NMR RELAXATION STUDIES OF THE INTERACTION OF THIOCYANATE WITH LACTOPEROXIDASE

The interaction of lactoperoxidase, LPO, with its substrate, thiocyana te, SCN-, has been investigated by C-13 and N-15 NMR relaxation measur ements. When 0.1 M SCN-, enriched with either C-13 or N-15, was titrat ed with native ferric lactoperoxidase a large change in the spin-latti ce relaxation time of the respective nucleus was observed. In the pres ence of saturating amounts of CN-, a high affinity ligand for the heme iron, a similar but much smaller change in the relaxation time for SC N was found. Studies of the rate of carbon relaxation as a function of temperature have shown that thiocyanate is in fast exchange between a site on the enzyme and bulk solution. When LPO in either the absence or presence of CN was titrated with SCN a linear increase in the relax ation time was observed. Dissociation constants (K(d) values) have bee n determined from a least-squares analysis of these data. Apparent dis tances between the heme iron of lactoperoxidase and either the carbon or nitrogen atoms of bound thiocyanate ion have been determined throug h application of the Solomon-Bloembergen equation. These distances dem onstrate that the observed association does not involve iron-thiocyana te coordination, suggesting the possibility of an anion binding site.