INTRODUCTION

Horseradish peroxidase (HRP)¹ (EC 1.11.1.7; donor H₂O₂ oxidoreductase) catalyzes the oxidation of a wide variety of organic and inorganic electron donors by H₂O₂ through intermediate formation of compound I and compound II (1-4). The oxidation of aromatic donors proceeds through these intermediates by two one-electron oxidations with the formation of the substrate free radicals (3). The enzyme also catalyzes one-electron oxidation of various plant electron donors such as indoleacetic acid and ascorbate (5, 6). Among inorganic substrates, HRP catalyzes the oxidation of iodide, thiocyanate, nitrite, and bisulfite (1, 7-9), of which the mechanism of oxidation of iodide has been extensively studied (7, 8, 10, 11). Iodide is oxidized through a two-electron transfer directly to compound I with the intermediate formation of enzyme-hypoiodous complex (7, 8, 10, 11). Electron donors appear to bind at the exposed heme edge close to the heme methyl C1H₃ and C18H₃ to promote electron transfer to the C20 heme edge for oxidation by heme ferryl group (12-20). Recently, the plausible role of some conserved residues in aromatic donor binding in the heme distal pocket has also been reported (18-22).

Thiocyanate, a pseudohalide, is oxidized by various mammalian peroxidases (10, 23-27). Lactoperoxidase-H₂O₂-SCN is a potent bacteriostatic-bactericidal system also (28-31). The antibacterial activity might be due to various oxidation products of SCN such as CN (32), (SCN)₂ (31), cyanosulfurous acid, or cyanosulfuric acid (33) or OSCN (23). Recently, OSCN has been identified by NMR studies as the major stable oxidation product at equimolar concentrations of H₂O₂ and SCN, but CN may be formed if the ratio of [H₂O₂]/[SCN] exceeds 1 (28). The pathway for SCN oxidation has been proposed (23) at equimolar concentrations of H₂O₂ and SCN as follows.

(Eq. 1)

(Eq. 2)

(Eq. 3)

Alternatively, SCN may be directly oxidized to OSCN as follows.

(Eq. 4)

As (SCN)₂ is unstable in aqueous solution and readily hydrolyzed to HOSCN (34), OSCN is the major oxidation product in LPO-catalyzed SCN oxidation (23, 28, 35). The binding of SCN to LPO is facilitated by protonation of an ionizable group of pK_a 6.4, which is presumably distal histidine (36). It binds with the K_d value of 90 ± 5 mM (36) and is oxidized by compound I through two-electron transfer to form OSCN (37). Although LPO-catalyzed SCN oxidation has been extensively studied (10, 23-37), the literature is very scanty on the mechanism of HRP-catalyzed SCN oxidation. ¹H and ¹⁵N NMR studies indicate that SCN binds to HRP away from the distal histidine, near the heme methyl C1H₃ and C18H₃ with the K_d value of 158 ± 19 mM and the binding is facilitated by protonation of an acid group with pK_a 4.0 (38, 39). The oxidation of SCN and identification of the oxidation product have been studied and compared with LPO (9). While HOSCN/OSCN is the major oxidation product in the LPO system (28), (SCN)₂ is the oxidation product in HRP as evidenced by NMR studies (9). However, the mechanism of formation of (SCN)₂ is not known yet. Moreover, the detailed kinetic and spectral evidence for SCN oxidation by HRP and its comparison with that of LPO are still lacking. In this paper, we present evidence to show that SCN is mainly oxidized by HRP through two one-electron transfer mechanisms to form thiocyanate radicals. The radicals may dimerize to form (SCN)₂ (9), which is hydrolyzed to yield CN. CN binds with the heme iron concurrent with oxidation and lowers the catalytic turnover of HRP. In contrast, LPO forms very little CN due to direct two-electron oxidation of SCN to form stable OSCN (28), and therefore it oxidizes SCN with much higher turnover.

