Binding of thiocyanate to lactoperoxidase: proton and nitrogen-15 nuclear magnetic resonance studies

Abstract

The binding of thiocyanate to lactoperoxidase (LPO) has been investigated by ¹H and ¹⁵N NMR spectroscopy. ¹H NMR of LPO shows that the major broad heme methyl proton resonance at about 61 ppm is shifted upfield by addition of the thiocyanate, indicating binding of the thiocyanate to the enzyme. The pH dependence of line width of ¹⁵N resonance of SC¹⁵N^- in the presence of the enzyme has revealed that the binding of the thiocyanate to the enzyme is facilitated by protonation of an ionizable group (with pK_a of 6.4), which is presumably distal histidine. Dissociation constants (K_D) of SC¹⁵N^-/LPO, SC¹⁵N^-/LPO/I^-, and SC¹⁵N^-/LPO/CN^- equilibria have been determined by ¹⁵N T₁ measurements and found to be 90 ± 5, 173 ± 20, and 83 ± 6 mM, respectively. On the basis of these values of K_D, it is suggested that the iodide ion inhibits the binding of the thiocyanate but cyanide ion does not. The thiocyanate is shown to bind at the same site of LPO as iodide does, but the binding is considerably weaker and is away from the ferric ion. The distance of ¹⁵N of the bound thiocyanate ion from the iron is determined to be 7.2 ± 0.2 A from the ¹⁵N T₁ measurements.