THE JOURNAL OF BIOLOGICAL CHEMISTRY
26129 26136, 2005
2005 by The American Society for Biochemistry and Molecular Biology, Inc.
Thiocyanate Modulates the Catalytic Activity of
Mammalian Peroxidases*
Received for publication, March 18, 2005, and in revised form, May 11, 2005
Published, JBC Papers in Press, May 13, 2005, DOI 10.1074/jbc.M503027200
Yahya R. Tahboub, Semira Galijasevic, Michael P. Diamond, and Husam M. Abu-Soud
From the Department of Obstetrics and Gynecology, The C. S. Mott Center for Human Growth and Development and the
Department of Biochemistry and Molecular Biology, Wayne State University, Detroit, Michigan 48201
We investigated the potential role of the co-substrate,
thiocyanate (SCN), in modulating the catalytic activity
of myeloperoxidase (MPO) and other members of the
mammalian peroxidase superfamily (lactoperoxidase
(LPO) and eosinophil peroxidase (EPO)). Pre-incubation
of SCN with MPO generates a more complex biological
setting, because SCN serves as either a substrate or
inhibitor, causing diverse impacts on the MPO heme
iron microenvironment. Consistent with this hypothe-
sis, the relationship between the association rate con-
stant of nitric oxide binding to MPO-Fe(III) as a function
of SCN concentration is bell-shaped, with a trough
comparable with normal SCN plasma levels. Rapid ki-
netic measurements indicate that MPO, EPO, and LPO
Compound I formation occur at rates slower than com-
plex decay, and its formation serves to simultaneously
catalyze SCN via 1e and 2e oxidation pathways. For
the three enzymes, Compound II formation is a funda-
mental feature of catalysis and allows the enzymes to
operate at a fraction of their possible maximum activi-
ties. MPO and EPO Compound II is relatively stable and
decays gradually within minutes to ground state upon
H2O2 exhaustion. In contrast, LPO Compound II is un-
stable and decays within seconds to ground state, sug-
gesting that SCN may serve as a substrate for Com-
pound II. Compound II formation can be partially or
completely prevented by increasing SCN concentra-
tion, depending on the experimental conditions. Collec-
tively, these results illustrate for the first time the po-
tential mechanistic differences of these three enzymes.
A modified kinetic model, which incorporates our cur-
rent findings with the mammalian peroxidases classic
cycle, is presented.
Myeloperoxidase (MPO)1 and other members of the mamma-
lian peroxidase superfamily (eosinophil peroxidase (EPO) and
lactoperoxidase (LPO)) display a crucial difference (within a
wide range of biological processes) in their unique ability in
catalyzing the H2O2-dependent peroxidation of halides and
pseudohalides to produce antimicrobial agents and hypohalous
acids (17). These heme-containing enzymes share 50 70%
* This work was supported by National Institutes of Health Grant
HL066367 to (H. M. A.-S.). The costs of publication of this article were
defrayed in part by the payment of page charges. This article must
therefore be hereby marked advertisement in accordance with 18
U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed: Dept. of Obstetrics
and Gynecology, C. S. Mott Center for Human Growth and Develop-
ment, Wayne State University, 275 E. Hancock, Detroit, MI 48201. Tel.:
313-577-6178; Fax: 313-577-8554; E-mail: habusoud@med.wayne.edu.
1
The abbreviations used are: MPO, myeloperoxidase; EPO, eosino-
phil peroxidase; LPO, lactoperoxidase; E, enzyme (MPO, EPO, or LPO);
H2O2, hydrogen peroxide; NO, nitric oxide (nitrogen monoxide).
This paper is available on line at http://www.jbc.org
Vol. 280, No. 28, Issue of July 15, pp.
Printed in U.S.A.
overall amino acid sequence homology but differ from each
other with respect to their sites of expression, primary se-
quences, and substrate specificities (1, 2, 8 10). MPO is a
150 165-kDa molecule synthesized during myeloid differenti-
ation that constitutes the major component of the neutrophil
azurophilic granules (11, 12). The enzyme is a homodimer
comprising of a pair of light and heavy chains derived from a
single gene product (12, 13) with its subunits joined by a single
disulfide bridge (12). The heavy chain contains an iron bound
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by a novel protoporphyrin IX derivative, which is covalently
linked to the heavy chain polypeptide (14 16). EPO is a mono-
meric molecule comprised of a light chain and a heavy chain
with molecular masses of 15.5 and 50 kDa, respectively (17).
This enzyme is stored in eosinophil granules and catalyzes the
formation of antimicrobial species from the oxidation of Br
and SCN (1719). LPO is a monomeric single polypeptide
chain with a molecular mass of 78.5 kDa (20 23). LPO has
been identified as an antimicrobial agent within exocrine gland
secretions such as milk, saliva, and tears through the oxidation
of thiocyanate by H2O2 to yield the intermediary oxidation
product hypothiocyanite (OSCN) (24, 25).