MATERIALS AND METHODS

Horseradish peroxidase (Type VI A, RZ = 3.0), lactoperoxidase (RZ = 0.88), PBN, diethylenetriamine pentaacetic acid, 5-5-dimethyl-1-pyrroline N-oxide (DMPO), indoleacetic acid, ascorbate, and guaiacol were obtained from Sigma. KSCN, KI, and KNO₃ were from Merck (India). Other chemicals were of analytical grade. The concentration of HRP and LPO was determined from ₄₀₃ = 102 cm¹ mM¹ (40) and ₄₁₂ = 112 cm¹ mM¹ (41), respectively.

Horseradish Peroxidase- and Lactoperoxidase-catalyzed Oxidation of SCN

SCN oxidation by HRP (0.5 µM) or LPO (0.01 µM) was measured in an incubation mixture containing 1 mM H₂O₂ and 1 mM SCN in 50 mM sodium acetate buffer, pH 4.5 or 5.6, respectively, in a final volume of 2 ml. The reaction was stopped by adding 100 nM catalase. To 0.2 ml of the reaction mixture, 1.6 ml of 0.1 M HCl and 0.2 ml of 0.1 M FeCl₃ were added and the absorbance of the FeSCN²⁺ complex was measured at 450 nm (42). The concentration of SCN was calculated from a standard curve.

Determination of H₂O₂ Consumption during SCN Oxidation

H₂O₂ consumption was assayed by measuring the concentration of H₂O₂ (43, 44). The assay system contained in a final volume of 1.2 ml: 50 mM sodium acetate buffer, pH 4.5, 2 mM SCN, 0.5 µM HRP, and 2.0 mM H₂O₂ added last to start the reaction. At different time intervals, a 0.1-ml aliquot was added to 3 ml of 80 mM HCl followed by the addition of 20 µl of 100 mM ferroammonium sulfate and 0.2 ml of 2.5 M KSCN to measure the absorbance at 480 nm (43). The concentration of H₂O₂ was calculated from a standard curve. The amount of SCN oxidized under identical conditions was determined (42) as already described.

Inactivation of HRP in the Presence of SCN and H₂O₂

HRP (2 nM) was incubated with varying concentrations (0-2 mM) of SCN in 50 mM sodium acetate buffer, pH 4.5, in the presence or absence of 0.6 mM H₂O₂ in a final volume of 1 ml in a spectrophotometric cuvette. At various times of incubation, the peroxidase activity of the whole reaction mixture was measured by I₃ assay at 353 nm after addition of 2.0 mM KI followed by 0.6 mM H₂O₂ to start the reaction (45). The increase in absorbance was followed for 1.5 min at an interval of 10 s, and the activity was calculated from the linear rate of the reaction. HRP-catalyzed I₃ formation in the presence of varying concentrations of SCN without preincubation served as control for the calculation of inactivation of HRP on preincubation with SCN and H₂O₂.

Inactivation of HRP by Varying Concentrations of H₂O₂ in the Presence of SCN

HRP (2 nM) was incubated with varying concentrations of H₂O₂ (0-0.6 mM) with a fixed concentration of SCN (1 mM) in 50 mM sodium acetate buffer, pH 4.5, in a final volume of 2 ml. After 5 min of incubation, 1 ml each of the reaction mixture was used for measurement of enzyme activity by I₃ assay and for the consumption of H₂O₂ as already described.

HRP (1.0 µM) was incubated for 10 min with 1 mM SCN and 1 mM H₂O₂ in 50 mM sodium acetate buffer, pH 4.5, for complete inactivation. Residual H₂O₂ was decomposed by a small amount of catalase. The reaction mixture was then passed through a GF/C filter, and the filtrate was mixed with LPO or HRP to get the final enzyme concentration of 1 µM. The optical absorption spectrum of the mixture was then recorded.

Quantitation of CN Production of HRP-H₂O₂-SCN and LPO-H₂O₂-SCN Systems

The amount of CN produced in the HRP or LPO system as recovered in the filtrate was determined from the absorbance of the peroxidase-cyanide complex at 428.5 nm using lactoperoxidase (23). The concentration of CN was calculated from a standard curve.

