Catalase prevents myeloperoxidase self-destruction in response to oxidative stress

Article abstract of DOI:10.1016/j.jinorgbio.2019.110706

Catalase (CAT) and myeloperoxiase (MPO) are heme-containing enzymes that have attracted attention for their role in the etiology of numerous respiratory disorders such as cystic fibrosis, bronchial asthma, and acute hypoxemic respiratory failure. However, information regarding the interrelationship and competition between the two enzymes, free iron accumulation, and decreased levels of non-enzymatic antioxidants at sites of inflammation is still lacking. Myeloperoxidase catalyzes the generation of hypochlorous acid (HOCl) from the reaction of hydrogen peroxide (H₂O₂) and chloride (Cl^?). Self-generated HOCl has recently been proposed to auto-inhibit MPO through a mechanism that involves MPO heme destruction. Here, we investigate the interplay of MPO, HOCl, and CAT during catalysis, and explore the crucial role of MPO inhibitors and HOCl scavengers in protecting the catalytic site from protein modification of both enzymes against oxidative damage mediated by HOCl. We showed that CAT not only competes with MPO for H₂O₂ but also scavenges HOCl. The protective role provided by CAT versus the damaging effect provided by HOCl depends in part on the ratio between MPO/CAT and the affinity of the enzymes towards H₂O₂ versus HOCl. The severity of such damaging effects mainly depends on the ratio of HOCl to enzyme heme content. In addition to its effect in mediating protein modification and aggregation, HOCl oxidatively destroys the catalytic sites of the enzymes, which contain porphyrin rings and iron. Thus, modulation of MPO/CAT activities may be a fundamental feature of catalysis, and functions to down-regulate HOCl synthesis and prevent hemoprotein heme destruction and/or protein modification.

Full text of DOI:10.1016/j.jinorgbio.2019.110706

Accepted Manuscript
Catalase prevents myeloperoxidase self-destruction in response to
oxidative stress
Iyad Ali, Sana N. Khan, Charalampos Chatzicharalampous, David
Bai, Husam M. Abu-Soud
PII:
S0162-0134(18)30707-4
DOI:
https://doi.org/10.1016/j.jinorgbio.2019.110706
Article Number:
Reference:
110706
JIB 110706
To appear in:
Journal of Inorganic Biochemistry
Received date:
Revised date:
Accepted date:
3 December 2018
17 April 2019
2 May 2019
Please cite this article as: I. Ali, S.N. Khan, C. Chatzicharalampous, et al., Catalase
prevents myeloperoxidase self-destruction in response to oxidative stress, Journal of
Inorganic Biochemistry, https://doi.org/10.1016/j.jinorgbio.2019.110706
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ACCEPTED MANUSCRIPT
Catalase prevents myeloperoxidase self-destruction in response to
oxidative stress
1
,2
1
1
1
Iyad Ali , Sana N. Khan , Charalampos Chatzicharalampous , David Bai , Husam M Abu-
1
,3
Soud *.
1
Department of Obstetrics and Gynecology, The C.S. Mott Center for Human Growth and
Development, Detroit, MI 48201, USA
2
Department of Biochemistry and Genetics, Faculty of Medicine and Health Sciences, An-Najah
National University, Nablus 7, Palestine
3
Department of Microbiology, Immunology and Biochemistry, Wayne State University School of
Medicine, Detroit, MI, 48201, United States.
*
Corresponding Author: Husam M. Abu-Soud, Ph.D, Department of Obstetrics and Gynecology,
Wayne State University School of Medicine, The C.S. Mott Center for Human Growth and
Development, 275 E. Hancock Detroit, MI 48201, Tel. 313 577-6178, Fax. 313 577-8554,
EMail: habusoud@med.wayne.edu
Running title: Hypochlorous acid and heme destruction
Keywords: MPO, Catalase, Respiratory inflammation, Heme destruction, Free iron,
Antioxidants.
1

