NMR relaxation studies of the interaction of thiocyanate with lactoperoxidase.

The interaction of lactoperoxidase, LPO, with its substrate, thiocyanate, SCN-, has been investigated by 13C and 15N NMR relaxation measurements. When 0.1 M SCN-, enriched with either 13C or 15N, was titrated with native ferric lactoperoxidase a large change in the spin-lattice relaxation time of the respective nucleus was observed. In the presence of saturating amounts of CN-, a high affinity ligand for the heme iron, a similar but much smaller change in the relaxation time for SCN- 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 relaxation time was observed. Dissociation constants (Kd values) have been determined from a least-squares analysis of these data. Apparent distances between the heme iron of lactoperoxidase and either the carbon or nitrogen atoms of bound thiocyanate ion have been determined through application of the Solomon-Bloembergen equation. These distances demonstrate that the observed association does not involve iron-thiocyanate coordination, suggesting the possibility of an anion binding site.