The properties of the heme in MPO, EPO, and LPO have
been characterized by a wide variety of spectroscopic tech-
niques, including optical absorption, stopped-flow, electron
paramagnetic resonance, and resonance Raman spectroscopy
(26 37). These techniques, with a combination of structure
analysis (16, 38 39) and advanced computer modeling (40),
have shown that the heme pockets of the mammalian peroxi-
dases are envisioned to form the catalytic site where the step-
wise reduction of H2O2 takes place. The simplified mechanism
that governs the catalytic activity of mammalian peroxidase
superfamily can be represented by the classic peroxidases cat-
alytic cycle. In this cycle, represented here by Equations 1 4,
E-Fe(III) H2O2 3 E-Fe(IV)O H2O
(Eq. 1)
Compound I
E-Fe(IV)O SCN H 3 E-Fe(III) HOSCN (Eq. 2)
E-Fe(IV)O AH2 3 E-Fe(IV)O AH
Compound II (Eq. 3)
E-Fe(IV)O AH2 3 E-Fe(III) AH (Eq. 4)
H2O2 reacts rapidly and reversibly with the ground state (E-
Fe(III) state) of mammalian peroxidases and generates a ferryl
cation radical (E-Fe(IV)O) intermediate named Com-
pound I (Equation 1) (33, 41). Compound I is capable of oxidiz-
ing either halides and pseudohalides through a 2e transition,
generating the ground state and the corresponding hypohalous
acid (Equation 2) or oxidizing multiple organic and inorganic
molecules (AH2) by two successive sequential 1e transitions
generating their corresponding cation (AH) and the peroxidase
intermediates Compound II (E-Fe(IV)O) and E-Fe(III), re-
26129
26130 Differences in the Mechanisms of Mammalian Peroxidases
spectively (Equations 3 and 4) (33, 41 43). Compound II is a
longer lived intermediate whose decay to ground state is con-
sidered to be the rate-limiting step during steady-state cataly-
sis (33, 44). Acceleration in Compound II formation and decay
has been noted with a series of organic and inorganic sub-
strates (28, 30, 33, 41 43) and physiological reductants like
nitric oxide (NO) and superoxide (O2. ) (28, 30, 33, 41 43).
Thiocyanate binds close enough to the heme iron of peroxi-
dase to influence both its reduction potential and binding af-
finity to small molecules, such as NO and CO, when these
molecules bind to the enzymes heme center (16, 28 30, 38, 39).
Recent x-ray crystal structure analysis and a variety of spec-
troscopic measurements have shown that SCN binding occurs
at distal heme cavity and serves as either a substrate or inhib-
itor, causing a diverse impact on the catalytic characteristic
behavior of mammalian peroxidases and function (16, 28 30,
38, 39). In some cases, these co-substrates bind to the peroxi-
dase heme iron and form the corresponding low spin, six-
coordinate complex that inhibits the catalytic activity of the
enzyme (45).
Despite the potential significance of mammalian peroxidases
to both human health and disease, little is known about the
factors that govern their substrate selectivity and specificity.
Previous kinetic studies with mammalian peroxidases have
been limited in scope by focusing primarily on the direct reac-
tion of SCN with Compound I (36, 37, 44, 46, 47). In the
present studies, we utilize a combination of optical absorbance,
rapid kinetic measurements, and NO binding studies to assess
the distinct conformational changes that occur upon SCN
binding and its influence on the catalytic activity of MPO and
other members of the mammalian peroxidase superfamily. Our
results indicate that SCN binding regulates the catalytic ac-
tivity of MPO, EPO, and LPO, which appears to result from
significant electronic and/or conformational alterations in the
catalytic sites of these three enzymes that are caused by SCN.
EXPERIMENTAL PROCEDURES
MaterialsNO gas was purchased from Matheson Gas Products, Inc.
and used without further purification. For each experiment, a fresh
saturated stock of NO was prepared under anaerobic conditions. The
extent of nitrite/nitrate (NO2/NO3) build-up in NO preparations over
the time course used for the present studies was 11.5% (per mole of
NO), as determined by anion exchange high performance liquid chro-
matography under anaerobic conditions (48). All other reagents and
materials were of the highest purity grades available and obtained from
Sigma or the indicated source.
Enzyme PurificationMPO was purified from detergent extracts of
human leukocytes as described (49). Trace levels of contaminating
eosinophil peroxidase were then removed by passage over a sulfopropyl-
Sephadex column (50). The purity of isolated MPO was established by
demonstrating a Reinheitszahl (RZ) value of 0.85 (A430/A280) by
means of SDS-PAGE analysis with Coomassie Blue staining and in-gel
tetramethylbenzidine peroxidase staining to confirm no observable con-
taminating eosinophil peroxidase activity (51). EPO was purified from
porcine whole blood as described previously (52). The purity of EPO was
confirmed by demonstrating an RZ of 0.9 (A415/A280) via SDS-PAGE
analysis with Coomassie Blue staining and in-gel tetramethylbenzidine
peroxidase staining (53). Enzyme concentrations were determined spec-
trophotometrically utilizing extinction coefficients of 89,000 and
112,000 M1 cm1 per heme of MPO (51) and EPO (54, 55), respectively.
LPO was obtained from Worthington Biochemical Corporation (Lake-
wood, NJ) and used without further purification. Purity was confirmed
by demonstrating an RZ of 0.75 (A412/A280) by means of SDS-PAGE
analysis with Coomassie Blue staining.
