301-311Interaction of thiocyanate with lactoperoxidase (LPO) and horseradish peroxidase (HRP) has been investigated by relaxation rate measurements (at 125.77 MHz) of 13C resonance of thiocyanate carbon. The apparent dissociation constant (KD) for thiocyanate binding to LPO at pH = 6.1 and to HRP at pH = 4.0 has been deduced to be 85 mM and 160 mM respectively from the relaxation rate measurements. The pH dependence of KD and 13C resonance line-width of thiocyanate has been used to calculate pKa value of amino acid residue on these enzymes where the thiocyanate is shown to be binding. From the pH dependence of KD and 13C resonance line-width, it is observed that thiocyanate binds to LPO and HRP only under acidic conditions (pH pH pKa = 6.1 for LPO and 4.0 for HRP. The pH dependence of 13C resonance line-width of thiocyanate as well as KD have been quantitatively analysed on the basis of a reaction scheme in which thiocyanate in deprotonated ionic form binds to the enzyme in protonated acidic form. KD for thiocyanate binding to the enzyme has also been evaluated in the presence of excess of exogenous substrates such as resorcinol, cyanide and iodide. The presence of cyanide (which binds to heme iron of enzyme at sixth coordination position) does not have any effect on the binding of thiocyanate, indicating that binding site of thiocyanate ion is located away from the ferric centre of these enzymes. The presence of resorcinol, has significant effect on KD for binding of thiocyanate to LPO but it has no effect on thiocyanate binding to HRP. The K D in the presence of iodide however shows that iodide competes with thiocyanate for binding at the same site in both the cases. Distance of the carbon atom of bound thiocyanate ion from ferric centre has been deduced from the 13C-TJ measurements and is found to be 8.4 Ǻ and 8.0 Ǻ for LPO and HRP respectively. This distance remains unchanged by the presence of cyanide ion at the sixth coordination site of the heme iron of these enzymes. Similarity in the mode of binding of iodide and thiocyanate suggests that the oxidation of thiocyanate ion by H2O2, like that of 1- by H2O2 may also proceed via two-electron transfer pathway under acidic conditions