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Abstract
Catalase (CAT) and myeloperoxiase (MPO) are heme-containing enzymes that have attracted
attention for their role in the etiology of numerous respiratory disorders such as cystic fibrosis,
bronchial asthma, and acute hypoxemic respiratory failure. However, information regarding the
interrelationship and competition between the two enzymes, free iron accumulation, and
decreased levels of non-enzymatic antioxidants at sites of inflammation is still lacking.
Myeloperoxidase catalyzes the generation of hypochlorous acid (HOCl) from the reaction of
-
hydrogen peroxide (H O ) and chloride (Cl ). Self-generated HOCl has recently been proposed
2
2
to auto-inhibit MPO through a mechanism that involves MPO heme destruction. Here, we
investigate the interplay of MPO, HOCl, and CAT during catalysis, and explore the crucial role
of MPO inhibitors and HOCl scavengers in protecting the catalytic site from protein
modification of both enzymes against oxidative damage mediated by HOCl. We showed that
CAT not only competes with MPO for H O but also scavenges HOCl. The protective role
2
2
provided by CAT versus the damaging effect provided by HOCl depends in part on the ratio
between MPO/CAT and the affinity of the enzymes towards H O versus HOCl. The severity of
2
2
such damaging effects mainly depends on the ratio of HOCl to enzyme heme content. In
addition to its effect in mediating protein modification and aggregation, HOCl oxidatively
destroys the catalytic sites of the enzymes, which contain porphyrin rings and iron. Thus,
modulation of MPO/CAT activities may be a fundamental feature of catalysis, and functions to
down-regulate HOCl synthesis and prevent hemoprotein heme destruction and/or protein
modification.
2

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Introduction
Catalase (CAT) and myeloperoxidase (MPO) are hemoproteins that have recently gained
increasing attention for their potential role in the etiology of respiratory conditions related to
oxidative stress [1-3]. Interestingly, many inflammatory disorders such as cystic fibrosis (CF),
bronchial asthma, and acute hypoxemic respiratory failure, in which MPO/CAT are known to be
elevated, and are also associated with significant free iron accumulation [4-9]. It has been
postulated that variations in the levels or function of these two enzymes could modulate
respiratory illness risk, especially under conditions of oxidative stress [1, 2]. However, a
detailed understanding of the interrelationship and competition between the two enzymes at sites
of inflammation is still lacking. Catalase is an anti-inflammatory enzyme that predominantly
competes with MPO by converting hydrogen peroxide (H O ) to H O and oxygen [10].
2
2
2
Myeloperoxidase, the most abundant protein in neutrophils and monocytes, catalyzes the
-
reaction between chloride (Cl ) and H O to form hypochlorous acid (HOCl) [11-13].
2
2
Hypochlorous acid is a powerful oxidant, and is extremely reactive with the heme moiety of
MPO and other hemoprotein model compounds causing heme damage and free iron release [14-
1
7]. Hypochlorous acid when released from the MPO cavity, first diffuses in the solution as a
whole, binds to MPO heme iron forming MPO-Fe(III)-OCl complex, and then medicates MPO
heme destruction, precluding the enzyme from functioning at the maximum activity (80-90%
inhibition) [14].
Accordingly, inhibition of MPO-chlorinating activity and/or factors that influence rates of HOCl
removal following its synthesis by MPO are of significant interest. In the biological system, a
major pathway for HOCl removal is through its interactions with antioxidant compounds such as
thiols (glutathione (GSH), taurine, and methionine), carotenoid (lycopene), derivatives of
3

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tetrapyrrole macrocyclic rings (heme and vitamin B ), and indole compounds (melatonin and
1
2
-
tryptophan) [18-24]. These molecules compete with Cl , the natural substrate of MPO, by serving
as a one electron substrate of MPO Compounds I (ferryl porphyrin  cation radical,
Fe(IV)=O(+•)) and Compound II (ferryl complex; Fe(IV)=O), and/or directly scavenging HOCl
[
18-24].
Several respiratory diseases with underlying inflammation have been linked to abnormal MPO to
CAT ratios, enhanced iron levels, and decreased levels of antioxidants [1, 2, 9, 25-27]. Increased
MPO activity combined with decreased CAT activity creates an environment of increased
reactive oxygen species (ROS) that are implicated in various cancers and in chronic respiratory
inflammation [28]. Synthetic compounds with superoxide dismutase (SOD) and CAT activities
have shown promising results in animal models against a variety of oxidant exposures including
cigarette smoke in the lung [27].
In this work, we utilized H O -selective electrode analysis and direct spectroscopic
2
2
measurements to determine the interdependence between CAT and MPO, and the role of HOCl
in down-regulating the catalytic activity of both enzymes. Our recently published [14, 16, 17]
and the current work, highlights the link between free iron accumulation, decreased levels of
antioxidant, and the catalytic activity of both enzymes.
Materials and methods
Materials
All the materials used were of the highest-grade purity and used without further purification.
Catalase powder from bovine liver, sodium hypochlorite (NaOCl), melatonin, L-methionine, and
taurine were obtained from Sigma–Aldrich (St. Louis, MO, USA).
MPO purification
4

