN)64−. X-ray diffraction analysis data show that the arrangement of Arg 143 and Glu 133 around the copper ion in the active cavity of human-copper, zinc-superoxide dismutase is very similar to that of bovine copper, zinc-superoxide dismutase. The low pKa value of the residue involved in adduct-formation suggests that the protonation group should be the carboxylate. Protonation of the carboxylate group of Glu 133 (Glu 131) results in neutralization of the minus charge of the carboxylate group and a decrease in the electronic repulsion for Fe(II)(CN)64−, so that the formation-constant of the adduct should increase with decreases in pH at lower pH values.