For measurements of the difference spectrum of the enzyme-SCN complex versus enzyme, both the reference and sample cuvettes were filled up with 1 ml of the enzyme solution (5.0 µM) for base-line tracing. This was followed by the addition of a small volume of the ligand to the sample cuvette with concomitant addition of equal volume of solvent into the reference cuvette (46). The apparent equilibrium dissociation constant (K_d) for the complex formation was calculated from

(Eq. 5)

where [S] and [E] are the concentrations of substrate and enzyme, A is the change in absorbance between the peak and trough of the spectrum, and is the difference in molar absorptivity. K_d was calculated from the plot of 1/A versus 1/[S]. Donors competing for binding to HRP at the same site as that of SCN affect the apparent dissociation constant, K_{d obs} of SCN in the presence of the inhibitor (I) and is related to the inhibitor concentration [I] by (38, 47, 48)

(Eq. 6)

where K_i is the apparent dissociation constant of the HRP-I complex in the absence of SCN and K_d is the apparent dissociation constant for the binding of SCN to HRP as defined in Equation 5. All kinetic and spectral studies were carried out in a Shimadzu UV-2201 computerized spectrophotometer at 28 ± 2 °C.

Detection of SCN Radicals by ESR Spectroscopy

Thiocyanate free radicals were detected as spin adduct with DMPO by ESR spectroscopy. The reaction mixture contained 100 mM sodium acetate buffer, pH 5.5, 100 mM SCN, 100 mM DMPO, 1 mM diethylenetriamine pentaacetic acid, 30 µM HRP, and 2 mM H₂O₂ added last to start the reaction. ESR spectra were recorded on a Varian E-112 spectrometer fitted with a TM-110 cavity operating at 9.45 GHz with 100 kHz modulation frequency.

RESULTS

HRP-catalyzed SCN Oxidation by H₂O₂

The catalytic turnover of HRP for SCN oxidation was compared with that of LPO at their optimum pH. From the initial rate of SCN oxidation, the catalytic turnover of HRP was found to be a hundredfold lower than that of LPO (Fig. 1). While studying the mechanism of this significantly lower turnover of HRP, we observed that preincubation of HRP with increasing concentrations of SCN in the presence of a fixed concentration of H₂O₂ resulted in concentration and time-dependent inactivation of the enzyme following pseudo-first order kinetics (Fig. 2A). Catalytic activity could be recovered by dilution, dialysis, or by passage through the Sephadex G-25 column indicating reversibility of the inactivation. K_obs values obtained from the slope of each line (Fig. 2A) when plotted against SCN concentrations yielded a straight line (Fig. 2A, inset) from which a second order rate constant for inactivation was calculated to be 400 M¹ min¹. Inactivation of HRP is also dependent on H₂O₂ concentration in the presence of a fixed concentration of SCN. A plot of the percent inhibition against the turnover number ([H₂O₂]/[HRP] ratio) as shown in Fig. 2B indicates that the percent inhibition is directly dependent on the number of turnovers of the enzyme. The enzyme is completely inactivated after 2 × 10⁴ turnovers consuming 20 nmol of H₂O₂/pmol of HRP.

Oxidation of SCN by HRP and LPO in the presence of H₂O₂.

Kinetics of the inactivation of HRP on preincubation with SCN and H₂O₂.

Effect of Spin Trap on the Catalytic Activity of HRP on SCN Oxidation

The kinetics of the HRP-catalyzed SCN oxidation was further studied in the absence or presence of the spin trap as shown in Fig. 3. No significant SCN oxidation and H₂O₂ consumption were evident in the absence of HRP. However, the initial rate of SCN oxidation or H₂O₂ consumption was significantly increased in the presence of the free radical traps such as PBN or DMPO. The inset shows a plot of varying concentrations of DMPO on the turnover of SCN which is 4-fold stimulated by 50 mM of DMPO. The data indicate that free radicals derived from the oxidation of SCN are involved in limiting the catalytic turnover of the enzyme.