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Myeloperoxidase was initially purified from detergent extracts of human leukocytes by
sequential lectin affinity and gel-filtration chromatography, as previously reported [29-31]. Trace
levels of contaminating eosinophil peroxidase were then removed by passage over a sulfopropyl
Sephadex column [30]. Purity of isolated MPO was established by demonstrating a Reinheitszahl
(
RZ) value of >0.87 (A /A ), SDS–PAGE analysis with Coomassie blue staining, and gel
430 280
tetramethylbenzidine peroxidase staining to confirm no contaminating eosinophil peroxidase
activity. Enzyme concentration was determined spectrophotometrically utilizing extinction
−
1
−1
coefficients of 89,000 M cm /heme of MPO at 430 nm [32]. Catalase, from the manufactures,
was used without further purification. Catalase concentration was determined
−
1
−1
spectrophotometrically using extinction coefficients of 120,000 M cm /heme at 406 nm [33].
H O -selective electrode measurements
2
2
Hydrogen peroxide consumption was measured using an H O -selective electrode (Apollo 4000
2
2
free radical analyzer; World Precision Instruments, Sarasota, FL, USA). Experiments were
o
performed at 25 C by immersing the electrode in 3 ml of 0.2 M sodium phosphate buffer, pH
7
.4. Hydrogen peroxide (10 µM) was added to a continuously stirred buffer solution containing
fixed amount of MPO (40 nM, final) and various concentrations of CAT (40-160 nM), or fixed
amount of CAT (40 nM final) and various concentrations of MPO (40–160 nM, final), and/or Cl
−
(
100 mM) during which the change of H O concentration was continuously monitored.
2 2
Absorbance measurements
The absorbance spectra were recorded using a Cary 100 Bio UV–visible photometer, at 25°C, pH
7
.4. Experiments were performed in 1 ml phosphate buffer solution supplemented with fixed
−
amounts of MPO (~0.6 μM) and Cl (100 mM) and increasing concentrations of H O (0–200
2
2
5

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μM), in the absence and presence of different ratios of CAT relative to MPO and/or various
antioxidants (100 μM methionine, taurine, or melatonin).
HOCl preparation
Hypochlorous acid was prepared as previously described, with some modifications [34]. Briefly,
a stock solution of HOCl was prepared by adding 1 ml of NaOCl solution to 40 ml of 154 mM
NaCl and the pH was adjusted to around 3 by adding hydrochloric acid. The concentration of
active total chlorine species in solution, expressed as [HOCl]T (where
−
−
[
HOCl] =[HOCl]+[Cl ]+[Cl ]+[OCl ]) in 154 mM NaCl, was determined by converting all the
T 2 3
−
active chlorine species to OCl by adding a bolus of 40 μl of 5 M NaOH and measuring the
−
−
concentration of OCl . The concentration of OCl was determined spectrophotometrically at 292
−
1
−1
nm (ε=362 M cm ) [35] As HOCl is unstable, the stock solution was freshly prepared on a
daily basis, stored on ice, and used within 1 h of preparation.
Results
Influence of HOCl generation on MPO/CAT catalytic functions
-
We first investigated if multiple rounds of H O addition to the MPO/Cl system could affect
2
2
MPO performance and if the generated HOCl would affect the MPO peroxidase activity. Fig 1,
depicts an experiment in which six cycles of MPO activity were promoted by repeatedly adding
a limiting amount of H O (10 µM) to trigger transient HOCl synthesis in a single sample of
2
2
MPO. The amount of H O accumulated in the solution mixture (amplitude H O signal) and the
2
2
2
2
time of its consumption (duration of the signal) were continually measured after the addition of
−
the H O solution to a continuously stirred buffer containing MPO (40 nM)/Cl (100 mM). The
2
2
first H O addition to the enzyme solution led to an instant disappearance in the H O signal due
2
2
2
2
6

