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 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.

The decameric structure of bovine pancreatic trypsin inhibitor (BPTI) crystallized from thiocyanate at 2.7 Å resolution

“…Most interactions of mineral anions such as chloride, nitrate (Galanter & Labotka, 1991) or thiocyanate (Modi et al, 1989b;Crull & Goff, 1993) are of the same order of magnitude in a number of different proteins. Again, these studies support the observation that, even though thiocyanate is a particular polarizable soft agent, it does not differ from others anions: in the particular case of BPTI, we have observed that sodium chloride is also able to form pentamers of BPTI in crystals,…”

Chemical, Spectroscopic and Structural Investigation of the Substrate‐Binding Site in Ascorbate Peroxidase

“…Under fast exchange condition$, the measured relaxation rate, R, <,h\ ( = I / TI,obJ, is the weighted average of the relaxation rates of the free and bound substrate (Rl,f and R, ,,, respectively), (Sakurada et al, 1986;Modi et al, 1989). Under the experimental conditions (substrate concentration much greater than protein concentration), pf = 1, and if a stoichiometry of 1 : 1 is assumed, Eqn (5) is derived, (5) where E, and S, represent the total concentrations of APX-CN and substrate, respectively, and Kd is the dissociation constant of the protein I substrate complex (Sakurada et al, 1986;Modi et al, 1989). In the present experiments, determination of R,,,,, which represents the paramagnetic contribution to the relaxation rate of the protons in the bound substrate arising from the unpaired electrons of the haem iron, is required.…”

Binding of Iodide to Arthromyces ramosus Peroxidase Investigated with X-ray Crystallographic Analysis, 1H and 127I NMR Spectroscopy, and Steady-state Kinetics

“…Because it is very difficult to study the binding to compound I that causes the reaction, the binding of these ions to peroxidases in the resting state was examined by spectrophotometric (4,7,8), kinetic (9,10), fluorometric (11), 127 I NMR (12,13), 15 N NMR (14 -17), 13 C NMR (17), 1 H NMR (13)(14)(15)(16)(17)(18)(19), and optical difference spectroscopy (20 -22) techniques. The actual site of the binding of these ions to the enzymes and the mechanism of electron transfer from these ions to the heme irons have yet to be clarified.…”