HRP-catalyzed SCN oxidation and concurrent consumption of H₂O₂ in the presence of PBN or DMPO.

Spectral Evidence for SCN Oxidation and Enzyme Inactivation

The spectral evidence for the oxidation of SCN by the HRP-H₂O₂ system is shown in Fig. 4A. Addition of a 5-fold excess of H₂O₂ to native HRP (trace a) produces a mixture of compound I and compound II as shown in trace b. Low concentrations of SCN (4 µM) immediately reduced compound I to compound II (trace c), which was then slowly (20 min) reduced to the native enzyme (trace h) as evidenced by a time-dependent spectral shift from 417 nm to 402 nm through an isosbestic point at 408 nm. It is interesting to note that the broad spectrum of the mixture of compound I and II (trace b) increases in height for the initial few minutes due to complete reduction of compound I to compound II (trace c), which is then slowly reduced to the native state. However, in the presence of higher concentrations of SCN (50 µM), a new Soret peak appears at 421 nm after the addition of H₂O₂ (Fig. 4B, trace b) with the visible peak at 540 nm (Fig. 4B, inset). This enzyme never returns to the native state in the presence of iodide (trace c) or guaiacol (trace d), indicating its inability to oxidize these electron donors. However, if SCN (50 µM) is added to a mixture of compound I and compound II produced by a 5-fold molar equivalent of H₂O₂ (Fig. 4C), it causes an immediate spectral shift from 412 nm (mixture of compound I and compound II) to 417 nm (not shown) as a result of reduction of compound I to compound II with the increase of its visible peaks (trace b to c) at 527 and 556 nm (49). After 3 min, the visible peaks are diminished (trace d) and a new peak appears at 540 nm (trace e) characteristic of the inactivated enzyme. Fig. 4D shows the effect of varying concentrations of H₂O₂ on the formation of the inactive enzyme at 421 nm. Addition of single equivalent of H₂O₂ to native HRP (trace a) does not cause the formation of the inactive enzyme (trace b). However, a gradual increase in H₂O₂ concentration causes a gradual decrease in the Soret peak at 402 nm with the increase in 421 nm peak for the inactive enzyme indicating its dependence on H₂O₂ concentration at a fixed concentration of SCN. As most of the studies were carried out with an excess of H₂O₂, the results may be interpreted as the observation under steady-state conditions at the particular time.

Spectral changes in HRP-catalyzed SCN oxidation by H₂O₂.

Spectral Evidence that Inactivation Proceeds through One-electron Oxidation of SCN

To study the mechanism of SCN induced inactivation of HRP in the presence of H₂O₂, spectral studies were carried out in the presence of the spin trap, DMPO. When SCN was added to the reaction mixture containing HRP and H₂O₂ in the presence of DMPO, it immediately reduced compound II to the native state absorbing at 402 nm (Fig. 5A). Only DMPO is ineffective in reducing compound II under this condition. Thus, in the presence of DMPO, the stable Soret and visible bands at 421 and 540 nm, respectively, for the inactivated enzyme, did not appear. This indicates that SCN is oxidized by one-electron oxidation to the thiocyanate radical and that either this radical or a product derived from it is involved in the inactivation. When the radical is scavenged by DMPO, the enzyme is protected and the catalytic cycle continues. In contrast, LPO compound II absorbing at 431 nm is immediately reduced by SCN to form native LPO absorbing at 412 nm (Fig. 5B) instead of formation of any stable complex. The inactive HRP which absorbs at 421 nm (Fig. 5C) when passed through a Sephadex G-25 column is converted back to the native active enzyme absorbing at 402 nm, indicating the reversible nature of the inactivation.

Protection of HRP by DMPO against inactivation by SCN-H₂O₂ system (A), spectral evidence for the oxidation of SCN by LPO compound II (B), and spectral evidence for the reversibility of the inactivated HRP (C).