ACCEPTED MANUSCRIPT
to its consumption as a substrate during catalysis, indicating that MPO is catalytically active,
similar to prior reports [14]. Sequential additions of the same amount of H O to the reaction
2
2
mixture inhibited MPO activity, as judged by the accumulation of H O (indicated by the
2
2
amplitude of the signal) from the decreased rate of H O consumption (indicated by the duration
2
2
of the signal) (Fig. 1A). This reduction in MPO activity required the buildup of sufficient
amounts of HOCl generated by multiple rounds of H O consumption. We also utilized the
2
2
H O -selective electrode to show that CAT alone can rapidly consume H O . As shown in Fig.
2
2
2
2
1
B, the first H O (10 µM) addition to the enzyme solution led to an instant disappearance of
2 2
H O signal due to its consumption as a substrate by the enzyme during steady-state catalysis,
2
2
indicating that CAT is catalytically active. However, sequential addition of the same amount of
H O to the reaction mixture demonstrated a similar instant consumption of H O (Fig. 1B).
2
2
2
2
To determine whether CAT possesses the ability to compete with MPO on H O and prevent
2
2
HOCl-mediated heme destruction of both enzymes, identical experiments were repeated in the
presence of both MPO and CAT. Experiments were carried out by either employing a fixed
amount of MPO (40 nM) and varying concentrations of CAT (40, 80, 120, 160 nM) (Fig. 2A) or
a fixed amount of CAT (40 nM) and varying concentrations of MPO (40, 80, 120, 160 nM) (Fig.
2
B). In each MPO/CAT cycle, H O , the common substrate of both enzymes, was the limiting
2 2
factor and would be reduced completely after each addition to the solution mixture by both
enzymes. Remarkably, addition of H O to a continuously stirred reaction mixture supplemented
2
2
-
with 40 nM MPO, 100 mM Cl , and 40 nM CAT, demonstrated a modest brief increase in H O
2
2
signal, indicating rapid H O disappearance (Fig. 1C). However, after several additions of H O
2
2
2
2
to the solution mixture the H O signal rose rapidly, achieved its maximum level after ~30 s, and
2
2
then gradually returned to baseline when all the H O was consumed (Fig. 1C). Thus, the
2
2
7

ACCEPTED MANUSCRIPT
accumulation of free H O in MPO/CAT milieu requires more addition of H O compared to the
2
2
2
2
MPO alone (Fig. 1B). Area-under-the-curves (AUC), which reflect the maximum signal
intensity and the duration of H O consumption by both enzymes, were determined and plotted
2
2
as a function of H O concentration. In both cases, no significant increase in the AUC was
2
2
observed when incremental addition of H O was added to CAT alone (Fig. 2 A&B). MPO was
2
2
not immediately inhibited by self-generated HOCl and remained active until a sufficient amount
of HOCl was built-up in the solution mixture (Fig. 2 A&B). When a 1:1 ratio of MPO:CAT was
used, this condition caused a right shift in the curve compared to MPO alone. Increasing CAT
concentrations caused a further shift in the curves to the right (Fig. 2A). A similar trend was also
observed by increasing the MPO to CAT ratio, but to a lesser degree (Fig 2B). Another factor
that caused the MPO inactivation curve to shift to the right in addition to MPO:CAT ratio, is the
presence of an HOCl scavenger.
Effect of MPO inhibitor and /or HOCl Scavengers
7
4
−1 −1
Melatonin, methionine, and taurine react with HOCl at rates from 3.4×10 to about 10 M s
and have been used to scavenge HOCl or inhibit MPO chlorinating activity and protect MPO
from inhibition and destruction by HOCl [36-38]. We therefore examined if these would protect
MPO/CAT from HOCl in our system. Fig. 3B shows the effect of melatonin (100 µM) on MPO
and CAT activities during HOCl synthesis. In this experiment, 10 µM (3µl) of H O was added
2
2
six consecutive times (at the arrow indicators in Fig. 3) to initiate six rounds of MPO/CAT
catalysis at 25°C. Hydrogen peroxide accumulation and its decay were monitored as an increase
and decrease in H O signal over time. The amount of H O accumulated in the solution mixture
2
2
2
2
(
amplitude of the H O signals) and the time of its exhaustion reaction (duration of H O signal)
2
2
2
2
8

