Evidence for different types of interaction between anions and the copper(II) site of superoxide dismutase

Abstract

It seems now well established that the two equivalent copper(II) ions in dimeric superoxide dismutase are exposed to solvent as well as to solute molecules, while the two zinc(II) ions are not capable of such interactions. [1–4]. Water proton relaxation studies [5] have shown that anions bind the copper atom by displacing the coordinated water molecule; the obtained affinity constants roughly compare with those calculated through inhibition measurements [measurements [5], suggesting the identity between the water and the inhibitor binding sites. The present report aims to show evidence for a second copper binding site for anions, not involving displacement of water; this site may also be of importance for the catalytic process. Previous investigations have shown that the thiocyanate ion is able to affect the ESR spectra of copper(II) in superoxide dismutase [6], whereas it has been subsequently shown not to affect the catalytic activity. Therefore, a detailed study of the interaction between the above ion and superoxide dismutase has been undertaken, by means of electronic, ESR, and 1H and 13C NMR spectroscopy. While the electronic spectra of the copper chromophore as well as the water proton relaxation of enzyme solutions remain substantially unchange up to 0.5 M KNCS concentrations, the ESR parameters and the 13C relaxation times of the anion are significantly affected. In particular, the 13C signal of NCS - dramatically broadens, consistently with a direct binding to the paramagnetic effect on the thiocyanate concentration an affinity constant of 50 ± 10 M -1 can be estimated. The ESR data on frozen solutions qualitatively agree with the above value, as judged from the progressive variation of A from 145 to 160 cm -1 × 10 4 upon anion addition. By increasing the thiocyanate concentration above 1 M a further range is observed in the ESR spectra, which show a decrease in the A value; a slight blue shift is observed in the electronic spectra and the water proton relaxation is reduced. Limit values of the above parameters could not be obtained; the affinity constant can be estimated to be in the range 0.1–1 M -1. All of the above results strongly suggest the existence of a second, non inhibitory anion binding site on copper in superoxide dismutase which, in the case of the thiocyanate anion, is preferred to the site of water. The electronic and ESR spectra of the adducts indicate that there are not major variations in the coordination geometry of the copper chromophore, which seems to be more consistent with the detachment of one of the four inplane imidazole donors that with the addition of the thiocyanate ion to a sixth coordination position.