Stopped-flow and Absorbance MeasurementsRapid kinetic meas-
urements were performed using a dual syringe stopped-flow instrument
obtained from Hi-Tech Ltd. (model SF-61). Measurements were carried
out at 10 C following rapid mixing of equal volumes of an H2O2-
containing buffer solution and a peroxidase solution. MPO Compound I
and Compound II formation and decay to ground state were monitored
at 435 and 455 nm, respectively. EPO and LPO Compound I and
Compound II formation and decay to ground state were monitored at
412 and 432 nm, respectively. For NO binding to MPO, measurements
were carried out by rapidly mixing equal volumes of the enzyme solu-
tions (0.86 M) with buffer solution supplemented with increasing con-
centrations of NO. The reactions for NO binding to MPO-Fe(III) were
monitored under anaerobic conditions at wavelengths determined
based on the spectral changes that occur upon NO binding to enzyme,
as indicated. The time course of absorbance change was fit to either
single (Y 1 ekt) or double exponential (Y Aek1t Bek2t)
functions as indicated. Signal-to-noise ratios for all kinetic analyses
were improved by averaging at least 6 8 individual traces. In some
experiments the stopped-flow instrument was attached to a rapid scan-
ning diode array device (Hi-Tech) designed to collect a multiple number
of complete spectra (200 800 nm) at specific time ranges. The detector
was automatically calibrated relative to a holmium oxide filter, as it has
spectral peaks at 360.8, 418.5, 446.0, 453.4, 460.4, 536.4, and 637.5 nm,
which were used by the software to correctly align pixel positions with
wavelength. Rapid scanning stopped-flow experiments involved mixing
solutions of MPO (3 M) with buffer solutions containing 40 M H2O2 in
the absence and presence of increasing SCN concentrations at 10 C.
SpectroscopyOptical spectra were recorded on a Cary 100 Bio UV-
visible spectrophotometer at 25 C. Anaerobic spectra were recorded
using septum-sealed quartz cuvettes that could be attached through a
quick-fit joint to an all-glass vacuum system. MPO samples were made
anaerobic by several cycles of evacuation and equilibrated with cata-
lyst-deoxygenated N2. Separate buffer solutions were evacuated, gassed
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with N2, and anaerobically transferred either to the stopped-flow in-
strument or to anaerobic cuvettes using gas-tight syringes. Cuvettes
were maintained under N2 or NO positive pressure during spectral
measurements.
Solution PreparationA fresh saturated stock of NO was prepared
under anaerobic conditions. Anaerobic 0.2 M sodium phosphate buffer
solutions, pH 7.0, containing various concentrations of NO were pre-
pared by mixing different volumes of buffer saturated with NO gas at
21 C with an anaerobic buffer solution. A saturating concentration of
NO at 21 C is 2 mM.
H2O2-selective Electrode MeasurementsH2O2 measurements were
carried out using a H2O2-selective electrode (Apollo 4000 free radical
analyzer; World Precision Instruments, Sarasota, FL). Experiments
were performed at 25 C by immersing the electrode in 10 ml of 0.2 M
sodium phosphate buffer, pH 7.0, under air. 20 M H2O2 was added to
a continuously stirred buffer solution during which the rise and fall in
H2O2 concentration was continuously monitored. Where indicated, 50
l of MPO (or EPO or LPO) (200 nM final) was added to the reaction
mixture. To determine the effect of SCN on H2O2 consumption by
mammalian peroxidases, similar experiments were repeated by adding
50 l of the enzyme solution (200 nM final) pre-incubated with 40 M
SCN to the reaction mixture.
RESULTS
Effects of SCN on NO Binding to MPO Heme IronTo
assess the influence of SCN on the catalytic activity of MPO,
the rates of NO binding to the Fe(III) form of MPO have been
determined in the presence of increasing SCN concentrations.
Investigations were carried out under two different circum-
stances, either a fixed amount of NO and varying concentra-
tions of SCN or a fixed amount of SCN and varying levels of
NO. As shown in Fig. 1, the plots of the apparent rate constants
for NO binding as a function of NO concentration were linear,
consistent with a simple one-step mechanism. As shown in
Equation 5,
kon
SCN-MPO-Fe(III) NO
O SCN-MPO-Fe(III)-NO (Eq. 5)
koff
the positive intercepts confirm that NO binds to MPO-Fe(III)
by a reversible process. These kinetic parameters suggest that
SCN modulates the affinity of MPO-Fe(III) toward NO. The
second-order combination rate constants (kon) calculated from
the slopes were plotted as a function of SCN concentration
and showed a bell-shaped relationship (Fig. 2), with a mini-
mum centered at biologically relevant levels of SCN (60 M).
Collectively, these results indicate that SCN binds within the
enzyme system and modulates the affinity of MPO-Fe(III)
toward NO.
 Differences in the Mechanisms of Mammalian Peroxidases
FIG. 1. SCN modulates NO binding to MPO heme iron. Plots of
the observed rates of NO binding to MPO-Fe(III) as a function of NO
and SCN concentrations. An anaerobic solution containing 0.86 M
MPO-Fe(III) supplemented with varying concentrations of SCN was
rapidly mixed with an equal volume of sodium phosphate buffer (200
mM at pH 7.0) supplemented with varying concentration of NO at 10 C.
The high concentration of the phosphate buffer is to keep the pH of the
solution unaltered after the addition of NO. The observed rates of
MPO-Fe(III)-NO were plotted as a function of NO concentration.
FIG. 2. Relationship between the second-order combination
rate (kon) of NO binding to MPO-Fe(III) obtained from Fig. 1 as
a function of the SCN concentration used.
