Binding of thiocyanate to lactoperoxidase: 1H and 15N nuclear magnetic resonance studies.
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 pKa of 6.4), which is presumably distal histidine. Dissociation constants (KD) 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 KD, 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.
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Chemical Physics Group, Tata Institute of Fundamental Research, Bombay, India.
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D000818:Animals; D001665:Binding Sites / Q000187:drug effects; D003486:Cyanides / Q000494:pharmacology; D004735:Energy Transfer; D006418:Heme / Q000032:analysis; D006639:Histidine / Q000032:analysis / Q000502:physiology; D006863:Hydrogen-Ion Concentration; D007455:Iodine / Q000494:pharmacology; D007784:Lactoperoxidase / Q000032:analysis*; D009682:Magnetic Resonance Spectroscopy; D008433:Mathematics; D009566:Nitrates / Q000494:pharmacology; D010544:Peroxidases / Q000032:analysis*; D011522:Protons; D013861:Thiocyanates / Q000032:analysis*
D003486:Cyanides; D009566:Nitrates; D011522:Protons; D013861:Thiocyanates; D006418:Heme; D006639:Histidine; D007455:Iodine; D007784:Lactoperoxidase; D010544:Peroxidases; C031760:thiocyanate