Chlorine-35 and proton NMR study of anion binding to reduced bovine copper-zinc superoxide dismutase

Abstract

Binding of chloride to reduced bovine copper-zinc superoxide dismutase (Cu{sub 2}Zn{sub 2}SOD) and chemically modified derivatives was monitored by the line width at half-height of the Cl{sup {minus}} resonance as measured by {sup 35}Cl nuclear magnetic resonance (NMR) spectroscopy. Reduced arginine-modified and reduced lysine-modified Cu{sub 2}Zn{sub 2}SOD (at concentrations of 2.14 {times} 10{sup {minus}4} M) caused less broadening of the Cl{sup {minus}} resonance line width of 0.1 M NaCl solutions than did reduced native protein when measured under the same conditions; Cl{sup {minus}} broadening with all protein derivatives decreased drastically in the presence of 0.05 M phosphate. The C-H and N-H proton resonances of histidyl imidazoles of reduced native and reduced lysine-modified Cu{sub 2}Zn{sub 2}SOD were shifted by addition of Cl{sup {minus}} (with apparent affinity constants of 12 and {approximately} 2 M{sup {minus}1}, respectively) whereas this anion had less effect in the {sup 1}H NMR spectrum of reduced arginine-modified Cu{sub 2}Zn{sub 2}SOD (affinity constant <2 M{sup {minus}1}) under the same conditions. phosphate caused relatively smaller changes on the {sup 1}H NMR resonances of all reduced protein derivatives. The competition measured by {sup 1}H NMR spectroscopy between chloride and phosphate for anion binding sites in the neighborhood of the Cu{sup 1}more » ion was much less than that for nonspecific Cl{sup {minus}} binding monitored by {sup 35}Cl NMR spectroscopy. It is concluded from these experiments that, in addition to the weak anion binding at or near the Cu{sup I} ion, Arg-141, Lys-120, and Lys-134 serve as major anion binding sites in the reduced native protein. 57 refs., 5 figs., 2 tabs.« less