Formation of MPO, EPO, and LPO Compound II during the
Metabolism of SCNWe next utilized diode array spectropho-
tometry to study the effect of SCN on MPO, EPO, and LPO
intermediate formation, duration, and decay, as these events
occur during steady-state catalysis. Investigation was carried
out by the rapid mixing of a buffer solution supplemented with
1.2 M MPO (or EPO or LPO) and 40 M SCN against an equal
volume of a buffer solution supplemented with 40 M H2O2, at
10 C. Spectral analysis indicated that the majority of MPO-
Fe(III) (80 90%) converted immediately to an MPO species
whose Soret absorbance peak, along with the visible bands, is
identical to that reported for MPO Compound II. This species
was stable and occurs immediately after initiating the reaction
without any indication of a prior accumulation of MPO Com-
pound I. Increasing SCN concentrations decreased the
amount of the MPO Compound II accumulation up to 50%, as
judged by the decrease in the amplitude of the Soret absorb-
ance peak at 455 nm. Similar results were obtained for EPO.
Indeed, the majority of EPO-Fe(III) converted immediately to a
stable EPO Compound II without any sign of the accumulation
of Compound I. Fig. 3 shows spectral traces collected at 0.027,
0.054, 0.081, 0.110, and 0.135 s after initiating the reaction.
Similarly, Compound II LPO accumulation during catalysis
occurs without prior accumulation of LPO Compound I, but to
a lesser extent, and decays immediately to ground state upon
H2O2 exhaustion. Fig. 4, panel A shows spectra collected at
0.035, 0.07, 0.105, 0.14, 0.175, and 0.244 s after initiating the
reaction, whereas panel B shows spectra collected at 0.45, 0.75,
0.90, 1.05, and 2.20 s after initiating the reaction. However,
when the similar reaction was repeated in the presence of 200
M SCN, ground state LPO-Fe(III) predominated with a small
a portion of Compound II (35% of the total LPO); its decay to
ground state occurred immediately after H2O2 exhaustion
(data not shown). Collectively, our results indicate for the first
26131
FIG. 3. Formation of EPO Compound II during steady-state
catalysis of SCN. Depicted is the absorbance change over time for the
reaction that was initiated by rapidly mixing a buffer solution contain-
ing 40 M H2O2 with an equal volume of buffer solution containing 1.2
M EPO and 40 M SCN at 10 C. Spectra traces were recorded at
0.027, 0.054, 0.081, 0.110, and 0.135 s after initiating the reaction.
Arrows in the panels indicate the direction of spectral change over time.
Experiments were carried out under anaerobic conditions in sodium
phosphate buffer (200 mM at pH 7.0).
time that Compound II is a major component in the catalytic
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activity of MPO, EPO, and LPO and that its formation allow
these three enzymes to operate at only a fraction of their
maximum activity.
Effect of SCN on the Formation, Duration, and Decay of
MPO and EPO Compound II Formation during Steady-state
CatalysisTo take a closer look at the reaction mechanism of
the metabolism of SCN by MPO, we next investigated the
influence of SCN on the kinetics of MPO compound II buildup,
duration, and decay using single wavelength stopped-flow
methods. Experiments were carried out under aerobic condi-
tions following rapid mixing of H2O2 (40 M) and enzyme solu-
tion (0.85 M) pre-incubated with various concentrations of
SCN ranging from 20 160 M. The time courses for the for-
mation, duration, and decay of MPO steady-state catalysis
were examined by following the absorbance changes at 434 and
455 nm. As shown in Fig. 5, we observed a rapid increase in
absorbance at 455 nm followed by a steady state in which the
intermediate formation remained relatively constant, with a
final decay to the origin after MPO had oxidized all of the
available substrate, H2O2. The decay of the steady-state reac-
tion is biphasic. There is a rapid initial decrease in absorbance
(Fig. 5) followed by a much slower decrease in absorbance over
the next 500 s (data is not included in Fig. 5). As summarized
in Fig. 6, the addition of SCN to the MPO reaction results in
dramatic effects on the duration and decay of the steady-state
accumulation rate. The rate of steady-state accumulation was
decreased in the presence of SCN in a concentration-depend-
ent and saturable manner (Fig. 6A). The rate of its initial decay
increased in a linear manner as a function of SCN concentra-
tion (Fig. 6B), yielding a second-order combination rate con-
stant (kon) of 1 104 M1 s1 calculated from the slope. In
addition, SCN influences the level of the steady-state accu-
mulation and stability as reflected by the amplitude and dura-
tion of the stopped-flow traces, respectively. As the concentra-
tion of SCN present in the reaction mixture increased, both
the steady-state level and the amount of the intermediates
generated progressively decreased (Fig. 6C). When the reac-
tions were monitored at 434 nm the direction of absorbance
change was reversed, and the signal amplitudes enhanced but
otherwise proceeded with identical kinetics (data not shown).
Collectively, the spectra of the intermediates initially formed
following initiation of the reaction indicate that the majority of
MPO exists as a Compound II and that its formation occurs
without the accumulation of Compound I. Similar results were
obtained for EPO (data not shown), indicating that both MPO
26132 Differences in the Mechanisms of Mammalian Peroxidases

FIG. 4. Absorbance changes for the

metabolism of SCN by LPO as it oc-
curs during steady-state catalysis.
Depicted is the absorbance change over
time for the reaction that was initiated by
rapidly mixing a buffer solution contain-
ing 40 M H2O2 with an equal volume of
buffer solution containing 1 M LPO and
40 M SCN using stopped-flow diode ar-
ray methods at 10 C. Panel A, spectra
collected at 0.035, 0.07, 0.105, 0.14, 0.175,
and 0.244 s after initiating the reaction.