ACCEPTED MANUSCRIPT
were nearly identical within experimental error for all six trials. The data showed that
consumption of H O can occur at multiple times without slowing the rate of H O consumption
2
2
2
2
-
by MPO and CAT. Panel 3A is the control reaction, in which both MPO/Cl (40 nM/100 mM)
and CAT (40 nM) were added to the reaction at time zero to initiate concurrent H O
2
2
consumption as H O is added to the reaction (at the arrow indicators in Fig. 3) and subsequent
2
2
-
HOCl synthesis. In this experiment (Fig. 3B), melatonin is a 1e substrate of MPO that competes
-
-
-
with Cl , and this shifts the MPO catalysis paradigm from a 2e to a 1e oxidation pathway. As
such, MPO consumed H O more slowly without producing HOCl, and the H O signal in the
2
2
2
2
presence of melatonin correspondingly had much higher amplitude and lower decay rate relative
to the control. Similar results have been observed for methionine and taurine (data not shown).
Thus, these compounds, such as melatonin, methionine, and taurine, could also prevent HOCl-
mediated heme destruction of both enzymes.
Effect of HOCl generation on MPO and CAT heme during steady-state catalysis
Our next experiments utilized light absorbance spectroscopy to examine if a significant amount
of the MPO and CAT heme contents would be destroyed during catalysis of HOCl synthesis
-
from MPO-Cl -H O system. Fig. 4 contains UV-Visible spectra of MPO alone (~0.6 µM)
2
2
(dashed line), CAT alone (~0.6 µM) (dotted line), and MPO and CAT in combination (solid
line). The starting MPO spectrum demonstrated a characteristic Soret peak at 430 nm with
absorbance shoulders at 500 and 631 nm, indicative of ferric heme, whereas the absorption
spectrum of the purified CAT exhibited a Soret peak at 406 nm and an additional minor peak at
6
10 nm. We next utilized the absorbance spectra to monitor MPO/CAT heme destruction during
HOCl synthesis to see how HOCl levels correlate with MPO/CAT heme destruction. Enzyme
9

ACCEPTED MANUSCRIPT
reactions reported here were run at 25°C in 1ml cuvettes, under conditions in which H O was
2
2
the limiting substrate and would be consumed completely by MPO/CAT after several minutes of
reaction. For clarity, a cuvette containing phosphate buffer supplemented with CAT (~0.6 µM)
-
and 100 mM Cl was mixed well and used to make the baseline of the absorbance spectra, from
3
00-500 nm, zero (Fig. 5 dotted line). The dashed line is the prominent Soret absorbance
centered at 430 nm after addition of MPO (~0.6 µM) to the solution mixture (Fig. 5). Sequential
incremental addition of 20 µM H O (3 µL) to the solution mixture caused MPO and CAT heme
2
2
destruction in a dose dependent manner, as judged by the loss of visible absorbance at 430 nm
MPO-Soret peak) and 406 nm (CAT-Soret peak) (Figs. 5A, 5B). Spectra were recorded
immediately after the addition of H O (Fig. 5A) as well as 10 min. after the addition of H O
2
(
2
2
2
(Fig. 5B) to demonstrate the slight accumulation of MPO Compound II as the stable form of
MPO. To examine the potential physiological significance of these observations, we also
determined the effect of HOCl scavengers on HOCl-mediated heme destruction utilizing similar
methods. Fig. 5C shows the same experiment in the presence of methionine (100 µM), an MPO
inhibitor and HOCl scavenger, which showed no alteration of MPO or CAT spectra, indicating
that methionine completely prevented MPO/CAT heme destruction and/or modification. When
similar experiments were performed with a MPO:CAT ratio of 5:1, less H O was needed to
2
2
achieve an increased degree of heme alteration, and at the conclusion of the experiment the MPO
heme was completely destroyed, as indicated by flattening of the MPO Soret absorption peak at
4
30 nm (data not shown). Additionally, the buildup of the absorbance shoulder at 450 nm is
identical to that partial formation of authentic MPO Compound II, a long lived intermediate,
where its accumulation is the first step in HOCl-mediated heme destruction in hemoprotein [14,
1
6, 17]. Furthermore, when MPO:CAT ratio was 5:1, HOCl-mediated heme destruction was, to a
1
0

