Vol. 264, No. 33, Issue of November 25, pp. 19677-19684,1989 THEJOURNAL OF BIOLOGICAL CHEMISTRY Printed in U.S.A. 0 1989 by The American Society for Biochemistry and Molecular Biology. Inc Interaction of Thiocyanate with Horseradish Peroxidase ‘H AND I5N NUCLEAR MAGNETIC RESONANCE STUDIES* (Received for publication, April 20, 1989) Sandeep Modi, Digambar V. Behere, and Samaresh MitraS From the Chemical Physics Group, Tata Institute of Fundamental Research, Homi Bhubha Road, Colaba, Bombay 400005,India Interaction of thiocyanate with horseradish peroxi- Horseradish peroxidase (HRP, EC 1.11.1.7, donor, HzOz, dase (HRP) was investigated by relaxation rate meas- oxidoreductase) is a plant heme protein enzyme that catalyzes urements (at50.68 MHz) of the “N resonance of thio- primarily the oxidation of a wide variety of oxidizable organic cyanate nitrogen and by following thehyperfine donor molecules by hydrogen peroxide (1, 2). The oxidation shifted ringmethyl proton resonances (at 500 MHz) of reaction for organic substrates generally proceeds through the heme group of SCN-oHRP solutions. At pH 4.0, the two distinct intermediates, HRP-I and HRP-11. The mecha- apparent dissociation constant (KO) for thiocyanate nism of oxidation involves initial binding of the donor to the binding to HRP was deduced to be 158 mM from the enzyme. Several studies have therefore been reported on the relaxation rate measurements. Chemical shift changes binding of oxidizable organic substrates to thenative enzyme of 1- and 8-ring methyl proton resonances in the pres- (2-5). HRP is also known to catalyze oxidation of inorganic ence of various amounts of thiocyanate at pH 4.0 substrates such as iodide, thiocyanate, nitrite, and bisulfite yielded KO values of 166 and 136 mM, respectively. ions (2, 6). Among the oxidizable inorganic substrates, the From the pH dependence of KO and the “N resonance interaction of iodide ion with HRP hasbeen studied to further line width, it was observed that thiocyanate binds to understanding of the mechanism of thyroid hormone biosyn- HRP only under acidic conditions (pH < 6). The binding thesis catalyzed by thyroid peroxidase and lactoperoxidase (7, was found to be facilitated by protonation of an acid 8). Kinetic (9), fluorometric (IO), and NMR (11) studies have group on the enzyme with pK, 4.0. The pH dependence suggested that iodide ion forms a 1:l complex with HRP and of the 16N line width as well as the apparentdissocia- binds near the heme group. The kinetic studies (9, 12) have tion constant were quantitativelyanalyzed on the basis suggested further that theoxidation of iodide with hydrogen of a reaction scheme in which thiocyanate in deproton- peroxide catalyzed by HRP occurs via two-electron transfer ated ionic form binds to the enzyme in protonated under acidic conditions. In this process, HRP-I is converted acidic form. The KD for thiocyanate binding to HRP to native enzyme directly by two-electron oxidation of the was also evaluated in the presence of an excess of substrate without formation of HRP-11. exogenous substrates such as resorcinol, cyanide, and Besides iodide, thiocyanate ion is also an attractive inor- iodide ions. It was found that the presence of cyanide ganic substrate because thiocyanate is classified as a pseudo- (which binds to heme iron at the sixth coordination halide and bears many resemblances to iodide in its chemical position) and resorcinol did not have any effect on the behavior. SCN-. H20,.lactoperoxidase provides a potent non- binding of thiocyanate, indicating that thebinding site specific bacteriostatic or bacteriocidal system (13, 14). Mag- of the thiocyanate ion is located away from the ferric nusson et al. (15) have studied the catalytic activity of lacto- center as well as from the aromaticdonor binding site. peroxidase using iodide and thiocyanate ions and have sug- The KDin the presence of iodide, however, showed that gested that the oxidation of iodide and thiocyanate with iodide competes with thiocyanate for binding at the hydrogen peroxide catalyzed by lactoperoxidase and thyroid same site. The distance of the bound thiocyanate ion peroxidase may also occur via two-electron transfer. We have from the ferric centerwas deduced from the “N relax- recently studied the interaction of thiocyanate with lactoper- ation time measurements and was found to be a 6.8 A. oxidase using 15N and ‘H NMR (16). Thiocyanateand iodide From the distance as well as the change in the chemical ions have been shown to bind to lactoperoxidase at a distal shifts and line width of 1- and 8-methyl proton reso- site of heme at thehistidyl residue with an apparentdissocia- nances, it is suggested that thebinding site of thiocya- tion constant of 90 and 38 mM, respectively at pH 6.1 (16, nate may be located near heme, placed symmetrically 17). The interaction of thiocyanate with HRP has, however, with respect