(Received for publication, August 17, 1996, and in revised form, January 19, 1997)
From the Department of Physiology, Indian Institute of Chemical Biology, Calcutta 700 032, India
The catalytic turnover of horseradish
peroxidase (HRP) to oxidize SCN is a hundredfold
lower than that of lactoperoxidase (LPO) at optimum pH. While studying
the mechanism, HRP was found to be reversibly inactivated following
pseudo-first order kinetics with a second order rate constant of 400 M
1 min
1 when incubated with
SCN
and H2O2. The slow rate of
SCN
oxidation is increased severalfold in the presence of
free radical traps, 5-5-dimethyl-1-pyrroline N-oxide or
-phenyl-tert-butylnitrone, suggesting the plausible role
of free radical or radical-derived product in the inactivation.
Spectral studies indicate that SCN
at a lower
concentrations slowly reduces compound II to native state by
one-electron transfer as evidenced by a time-dependent spectral shift from 418 to 402 nm through an isosbestic point at 408 nm. In the presence of higher concentrations of SCN
, a
new stable Soret peak appears at 421 nm with a visible peak at 540 nm,
which are the characteristics of the inactivated enzyme. The
one-electron oxidation product of SCN
was identified by
electron spin resonance spectroscopy as 5-5-dimethyl-1-pyrroline N-oxide adduct of the sulfur-centered thiocyanate radical
(aN = 15.0 G and a
H = 16.5 G). The inactivation of the enzyme in the presence of SCN
and H2O2 is prevented by
electron donors such as iodide or guaiacol. Binding studies indicate
that both iodide and guaiacol compete with SCN
for
binding at or near the SCN
binding site and thus prevent
inactivation. The spectral characteristics of the inactivated enzyme
are exactly similar to those of the native HRP-CN
complex. Quantitative measurements indicate that HRP produces a 10-fold
higher amount of CN
than LPO when incubated with
SCN
and H2O2. As HRP has higher
affinity for CN
than LPO, it is concurrently inactivated
by CN
formed during SCN
oxidation, which is
not observed in case of LPO. This study further reveals that HRP
catalyzes SCN
oxidation by two one-electron transfers
with the intermediate formation of thiocyanate radicals. The radicals
dimerize to form thiocyanogen, (SCN)2, which is hydrolyzed
to form CN
. As LPO forms OSCN
as the major
stable oxidation product through a two-electron transfer mechanism, it
is not significantly inactivated by CN
formed in a small
quantity.
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) |
![]() |
(Eq. 4) |
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 cm
1
mM
1 (40) and
412 = 112 cm
1 mM
1 (41), respectively.
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.
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.
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.
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.
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
(Kd) for the complex formation was calculated
from
![]() |
(Eq. 5) |
![]() |
(Eq. 6) |
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.
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. Kobs 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 M
1 min
1. 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.
Effect of Spin Trap on the Catalytic Activity of HRP on SCN
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.
Spectral Evidence for SCN
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 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 against SCN
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.
|
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, Kd, 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
Kd obs of SCN
as a
function of iodide concentration. Using Equation 6, the Kd of iodide at this site was calculated to be 110 mM. Similarly, guaiacol also competitively inhibits
SCN
binding (Fig. 6C) with the
Kd value of 16 mM at or near this site
(Fig. 6C, inset).
Formation of Thiocyanate Radical in HRP-catalyzed SCN
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 aN = 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 O
2 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.
Identification of the Inactivating Species during SCN
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
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.
|
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 pKa
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) |
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
pKa 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 pKa 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 Kd 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 Kd
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 (Kd = 2.3 µM) (58) for
inactivation. This somewhat higher concentration of CN
(13.3 µM) over the Kd value might be
explained as due to its competition with the
H2O2 for reaction with the heme iron.
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).