ACCEPTED MANUSCRIPT
large extent, prevented in the presence of excess amounts of methionine (100 µM) with slight
formation of MPO compound II. Methionine, like taurine and melatonin, is an important
scavenger for HOCl that eliminates the impact of it on both CAT and MPO [36-38]. Similar
experiments were also done using exogenously added HOCl to the enzyme solution mixtures; it
showed the same effect, confirming that the alteration in the spectra of Fig. 5 comes from HOCl.
Together, these results suggest that a significant amount of MPO and CAT were destroyed
during aerobic HOCl synthesis under these conditions, and increasing the ratio between MPO
and CAT increased the degree of heme destruction and/or modification, a process that was
substantially inhibited by the presence of HOCl scavengers.
Finally, we utilized H O - selective electrodes to determine whether HOCl mediated MPO heme
2
2
destruction occurs first and CAT destruction/modification second or vice versa. During
continuous monitoring of H O levels with an H O -selective electrode, rates of H O
2
2
2
2
2
2
consumption from the reaction were dramatically attenuated by addition of HOCl (10 µM) to
MPO/CAT mixture with prior H O addition to the solution mixture. Under these experimental
2
2
conditions, the H O signal rose rapidly, achieved a maximum after ~30 s, and fell gradually to
2
2
the base line as H O was depleted. To ensure MPO/CAT inactivation, an additional consecutive
2
2
two HOCl aliquots (10 µM)/H O (10 µM) were added to a continuously stirred phosphate buffer
2
2
(
200 mM, pH 7.4) supplemented with 40 nM MPO and 40 nM CAT at 25ºC. Here, the H O
2 2
signal rose rapidly, achieved a maximum after ~30 s, and fell very slowly not reaching the base
-
line as H O was depleted slowly. The reaction mixture then received 100 mM Cl followed by
2
2
-
two consecutive H O additions (denoted by the arrows). The fact that the addition of Cl led to
2
2
acceleration of H O consumption (Fig. 6) indicates that MPO is still active and that the slow
2
2
reaction could be due to the formation of Compound II, the stable form of MPO, partial MPO
1
1

ACCEPTED MANUSCRIPT
heme destruction, or a combination of both. Therefore, our experiment shows that CAT is
inactivated prior to MPO in the presence of HOCl. Collectively, these results may suggest that
CAT is more susceptible to HOCl-mediated destruction than MPO under these experimental
conditions.
1
2

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Discussion
Our central finding is that CAT competes with MPO for the substrate H O and thus prevents
2
2
HOCl-mediated heme degradation and/or protein modification. The protective role provided by
CAT versus the damaging effect of HOCl depends on both the MPO/CAT ratio and the
enzymes’ affinity towards H O versus HOCl. In addition to mediating protein modification and
2
2
protein aggregation [39-41], HOCl’s damaging effect is also due to its ability to oxidatively
destroy the catalytic sites, in this case the heme group, of the enzymes to different degrees. The
severity of this process depends mainly on the HOCl level in the solution milieu. Collectively,
increased MPO/CAT ratio may lead to accumulation of free iron, in addition to modifications of
the structure and function of proteins that are biologically relevant to the initiation and
maintenance of inflammation. On the other hand, increasing CAT:MPO ratio, scavenging
HOCl/H O , and/or inhibiting MPO minimize instant HOCl-mediated damage, thereby keeping
2
2
both enzymes catalytically active. Understanding the mechanism of the mutual influences of
individual enzyme activities should be useful for elucidating disease mechanisms and developing
clinically useful therapy.
Catalase modulates the toxicity of the MPO-H O system
2
2
The toxicity of the MPO/HOCl system could be controlled by multiple factors including CAT
and other related enzymes that catalyze the conversion of H O to H O, HOCl scavengers, and
2
2
2
MPO inhibitors (e.g. taurine, methionine, and melatonin). Disturbance of this balance may play
a crucial role in catalytic function of both MPO and CAT. HOCl may influence MPO/CAT
steady-state catalysis by acting either as a heme ligand [14], or as a mediator of heme destruction
-
[
14, 15], protein modification [40, 41], and protein aggregation [39]. The interaction of OCl
1
3