Protection of HRP against SCN Inactivation by Various Electron Donors

Since iodide is optimally oxidized at a pH of about 4, where SCN is oxidized, the effect of iodide on the formation of the inactive enzyme was studied spectrally. Table I shows that in the presence of I, SCN cannot inactivate the enzyme as evidenced by the absence of 421- and 540-nm peaks for the inactive enzyme. Instead, the enzyme remains in the native state with peaks at 402, 500, and 650 nm. The enzyme is also protected by aromatic electron donors such as guaiacol and other natural substrates such as ascorbate (6). Indoleacetic acid is, however, ineffective and causes its conversion to compound III with a visible peak at 670 nm (49). Protection studies could not be done kinetically, as the colored oxidation products of iodide and guaiacol during preincubation interfere with the final enzyme assay.

Table I. Spectral evidence for the protection of HRP by electron donors against inactivation by SCN-H2O2 system Electron donors were added before the addition of H2O2 (15 µM) in the preincubation system containing HRP (1.5 µM) and SCN (25 µM) in 50 mM sodium acetate buffer, pH 4.5, in a final volume of 1 ml. In each case the spectrum of the preincubated mixture was taken 3 min after the addition of H2O2. The concentration of the electron donor used was 1 µM guaiacol, or 10 µM iodide, 50 µM ascorbate, or 50 µM indoleacetic acid. Incubation conditions Soret peak Visible peak nm nm HRP 402 500, 650  HRP + SCN 402 500, 650  HRP + H2O2 418 527, 556  HRP + SCN + H2O2 421 540 HRP + I + SCN + H2O2 402 500, 650  HRP + guaiacol + SCN + H2O2 402 500, 650  HRP + ascorbate + SCN + H2O2 402 500, 650  HRP + IAA + SCN 410 555, 670

Binding of SCN by Optical Difference Spectroscopy in the Presence or Absence of Iodide or Guaiacol

The binding of SCN gives a characteristic difference spectrum of HRP-SCN complex versus HRP, having a maximum at 416 nm and a minimum at 395 nm (Fig. 6A). The equilibrium dissociation constant, K_d, for the HRP-SCN complex as calculated from the plot of 1/A versus 1/[SCN] was 125 mM. SCN binding was also studied in the presence of varying concentrations of iodide. The plot (Fig. 6B) indicates that iodide competitively inhibits SCN binding. The inset shows the plot of K_{d obs} of SCN as a function of iodide concentration. Using Equation 6, the K_d of iodide at this site was calculated to be 110 mM. Similarly, guaiacol also competitively inhibits SCN binding (Fig. 6C) with the K_d value of 16 mM at or near this site (Fig. 6C, inset).

SCN binding to HRP by optical difference spectroscopy.

Formation of Thiocyanate Radical in HRP-catalyzed SCN Oxidation

From the kinetic and spectral studies, it is evident that DMPO or PBN prevents inactivation of HRP during SCN oxidation, suggesting involvement of a free radical species in the inactivation process. Fig. 7A shows the ESR spectrum obtained when HRP was incubated with SCN and H₂O₂ in the presence of DMPO as a spin trap. The ESR spectrum was due to the formation of spin-trapped sulfur-centered thiocyanate radical with the hyperfine splitting constants of a^N = 15.0 G and a^H = 16.5 G, which are consistent with the splitting constants of the known DMPO-sulfur centered radicals (50, 51). In the absence of H₂O₂ (Fig. 6B) or HRP (Fig. 6C), no significant ESR signal was detected. The signal characteristic of O₂ or OH^· (52) was not observed. This result indicates the formation of sulfur-centered thiocyanate radical through one-electron oxidation of SCN by the catalytic intermediates of HRP.

ESR spectra of DMPO-thiocyanate radical adduct in the HRP-H₂O₂-SCN system.