Panel B, spectra collected at 0.45, 0.75,
0.90, 1.05, and 2.20 s after initiating the
reaction. Arrows indicate the direction of
spectral change over time.

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FIG. 5. Effect of SCN concentration on MPO Compound II
formation, duration, and decay as they occur during steady-
state catalysis. Formation, duration, and decay of steady-state catal-
ysis of MPO were monitored as a function of time at 455 nm. An aerobic
solution containing 0.86 M MPO-Fe(III) pre-incubated with different
concentrations of SCN was rapidly mixed with an equal volume of
sodium phosphate buffer (200 mM at pH 7.0) supplemented with 40 M
H2O2 at 10 C. The initial concentration of SCN in the mixtures is 20,
40, 80, and 160 M, from bottom to top.

and EPO display a similar kinetic mechanism in metabolizing

SCN.
Effect of SCN on the Formation, Duration, and Decay of
LPO Steady-state CatalysisThe time courses for the forma-
tion, duration, and decay of LPO steady-state catalysis were
examined by following the decrease in absorbance changes at
412 nm. At each SCN concentration tested, there is a rapid
decrease in absorbance followed by a steady state in which the
intermediate formation remained relatively constant and fi-
FIG. 6. Rate of MPO Compound II formation, duration, and
nally decayed exponentially to the origin after LPO had oxi- decay as a function of SCN concentration. The observed rates of
dized all of the available substrate, H2O2. However, it is ap- MPO Compound II formation (panel A), decay (panel B), and duration
parent that as the SCN concentration increased there was a (panel C) (monitored at 455 nm) observed in Fig. 5 were plotted as a
decrease in rate of formation, a decrease in the duration of the function of SCN concentration. The percent absorbance change at 455
nm is also shown for MPO steady state as a function of SCN concen-
steady state, and an increase in the decay rate (Fig. 7). When tration (panel C).
the reactions were monitored at 434 nm the direction of ab-
sorbance change was reversed, and the signal amplitudes were
reduced but otherwise proceeded with identical kinetics (data SCN in a concentration-dependent and saturable manner
not shown). This behavior suggests that the spectral changes (Fig. 8A). The rate of its decay increased in a linear manner as
observed should reflect the change in LPO Compound II during a function of SCN concentration (Fig. 8B), yielding a second-
catalysis. As summarized in Fig. 8, the addition of SCN to order combination rate constant (kon) of 7 103 M1 s1 and a
LPO reaction results in dramatic effects on the duration and first order dissociation rate constant of 1 s1 calculated from
decay of the steady-state accumulation rates. The rate of the slope and the intercept, respectively. In addition, SCN
steady-state accumulation was decreased in the presence of influences the level of the steady-state accumulation and sta-
 Differences in the Mechanisms of Mammalian Peroxidases
FIG. 7. Spectral changes of SCN metabolism by LPO as they
occur during steady-state catalysis at four different SCN con-
centrations. Formation, duration, and decay of the steady-state catal-
ysis of LPO were monitored as a function of time at 412 nm. An aerobic
solution containing 0.86 M LPO-Fe(III) pre-incubated with different
concentrations of SCN was rapidly mixed with an equal volume of
sodium phosphate buffer (200 mM, pH 7.0) supplemented with 40 M
H2O2 at 10 C. The initial concentration of SCN in the mixtures is 10,
20, 40, 80, and 160 M from bottom to top.
FIG. 8. Rate of LPO steady-state formation, duration, and de-
cay as a function of SCN concentration. The observed rates of
LPO steady-state formation (panel A), decay (panel B), and duration
(panel C) (monitored at 412 nm) observed in Fig. 7 were plotted as a
function of SCN concentration. The percent absorbance change at 412
nm is also shown for the LPO steady state as a function of SCN
concentration (panel C).
bility as reflected by amplitude and duration of the stopped-
flow traces, respectively. As the concentration of SCN present
in the reaction mixture was increased, both the steady-state
level and the amount of the intermediates generated progres-
sively decreased (Fig. 8C).
Direct H2O2 Consumption by MPO Using a H2O2-selective
ElectrodeFollowing the addition of 20 M H2O2 to a continu-
ously stirred phosphate buffer, the H2O2 signal rose rapidly,
achieved a maximum after 30s, and fell gradually to the
origin as H2O2 was depleted by autooxidation. The addition of
MPO to the reaction mixture caused an immediate rapid decay
in the level of free H2O2 followed by a slow decay, indicating
26133
FIG. 9. A typical recording by a H2O2-selective electrode for
H2O2 consumption by MPO. Top, the addition of 20 M H2O2 followed
by 0.2 M MPO to a stirred 0.2 M sodium phosphate buffer (pH 7) results
in dramatic biphasic acceleration in the rate of H2O2 consumption at
25 C. Bottom, the addition of 20 M H2O2 followed by 0.2 M MPO
pre-incubated with 40 M SCN; only the fast phase has been seen.
Tracings shown are from typical experiment performed at least three
times. The H2O2 traces were corrected by subtracting the autooxidation
of H2O2.