to 1- and 8-methyl groups of the heme of not been studied in any detail. The only study appears to be HRP. Similarity in themodes of binding of iodide and that of Lukat andGoff (18), who investigated the interaction thiocyanate suggests that theoxidation of thiocyanate by optical spectroscopy and reported that the interaction of ion by HzOz may also proceed via the two-electron thiocyanate with HRP is optically inoperable even at a high transfer pathway under acidic conditions, as is the case concentration of thiocyanate. Thus, in spite of the fact that for iodide. the oxidation of thiocyanate ion by Hz02is catalyzed by HRP like that of iodide ion, it is still not known where thiocyanate The abbreviations used are: HRP, horseradish peroxidase; SCN-, deprotonated thiocyanate; Avih(obs,,observed line width; KSH,protoly- * The costs of publication of this article were defrayed in part by tic dissociation constant of substrate; TI,relaxation time; TI*,TI of the payment of page charges. This article must therefore be hereby the HRP-substrate complex; TI,, TIof substrate in the absence of marked “aduertisement” in accordance with 18 U.S.C. Section 1734 enzyme; Tld,diamagnetic contribution to TI*;T,, paramagnetic con- solely to indicate this fact. tribution to Tl; T,, autocorrelation time, T,, lifetime of the enzyme- $ To whom correspondence should be sent. substrate complex. 19677 19678 Interaction of Thiocyanate with Horseradish Peroxidase ion binds and how electrons are transferred from thiocyanate to the heme iron of HRP-I. KD = K(l + K./H+)(l + H+/Ksd (1) In the present study, the interaction of thiocyanate with The variation of the observed line width as a function of pH is HRP was investigated using 15N and 'H NMR spectroscopy. given by Equation 2 (23) From the measurements of relaxation times of (SC1'N)- in the presence and absence of HRP, the apparentdissociation constant (KD)was evaluated, and the distance of the 15N of SCN- from the ferric ion of HRP was estimated. The line where A& and A& denote the line widths of the enzyme-bound and unbound substrate I5N resonances, respectively. Eoand So represent width measurements on "N resonance were utilized to deduce the initial enzyme and substrate concentrations, respectively. the autocorrelation time (re).The pHdependence of the line Relaxation Rate Measurements-Since the optimum pH for the width gave a pK, of the ionizable group in the heme crevice, binding of SCN- to HRP is between 3.0 and 5.0 (see "Results"), the which is responsible for the binding of thiocyanate. The KO relaxation rate measurements were done at pH4.0. For the relaxation was also estimated from the chemical shift changes of the time measurements, HRP was treated with Chelex 100 (Bio-Rad) to remove any traces of free metal ions (24). Deionized double-distilled heme methyl resonances of HRP with varying concentrations water was used to prepare 0.1 M phosphate buffer (pH 4.0). Filtrates of thiocyanate. The results of these studies have been used to were lyophilized and redissolved in DzO for NMR studies. Titrations determine the strength andsite of the binding of thiocyanate were carried out in the enzyme concentration range of 100 pM-4 mM, to HRP andare compared with the results on the interaction and titrations of the substrate were in the range of 20-470 mM. To of iodide with HRP (11) and the interaction of thiocyanate obtain the longitudinal relaxation time ( Tl(ob)),the inversion recovery method with a 180"-~-90'pulse sequence was used (16, 23, 25). with lactoperoxidase (16). Determination of the Apparent Dissociation Constant of Thiocya- nate Binding to HRP Using l5N-TI Measurements-T1(,b,) can be MATERIALS ANDMETHODS considered as the sum of the relaxation rates of the bound and free Horseradish peroxidase was purified from crude HRP (Sigma, Rz substrate fractionsand is related to KD, Tlb, and Tl/through Equation = A403/Apm = 0.8) by DEAE and CM-cellulose column chromatogra- 3 for the binding of one molecule of thiocyanate to one protonated phy (19). The B and C HRP isoenzymes were collected (Rz = 3.2) molecule of HRP (16, 23) after elution with acetate buffer (100 mM, pH 4.4). The concentration of the enzyme was determined spectrophotometrically using a molar extinction coefficient of 1.02 X lo6cm-' M" at 403 nm for HRP (20). Deuterium oxide (>99.85%) was purchased from Aldrich. Enriched where T1b is the Tl of the HRP-substrate complex, and TI/ is the T1 I5N sodium thiocyanate (NaSC"N, atom % of I5N 99) was pur- > of the substratein the absence of the enzyme. KD and T1b for chased from MSD Isotopes. All other reagents wereof analytical thiocyanate binding to HRP can be obtained by least squares fit of grade. the data toEquation 3. NMR Measurements-Proton NMR measurements were carried Determination of the Apparent Dissociation Constant of Thiocya- out on a Bruker AM 500"Hz FT NMR spectrometer at 23 'C.