Identification of the Inactivating Species during SCN Oxidation

The stable oxidation product of SCN as recovered from the reaction mixture after filtration when mixed with the native HRP, native LPO, or ferrous LPO shifts the Soret peak of the enzyme to 421, 430, and 434 nm, respectively, with the corresponding visible peak at 540, 555, and 570 nm (Table II). These are identical to the peaks obtained by the addition of CN to the corresponding enzyme preparations. The spectrum obtained after addition of filtrate or CN to HRP is also exactly similar to the spectrum (Fig. 4B) obtained during inactivation of HRP in the presence of SCN and H₂O₂.

Table II. Optical spectra of HRP and LPO in presence of the oxidation product of HRP-SCN-H2O2 system Preparation of the filtrate from HRP-SCN1-H2O2 system and the addition of enzyme to it for measurement of optical spectrum were described under "Materials and Methods." For obtaining the cyanide spectrum, 10 or 100 µM KCN was added to 1 µM HRP or LPO, respectively. Native LPO was reduced to ferrous form by the addition of a few crystals of sodium dithionite. Incubation conditions Soret peak Visible peak nm nm HRP 402 500, 650  HRP + filtrate 421 540 HRP + CN 421 540 LPO 412 500, 640  LPO + filtrate 430 555 LPO + CN 430 555 LPO (ferrous) + filtrate 434 570 LPO (ferrous) + CN 434 570

Quantitation of CN production during SCN oxidation by the HRP or LPO System

Table III shows that CN production in the HRP-H₂O₂-SCN system is 98 ± 10 µM, which is significantly inhibited by PBN, indicating its generation from thiocyanate radical. In contrast, LPO produces only 10 ± 5 µM CN, which is 10-fold lower than the HRP system.

Table III. Quantitative estimation of CN in HRP or LPO-catalyzed SCN oxidation system HRP (0.5 µM) or LPO (0.01 µM) was incubated with 1 mM SCN and 0.6 mM H2O2 for 30 min at their optimum pH- of 4.5 or 5.6, respectively, using 50 mM acetate buffer. CN concentration in the reaction mixture was measured as described under "Materials and Methods." Incubation conditions CN production µM HRP + SCN 0 HRP + SCN + H2O2 98  ± 10 HRP + SCN + PBN + H2O2 15  ± 5 LPO + SCN 0 LPO + SCN + H2O2 10  ± 5

DISCUSSION

The results of this study indicate the following. (a) HRP-catalyzed SCN oxidation occurs at a significantly lower rate than LPO due to concurrent inactivation. (b) HRP catalyzes SCN oxidation through a one-electron transfer mechanism forming sulfur-centered thiocyanate radicals which finally give rise to an inactivating species. (c) The inactivating species has been identified as CN. (d) CN production is 10-fold higher in the HRP-H₂O₂-SCN system than LPO. (e) SCN oxidation by HRP is under the major constraint of product inhibition by CN, which is not observed in LPO.

The catalytic turnover for SCN oxidation by HRP is a hundredfold lower than that of LPO. Although the electrochemical potential of LPO compound I is much higher than HRP compound I (10) and LPO has a higher affinity for binding of SCN compared with HRP (9), the third factor which controls the oxidation of SCN is the mode of oxidation on which the nature (inactivating or not) of the product formation is dependent. LPO catalyzes SCN oxidation by a two-electron transfer mechanism (37) leading to the formation of the stable oxidation product, OSCN (23, 28, 35, 37), leaving very little possibility for the generation of CN unless the concentration ratio of H₂O₂ and SCN exceeds 1 (28). Our spectral studies indicate that HRP catalyzes SCN oxidation through a one-electron transfer mechanism with the formation of sulfur-centered thiocyanate radical which is detected by ESR spectroscopy. Thiocyanate radicals may dimerize to form thiocyanogen, (SCN)₂, which, being highly unstable in aqueous solution in the pH range of 5-8, is hydrolyzed to give rise to HOSCN (32, 34). As HOSCN has a pK_a value of 5.3 (35), at pH above 5.3, OSCN is the major stable oxidation product. Recently, Modi et al. (28) have shown that LPO catalyzes SCN oxidation at pH 6.1 to produce HOSCN and OSCN. However, as HRP-catalyzed SCN oxidation occurs optimally at pH 4.0, (SCN)₂ is the predominant oxidation species (9). (SCN)₂ is hydrolyzed to yield CN (32, 34) without the formation of HOSCN (9). The entire sequence of HRP-catalyzed SCN oxidation may thus be represented as follows.