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that H2O2 is consumed as a substrate by MPO during steady-
state catalysis (Fig. 9, top). The first step occurs immediately
after the enzyme addition and is attributable to the formation
of MPO Compound I. The second step is much slower and is
attributable to the reaction of MPO with H2O2 after the con-
version of MPO Compound II to ground state. When the same
reaction was repeated by adding an MPO sample saturated
with SCN solution to a stirred H2O2 solution, only the first
step was observed (Fig. 9, bottom). Similar results were ob-
tained for both EPO and LPO.
DISCUSSION
Pre-incubation of SCN with MPO and other members of the
mammalian peroxidase superfamily (e.g. EPO and LPO) causes
multiple and sequential reactions and suggests a multi-func-
tional role for SCN before and during steady-state catalysis.
Indeed, pre-incubation with SCN prior to initiating peroxida-
tion generates a more complex biological setting through its
potential capacity to bind within the enzyme systems, above
the heme prosthetic group, or directly to the heme iron, gener-
ating a six-coordinate low spin complex. SCN binds within the
MPO system, serves as a substrate or inhibitor, and modulates
the heme iron microenvironment to cause a significant alter-
ation in its catalytic site, thereby altering the enzymes affinity
toward H2O2. Rapid kinetic measurements utilizing sequential
stopped-flow methods have indicated that the direct reaction
between MPO, EPO, and LPO Compound I and SCN is ex-
tremely fast and occurs with second-order rate constants rang-
ing from 9.6 107 to 2 108 M1 s1 (33, 36, 37, 44, 46, 47).
Thus, the order of addition (the enzyme and H2O2 mixed first
and SCN second, or the enzyme and SCN mixed first and
H2O2 second) may have created a considerable degree of con-
troversy, ambiguity, and diverse results in predicting the pre-
ferred biological substrate of the mammalian peroxidase
superfamily.
Because MPO, EPO, and LPO Compound I formation rates
are slower than the 2e oxidation of SCN, Compound I accu-
mulation cannot be detected during steady-state catalysis (27,
33, 36, 37, 44, 46, 47). Thus, the observed absorbance changes
during SCN metabolism should reflect the alteration in Com-
pound II accumulation, duration, and decay. Rapid kinetic
measurements indicated that MPO, EPO, and LPO Compound
II is the predominant species formed, providing the first direct
evidence for the involvement of Compound II in the catalytic
inhibition of mammalian peroxidases. The kinetic parameters
26134 Differences in the Mechanisms of Mammalian Peroxidases
FIG. 10. Modified working kinetic model for SCN interaction
with mammalian peroxidases (E, enzyme MPO, EPO, or LPO).
of Compound II formation, duration, and decay for MPO and
EPO were similar, but they differed from those obtained for
LPO Compound II. The degree of MPO and EPO Compound II
complex accumulation was surprisingly high in that 80 90% of
the total enzymes were estimated to be in Compound II form
and attenuated in a saturable manner to a level of saturation
approaching 50% upon increasing SCN concentration. Be-
cause Compound II is a catalytically inactive complex, a steady
but suboptimal rate of SCN oxidation and HOSCN synthesis
is maintained that is proportional to the percentage of MPO
and EPO cycling through the 2e oxidation pathway. Indeed,
the presence of SCN in the milieu resulted in a dramatic
acceleration in H2O2 consumption by both MPO and EPO, as
detected by continuous monitoring with a H2O2-selective elec-
trode. In contrast, the LPO Compound II was unstable and
converted to ground state immediately after H2O2 exhaustion,
indicating that SCN may serve as a 1e substrate for LPO
Compound II. At a low SCN concentration, LPO Compound II
was also maintained at a constant level, whereas the enzyme
continued to catalyze SCN oxidation and HOSCN synthesis.
We observed that increasing the SCN concentration was ac-
companied by a modest decrease in the rate of Compound II
formation, but with a significant decrease in accumulation, a
decrease in duration, and a significant increase in the decay
rate. LPO Compound II formation and the accompanying inhi-
bition did not occur at a higher SCN concentration. This
behavior can be interpreted by either or both of the following
statements. 1) The Compound II formation rate becomes slower
than the complex decay; and 2) the majority of the reaction
proceeds through a 2e oxidation pathway.
A general kinetic scheme of how SCN interacts with mam-
malian peroxidase intermediates incorporated into the classic
catalytic cycle is illustrated in Fig. 10. This working kinetic
model consists of two major pathways, namely the classical
peroxidase cycle (pathway A) and the binding of a co-substrate
to E-Fe(III), the formation of E(inh)-Fe(II) complex, and the
effect of these bindings on the formation of Compound I (path-
way B) (Fig. 10). Compound I formation is rapid and occurs
with a second-order rate constant ranging from 1 107 to 4.3
107 M1 s1 (27, 33, 36, 37, 44, 46, 47). Mammalian peroxidase
Compound I oxidizes halides and pseudohalides by a 2e oxi-
dation process yielding the corresponding hypohalous acid and
the ground state (E-Fe(III)) (33, 41 43). Alternatively, Com-
pound I oxidizes organic and inorganic substrates by two se-
quential 1e oxidations to ground state through the formation
of Compound II, the rate-limiting step in the classic cycle of
peroxidases (33, 44).
NO, like CN, binds to the MPO heme iron and displaces the
water molecule (W1), which is hydrogen-bonded to the distal
FIG. 11. Stereo view of the MPO distal heme cavity of human
MPO with bound thiocyanate. The figure was created using the
coordinates deposited in the Protein Data Bank (accession code 1D7W).
