Reaction Mechanism of Electron Transfer from FeII(CN)4−6 or WIV(CN)4−8 to the Cupric Ions in Human Copper, Zinc Superoxide Dismutase

The electron transfer reactions from FeII(CN)4−6 and WIV(CN)4−8 to the cupric ions in human copper, zinc superoxide dismutase were followed by the micro-stopped-flow method. The kinetic rate data clearly indicate that FeII(CN)4−6 or WIV(CN)4−8 first forms an adduct with the enzyme through the interaction with Arg143 of the active cavity and then an electron from FeII(CN)4−6 or WIV(CN)4−8 of the adduct transfers to the cupric ion in the enzyme. The dissociation constants of the adducts of FeII(CN)4−6 and WIV(CN)4−8 were 4.0(±0.3) × 10−3 and 2.2(±0.3) × 10−3 M, respectively. In spite of the difference between the standard redox potentials of FeIII(CN)3−6/FeII(CN)4−6 (468 mV) and WV(CN)3−8/WIV(CN)4−8 (556 mV), the electron transfer rate constant (0.148(±0.005) s−1) of FeII(CN)4−6 at 25°C is very similar to that of WIV(CN)4−8 (0.072(±0.011) s−1). The entropy values of the adduct formations and the activation energies of the electron transfer rates were determined by the temperature dependence of the dissociation constants of the adducts and the electron transfer rates. The enthalpy values of the formation of adducts are almost zero, so that the driving forces to form the adducts are mainly derived from the entropy. The activation energy of the electron transfer rate of FeII(CN)4−6 is very similar to that of WIV(CN)4−8. The formation of the adduct between FeII(CN)4−6 and the enzyme was inhibited by the presence of various anions (ClO−4, SO2−4, SCN−, and N−3). The bulky anions SO2−4 and ClO−4 behave as competitive inhibitors for FeII(CN)4−6; these anions should interact mainly with Arg143, as it has a positive charge at the entrance of the active cavity. The competitive inhibition constants of ClO−4, SO2−4, and SCN− were 0.010, 0.012, and 0.008 M. The azide ion, which is smaller than SO2−4 or ClO−4, shows mixed inhibition, because N−3 can interact with Arg143 (competitive inhibition) and also directly binds to the cupric ion in h-SOD (noncompetitive inhibition). The competitive and noncompetitive inhibition constants of N−3 were 0.004 and 0.016 M, respectively.