Horseradish peroxidase catalyzed oxidation of thiocyanate by hydrogen peroxide: comparison with lactoperoxidase-catalysed oxidation and role of distal histidine

Abstract

Horseradish peroxidase-catalysed oxidation of thiocyanate by hydrogen peroxide has been studied by ¹⁵N-NMR and optical spectroscopy at different concentrations of thiocyanate and hydrogen peroxide and at different pH values. The extent of the oxidation and the identity of the oxidized product of the thiocyanate has been investigated in the SCN^-/H₂O₂/HRP system and compared with the corresponding data on the SCN^-/H₂O₂/LPO system. The NMR studies show that (SCN)₂ is the oxidation product of thiocyanate in the SCN^-/H₂O₂/HRP system, and its formation is maximum at pH≤4 and that the oxidation does not take place at pH≥6. Since thiocyanate does not bind to HRP at pH≥6 (Modi et al. (1989) J. Biol. Chem. 264, 19677-19684), the binding of thiocyanate to HRP is considered to be a prerequisite for the oxidation of thiocyanate. It is further observed that at [H₂O₂]/[SCN^-] = 4, (SCN)₂ decomposes very slowly back to thiocyanate. The oxidation product of thiocyanate in the SCN^-/H₂O₂/LPO system has been shown to be HOSCN/OSCN^- which shows maximum inhibition of oxygen uptake by Streptococcus cremoris 972 bacteria when hydrogen peroxide and thiocyanate are present in equimolar amounts (Modi et al. (1991) Biochemistry 30, 118-124). However, in case of HRP no inhibition of oxygen uptake by this bacteria was observed. Since thiocyanate binds to PO at the distal histidine while to HRP near 1- and 8-CH₃ heme groups, the role of distal histidine in the activity of SCN^-/H₂O₂/(LPO, HRP) systems is indicated.