His-95 in the native enzyme. The bent mode of the Fe-N-O may
allow a perturbation in the hydrogen bonding within the distal
cavity. Formation of such a complex is accomplished with the
movement of the iron atom into the porphyrin ring, generating
its respective low spin, six-coordinate complex. SCN is a rel-
atively bulky molecule; its binding above the heme moiety of
MPO may constrain NO binding by either filling the space
directly above the heme or by promoting a protein conforma-
tional change that constricts the distal heme pocket (Fig. 11).
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The ability of SCN to modify the heme pocket of MPO and/or
shield the catalytic site of the enzyme is directly reflected by
the modulation of NO binding to the enzyme heme moiety.
Varying the concentration of SCN displays different types of
impact on the MPO heme iron microenvironment. Indeed, the
plot of the second-order combination rate constant (kon) of NO
binding to MPO-Fe(III) as a function of SCN concentration
displayed a bell-shaped curve with a negative slope over the
range where SCN concentrations were 60 M and a positive
slope over the range where SCN concentrations were 60 M,
with saturation 300 M. This behavior may have a broader
link effect on MPO activity, because the initial decrease in the
second-order rate constant of NO combination (kon) occurs
within the range where SCN binds to a distal cavity located in
MPO Compound I and allows the direct contact with the oxy-
ferryl oxygen. Under these circumstances, Compound I appears
to act favorably in triggering electron transfer to the heme with
subsequent conversion to hypothiocyanate as a final reaction
product (16, 38, 39, 73). This decrease was reversed and
reaches saturation at higher SCN concentrations, where
SCN binds to MPO at both the distal cavity and the proximal
helix sites and forms an inactive complex (SCN-E-Fe(III)(inh)).
Indeed, Blair-Johnson et al. (73) have shown that the inhibitory
complex with thiocyanate indicates replacement of chloride at
the proximal helix halide binding site in addition to binding in
the distal cavity in an orientation parallel with the heme (Fig.
11). It was thought that halides binding at the distal cavity site
inhibit the enzyme by interfering with the deprotonation of
H2O2 by the adjacent distal His-95 (16, 38, 39, 73), a mecha-
nism consistent with previous reports indicating that inhibi-
tion by halides is competitive with respect to H2O2 (56 59).
Thus, the saturation in a bell-shaped curve occurs, at a high
concentration of SCN (300 M), when the two sites are
occupied. Therefore, the plasma levels of SCN (60 M) are
crucial in saturating the substrate site of the enzyme, as judged
by the inflection point in the bell-shaped curve that is illus-
trated in Fig. 2. Consequently, the plasma level of SCN may
govern the catalytic reaction of MPO both in vivo and in vitro.
Identifying similarities and differences in the interactions
between MPO and various members of the mammalian perox-
idase superfamily yields valuable mechanistic insights into the
role of Compound II formation in modulating catalytic activity
and function. The Compound I of MPO, EPO, and LPO simul-
taneously catalyzes SCN via 2e and 1e oxidation pathways,
which leads to the formation of HOSCN and sulfur-centered
 Differences in the Mechanisms of Mammalian Peroxidases
thiocyanate radicals (SCN), respectively (60, 61). The parti-
tioning between the two pathways depends in part on the rate
of 2e versus 1e oxidation of SCN and on the concentration
of SCN.
It appears that as the SCN concentration increases, Com-
pound I is converted rapidly and more efficiently to both
ground state and Compound II. This behavior is illustrated by
the significant alteration in the intermediate distributions dur-
ing steady-state catalysis, as reflected by the decrease in the
amplitude of Compound II formation observed in an SCN-de-
pendent and saturable manner (Fig. 6C). Thus, the plateau in
the curve is governed by the rate of the 1e oxidation reaction
of SCN by Compound I, which would then result from the
cleavage of SCN from the SCN-MPO(inh)-Fe(III) complex.
Thus, the plot of the formation rate of Compound II plateaus at
a rate that should be comparable with the rate of SCN-E(inh)-
Fe(III) conversion to the active form, which, under these cir-
cumstances, is the formation of E-Fe(III) and/or SCN-E-Fe(III)
(Fig. 10). The low conversion rate constant limits E-Fe(III)
bioavailability and subsequently attenuates the turnover num-
ber of peroxidation.
The time course for MPO Compound II decay monitored in
the absence of SCN, detected by monitoring the absorbance
changes at 455 nm, is extremely slow and occurs with a rate
constant of 0.008 s1 at 10 C (28 30). In the presence of SCN cyanate (OCN) (19, 67, 68).
and after the reaction had ceased, there was an initial rapid
decay that was followed by a slower decay regenerating ferric
MPO. The initial rapid phase may be attributed to a rapid
decay of Compound I to ground state, which increases in a
linear manner as a function of SCN concentration with a
second-order rate constant of 1 104 M1s1, whereas the
second phase was attributed to the decay of MPO Compound II
to ground state. Compound II does not possess suitable redox
potential to oxidize halides and pseudohalides (33, 41). Rather,
Compound II uses a variety of organic and inorganic com-
pounds as substrates (33). Similar results were observed for
EPO, indicating that both enzymes display similar mechanism
in metabolizing SCN. Van Dalen and Kettle have recently
reported similar spectral changes during the turnover of the
metabolism of SCN by EPO and MPO (62). Like them, we also
noted that the addition of GSH, a hypothiocyanite scavenger,
prevented the buildup of the Compound II intermediate; but, at
the same time GSH would not have contributed to the turnover
of Compound II, because it did not reduce preformed MPO
Compound II (data not shown). Furthermore, Compound II
formation can be destabilized in the presence of organic and
inorganic substrates (e.g. low levels of NO) (data not shown).