(Eq. 7)

(Eq. 8)

(Eq. 9)

(Eq. 10)

(Eq. 11)

Equation 10 is the well known coupling reaction for the formation of stable oxidation product from the free radicals by dimerization (3, 53), and (SCN)₂ has been shown to be formed in HRP-catalyzed SCN oxidation (9). Equation 11 is consistent with the reaction shown for the hydrolysis of (SCN)₂ and may occur through the formation of some intermediates (42, 54). From the reaction sequences, thiocyanate radical, (SCN)₂, SO₄², or CN may be considered for the plausible inactivating species for HRP. Our studies indicate that HRP-catalyzed SCN oxidation is increased severalfold in the presence of the free radical traps DMPO or PBN. This indicates that either thiocyanate radical or the radical-derived product is responsible for the inactivation. As the inactivation is reversible, it is unlikely that thiocyanate radical inactivates the enzyme through covalent interaction at or near the active site similar to the suicidal substrates (55, 56). Since (SCN)₂ is unstable at pH 4.5 due to hydrolysis (32, 34, 54), the role of this compound in inactivation is excluded. As sulfate has no significant effect on the catalytic activity of HRP, the only stable reactive product present in the system is CN, which can inactivate peroxidases by reversible interaction with the heme iron (49). Our spectral studies clearly indicate the formation of HRP-CN complex in the presence of SCN and H₂O₂ as evidenced by the Soret peak at 421 nm and visible peak at 540 nm (49). However, due to remarkable similarity of the spectrum of compound II and CN complex (both low spin) at the Soret region, one cannot really distinguish between the two (9) unless their visible spectra are observed, where absorption at 540 nm is convincing evidence for the formation of the enzyme-CN complex (49). More convincing evidence for CN production comes from the observation that the enzyme-free reaction mixture when added to native HRP or LPO yields HRP-CN or LPO-CN complex having characteristic absorption maxima (49, 57). Moreover, quantitative measurement demonstrates that CN is the major reactive product in the HRP system as compared with LPO. Our kinetic and spectral studies as well as measurement of CN production indicate that in the presence of free radical trap, the enzyme remains in the highly active state because of the absence of CN production. Thus, for CN production, HRP must oxidize SCN by one-electron transfer to generate thiocyanate radical as intermediate which, when scavenged by the radical trap, relieves inhibition. LPO cannot generate sufficient CN for inactivation, as it catalyzes SCN oxidation by a single two-electron transfer (37) to form stable OSCN as the major oxidation product (9, 23, 28, 54).

