Magnetic Resonance Studies of the Interaction of Cupric Ion with Native and Modified Forms of Ribonuclease

Abstract The binding of copper to bovine pancreatic ribonuclease A, its carboxymethylated histidine-119 and histidine-12 derivatives, ribonuclease S and S-protein, has been investigated at pH 5 and 6 by measuring the enhancement of the magnetic proton relaxation rate (PRR) of water due to bound copper. The magnitude of the PRR enhancement factor is characteristic for the binding site and reflects its protein environment. Titrations of copper and protein yield values of the binding constants and the number of sites and reveal interaction between binding sites. At pH 5, RNase A and RNase S exhibit three independent binding sites, one strong binding site with a dissociation constant, Kd, of 6 to 7 x 10-4 m and two weaker ones with Kd of approximately 8 x 10-3 m. The 1-carboxymethylhistidine-119-RNase and Sprotein have only two binding sites, one strong and one weak, and 3-carboxymethyl histidine-12-RNase has one strong site. The enhancement of the strong site, ebb1, is 6.1 for RNase A, almost the same for the 1-carboxymethylhistidine119-RNase, 5.9, but significantly lower for RNase S, 5.1, suggesting a more flexible structure for RNase S at the first Cu(II)-binding site. Modification of histidine-12 by carboxymethylation results in a drastic change in the environment of this Cu(II)-binding site, ebb1 = 30. To a lesser extent, removal of S-peptide also results in an increased value of ebb1 at the tight binding site, ebb1 = 9.0 in S-protein. At pH 6.0, the binding patterns become much more complex; all species except the histidine-12 derivative exhibit cooperativity in Cu(II) binding and some association, rendering it impossible to extract the number of sites or values of Kd and eb for individual sites from the data. 3-Carboxylmethylhistidine-12-RNase is exceptional in that it reveals two equivalent noninteracting binding sites at pH 6, Kd equals 4 x 10-4 m, and eb equals 24.3. Measurements of the enhancement as a function of temperature indicated that the chemical exchange of water ligands in the Cu(II) ribonuclease complexes is so fast that the observed relaxation rates of all of the complexes are determined by the dipolar correlation time in the first hydration sphere of Cu(II). The magnitude of the energies of activation of 3 to 5 kcal per mole indicates that rotational motion determines the relaxation rate. The inhibitor constants for Cu(II) were determined at pH 5 for RNase A and RNase S, respectively, with cytidine2', 3'-cyclic phosphate as substrate and agreed within a factor of 2 or less with the Kd of the tight binding site determined from PRR enhancement data. In electron paramagnetic resonance spectra at pH 5 of solutions of Cu(II) and RNase A, two peaks were observed corresponding to free copper and bound copper. Titrations of RNase A with Cu(II) by electron paramagnetic resonance yielded the same values for free Cu(II) as those calculated from the PRR enhancement data, indicating that all of the Cu(II)-binding sites were enhanced and that the PRR data accounted for the total Cu(II) bound. On the basis of the enhancement data and other known properties of RNase A, the strong Cu(II)-binding site is tentatively assigned to histidine-12 and the two weaker sites to histidine-105 and histidine-119.