In contrast, LPO Compound II formation and the accompa-
nying LPO inhibition did not occur under conditions where
SCN is in large excess. Thus, a majority of Compound I
quickly generates its ferric complex upon initiating the reac-
tion. The apparent ability of LPO to oxidize SCN through two
successive 1e transfers generating Compound II and LPO-
Fe(III) is unprecedented. Increasing SCN concentration accel-
erates the decay of the Compound II, a rate-limiting step in the
peroxidase cycle, and enhances the overall rate of catalysis.
Enhancement of peroxidase catalysis due to the reduction of
LPO Compound II to LPO-Fe(III) has been noted with other
physiological reductants such as NO, superoxide, and ascorbic
acid (28 30,33). That the rate of LPO Compound II formation
remained relatively unchanged at the high SCN concentra-
tions studied (50 M SCN) is consistent with the reaction
being limited by the release of SCN from the SCN-LPO(inh)
complex. Consistent with this interpretation, the failure of the
higher SCN concentration to alter the LPO spectrum ap-
peared to be linked to the fact that the formation of Compound
26135
II becomes slower than the complex decay. Collectively, the
formation of LPO Compound II during steady-state catalysis is
a fundamental feature of catalysis and functions to down-reg-
ulate the peroxidation process by the enzyme.
The ability of MPO, EPO, and LPO to employ SCN as a 1e
substrate influences the nature of the end products of the
SCN oxidation reaction. For example, 1e oxidation of SCN
generates a thiocyanate radical, which dimerizes to yield a
labile short-lived intermediate, thiocyanogen (SCN)2 (61, 63).
This intermediate is rapidly hydrolyzed to generate a combi-
nation of HOSCN and OSCN as final end products (61, 63).
These observations are consistent with earlier studies by Modi
et al., which suggested that LPO catalyzes SCN oxidation at
pH 6.1 to produce HOSCN and OSCN (60). Alternatively,
(SCN)2 is hydrolyzed to produce CN as an end product with-
out the formation of HOSCN (64, 65). In the presence of the
plasma level of SCN, SCN may be directly oxidized to
OSCN (60, 63, 66). It has been suggested that other products
besides hypothiocyanite and CN might be formed during the
metabolism of SCN. For example, Pruitt et al. have proposed
that hypothiocyanous acid might be oxidized by excess H2O2 or
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MPO to yield two short-lived intermediate oxidants, cyanosul-
furous acid (HO2SCN) and cyanosulfuric acid (HO3SCN),
which then break down in the presence of H2O2 to generate
In related studies, Furtmuller et al. have demonstrated that
the apparent second-order rate constants of the reactions be-
tween the LPO, EPO and MPO Compound I and SCN are
extremely high, with apparent second-order constants of 2
108, 1 108 and 9.6 107 M1 s1, respectively (36, 44, 47).
These results are consistent with the direct reaction between
Compound I and SCN, which, to a large extent, prevents the
potential alteration in the heme pocket and, thereby, the for-
mation of the inactive form of the enzyme that occurs upon the
prior incubation of SCN with the three enzymes.
Binding of the co-substrate to the heme moiety generating
the E-Fe(III)-SCN complex (Fig. 10) may considerably restrict
H2O2 access to the heme iron, and the slow rate of H2O2
binding observed would then result from ligand replacement
processes. There is no data available to support the notion that
SCN serves as a ligand in mammalian peroxidase when pres-
ent at plasma levels. However, the presence of higher levels of
SCN (e.g. 1 mM) may promote the formation of such a com-
plex (45). Formation of E-Fe(III)-SCN can be characterized
based on its distinctive absorbance feature, which displays a
large red shift in the Soret absorbance region compared with
native enzyme (45). Whether the slow binding of H2O2 to the
enzyme occurs through conformational changes in the heme
pocket geometry or because of the replacement reaction, the
rate-limiting step becomes the binding of H2O2 to the enzymes.
Under all circumstances, the catalytic activity of the peroxi-
dases will depend on multiple factors, including the concentra-
tion of SCN versus H2O2, the affinity of the enzymes toward
H2O2 versus SCN, and the rate of SCN-E-Fe(III) and
E-Fe(III)-SCN breakdown (Fig. 10). Changes in heme pocket
geometry upon ligand binding have been described for cyto-
chrome C peroxidase (70), and the slower rates of NO binding
to the Fe(III) forms of a number of heme proteins have been
attributed to ligand replacement (69, 71, 72). Ligation of SCN
to the distal heme would prevent access of H2O2 to the catalytic
site of the enzymes.
In summary, our current studies reveal for the first time that
the formation of Compound II during steady-state catalysis is a
fundamental feature of the kinetic reaction of mammalian per-
oxidases. Its formation during steady-state catalysis operates
to down-regulate the catalytic activity of MPO, EPO, and LPO.
26136 Differences in the Mechanisms of Mammalian Peroxidases
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 Thiocyanate Modulates the Catalytic Activity of Mammalian Peroxidases
Yahya R. Tahboub, Semira Galijasevic, Michael P. Diamond and Husam M. Abu-Soud
J. Biol. Chem. 2005, 280:26129-26136.
doi: 10.1074/jbc.M503027200 originally published online May 13, 2005

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