UTILIZATION OF COPPER AS A PARAMAGNETIC PROBE FOR THE BINUCLEAR METALCENTER OF PHOSPHOTRIESTERASE

Bacterial phosphotriesterase catalyzes the hydrolysis of organophospha te triesters. To be active, the enzyme requires that two divalent cati ons are bound. These metal ions are bound in close proximity to one an other as a binuclear center. To characterize the structure and functio n of the binuclear metal binding sites, we have prepared the copper-su bstituted enzyme. The kinetic data indicate that this enzyme is essent ially inactive toward the hydrolysis of phosphotriesters. The EPR sign al arising from the copper-substituted enzyme is nearly axial, with g( parallel to) = 2.24 and g(perpendicular to) = 2.05 and shows at least seven superhyperfine transitions in the gi region with A(perpendicular to) = 1.45 X 10(-3) cm(-1). These splittings are consistent with the direct ligation of more than one nitrogen to the metal center. The ave rage spin quantitation of copper-substituted enzymes are 0.6 spin/Cu, approximately half of that observed for noninteracting Cu2+ ions. The spin intensity increases to ca. 1 spin/Cu when samples are denatured w ith acid. The binding of metal ions to the designated alpha and beta s ites is highly synergistic (i.e., the metal ions bind in pairs). Mixed metal complexes of the type Cu/X and X/Cu were prepared. When X is a diamagnetic ion (Zn2+ or Cd2+), the spin quantitation increases, but w hen X is the paramagnetic Co2+ ion, the spin quantitation decreases. T his behavior indicates that the low spin intensities observed for copp er-substituted phosphotriesterase arise from spin-coupling of the two adjacent Cu2+ ions. The addition of dithiothreitol, ascorbate, or dith ionite to the copper-substituted phosphotriesterase results in nearly the complete loss of spin intensity. This indicates that the bound cop pers can be reduced to the cuprous state. (C) 1995 Academic Press, Inc .