It is intriguing as to why HRP and LPO catalyze SCN oxidation by two different mechanisms leading to two different oxidation products. Modi et al. (9) have suggested that this might be due to a different binding site of SCN in the heme distal pocket. HRP binds SCN at a site close to the heme peripheral C1H₃ and C18H₃ groups, having a pK_a of 4.0 (38), which might favor one-electron transfer, whereas the binding of SCN to LPO is facilitated by protonation of a group at pK_a 6.1, presumably contributed by the distal histidine which might favor two-electron transfer via the imidazole ring (36). However, further studies are required to substantiate it. Moreover, LPO binds CN with a K_d of 60 µM (57), which is much higher than the concentration of CN (10 µM) formed in the reaction mixture. In contrast, HRP binds CN with very high affinity of K_d of 2.3 µM (58), which is much lower than the concentration of CN (98 µM) formed in the system, making it more susceptible to inactivation by CN. However, the difference in the mode of oxidation of SCN by two different peroxidases appears to be the fundamental mechanism for the differential sensitivity to CN. The mechanism of SCN-induced inactivation of HRP is shown in Scheme 1. The essential feature of the scheme is the one-electron oxidation of SCN to thiocyanate radicals. This is unlike iodide, which is oxidized by a direct two-electron transfer to compound I (7, 8) but is similar to the one-electron oxidation of thiol, bisulfite, and nitrite (2, 59-61). The hyperfine splitting constants of thiocyanate radicals are comparable to the sulfur-centered thiol and bisulfite radicals formed in the HRP system (50, 51, 62-64), indicating that the radical is centered on the sulfur atom of thiocyanate. Also, the stoichiometry indicates that 1 mol of CN should be formed from the oxidation of 6 mol of SCN of which 5 mol are regenerated with the consumption of 3 mol of H₂O₂. In other words, 3 mol of H₂O₂ should be consumed with the net oxidation of 1 mol of SCN, which is evident from Fig. 3, and three catalytic cycles are thus required for the production of 1 mol of CN. Thus, the formation of HRP-CN complex will mainly depend on the H₂O₂ concentration at fixed enzyme and SCN concentrations, which is evident from the kinetic and spectral studies. As 2 × 10⁴ turnovers are required for complete inactivation of the enzyme (Fig. 2B), 40 nmol of H₂O₂ will be consumed by 2 pmol of HRP/ml of the reaction mixture with the formation of 13.3 nmol of CN. Thus, 13.3 µM CN could be formed in the system, which is compatible with the dissociation constant of the enzyme-CN complex formation (K_d = 2.3 µM) (58) for inactivation. This somewhat higher concentration of CN (13.3 µM) over the K_d value might be explained as due to its competition with the H₂O₂ for reaction with the heme iron.

Proposed mechanism for the inactivation of HRP by SCN and H₂O₂

Peroxidases are abundant in animal systems as well as in plants (65), which also contain SCN (66). It is evident from this study that CN produced from the oxidation of SCN by HRP blocks the peroxidative activity and may thus affect plant physiology. However, the enzyme is protected against inactivation by iodide or the aromatic electron donor guaiacol. Although iodide is present in traces, various aromatic electron donors, including phenolic compounds, are rich in plants. It is thus highly probable that the phenolic compounds protect the enzyme against SCN-induced inactivation. We have shown that iodide protects the enzyme by competing with SCN for binding at the same site. This is consistent with the earlier findings that both iodide and SCN bind to HRP at the same site (13, 38). However, inactivation of the enzyme is also prevented by guaiacol, which also competes with SCN for binding at the same site or very close to it, as shown by our competitive binding studies. Although earlier studies indicate that aromatic donors may bind near the heme methyl C18H₃ group (12, 18), which is away from the iodide or SCN binding site (12), our competitive binding data indicate that these sites are very close to each other, if not the same. Recently, we have shown that an active site arginine residue plays an obligatory role in aromatic donor binding (22) and mutant studies (21) have established that arginine-38 controls the binding of the aromatic donor in addition to its role in compound I formation. Since the positively charged arginine residue may also interact with the negatively charged substrates or cofactors (67), it is probable that the same arginine residue also controls SCN binding, and in that case the competition of guaiacol with SCN for binding at the same site is comprehensible. From the competitive binding studies it is, however, clear that the ratio of the concentration of the aromatic donors to SCN is the determining factor for the normal functioning of the peroxidase in plant physiology. Although indoleacetic acid is the endogenous substrate of HRP (5), it cannot protect the enzyme because of the formation of compound III (5). Recently, ascorbate has been suggested to be the physiological substrate of the plant peroxidases (6), and it can completely protect HRP against SCN-induced inactivation by CN. It is also possible that iodide, guaiacol, and ascorbate, being better substrates (high turnover) than SCN, can consume H₂O₂ at a very high rate and thereby limiting the production of CN. However, ascorbate might play an important role in keeping the enzyme in a fully active state in the presence of SCN and thus helps in the decomposition of cellular H₂O₂, especially in the acid compartments such as vacuoles and apoplastic space (68).