ACCEPTED MANUSCRIPT
with both MPO and CAT includes its ability to form the Fe(III)-OCl complex and subsequently
generate compounds I and II [14, 39, 42]. Moreover, the ability of both MPO-Fe(III)-OCl and
CAT-Fe(III)-OCl complexes to decay to higher oxidative states, namely Compounds I and II,
was also illustrated by diode array stopped-flow measurements [14, 42-44].
A model showing competition between MPO and CAT for H O and resulting HOCl-
2
2
mediated heme destruction and/or modification
HOCl is a potent oxidant that under normal physiological conditions operates as an antimicrobial
agent [14, 39, 43, 44]. However, under pathological conditions, the rate of HOCl production
becomes excessive, and the compound may mediate protein modification and/or hemoprotein
heme destruction leading to the generation of free iron [14, 39, 43, 44]. A kinetic model that
describes the interaction of HOCl with MPO/CAT intermediates is shown in Fig. 7. Under
-
normal conditions, CAT catalyzes the conversion of H O to H O, whereas MPO employs Cl in
2
2
2
the presence of H O to generate HOCl through the formation of MPO Compound I (ferryl
2
2
porphyrin  cation radical, Fe(IV)=O(+•)). The partitioning of H O consumption between the
2
2
two pathways depends mainly on two enzymes’ concentration and their relative affinity to H O .
2
2
Rapid kinetic measurements have shown that the reaction between CAT and H O followed a
2
2
6
-1
-1
second order reaction with a rate constant of 5.0×10 s M [45]. In parallel, the second order
rate constant for the reaction of MPO−Fe(III) with H O to form MPO Compound I was found to
2
2
7
-1 -1
-
be 1.8×10 M s at 25ºC, whereas the second order rate constant of Compound I with Cl to
6
-1 -1
form HOCl was found to be 4.7×10 M s [46, 47]. HOCl, when accumulated in the
MPO/CAT solution mixture, competes with H O for the catalytic sites of both enzymes and
2
2
binds to the heme iron at a near diffusion rate forming the corresponding Fe(III)-OCl complexes.
1
4

ACCEPTED MANUSCRIPT
Fe(III)-OCl complexes then decay to Compound II through the formation of Compound I.
Compound II is formed through the reaction of HOCl with CAT-Fe(III)/MPO-Fe(III) and is the
prime target of HOCl-mediated heme destruction and subsequent free iron release [14-17].
Alternatively, CAT Compound I reacts with H O to complete its natural catalytic cycle that
2
2
could partially protect against HOCl-mediated heme destruction but not against protein
aggregation [48]. Consistent with this notion, Bonini et al. have demonstrated that the reaction of
CAT with HOCl leads to protein oxidation both in vitro and in cell cultures, which is a primary
event leading to CAT aggregation and consequent loss of function [39]. Taking into
consideration that heme groups in the catalase molecule are deeply buried [49], Krych-Madej
and Gebicka have shown that the sulfur-containing amino acid residues on the protein surface are
the main targets for HOCl modification [42]. Irreversible CAT heme damage was detected by
the authors when the enzyme was treated with higher concentrations of HOCl [42]. Regardless of
enzymatic inactivation through either heme destruction, protein aggregation, or a combination of
both, HOCl mediates the inhibition of both enzymes [39, 42, 50]. The degree of CAT/MPO
inactivation is significantly higher at lower CAT: MPO ratios because only a small portion of
total enzymes is estimated to remain active after sequential addition of H O .
2
2
With H O serving as a limiting substrate of HOCl synthesis under such circumstances,
2
2
modulation in the ratio between both enzymes provides significant protection against HOCl
insults. This protection is due to the increase in H O -consumption by CAT and CAT compound
2
2
I, increase in the heme content provided by MPO and CAT, or a combination of both. This
process is reflected by the shift to the right in the MPO inactivation curve (Fig. 2A). In marked
contrast, increasing the MPO/CAT ratio could lead to more HOCl production, but the MPO
inactivation curve still shifts to the right (Fig. 2B), indicating that the HOCl/heme content ratio
1
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plays a crucial role in hemoprotein inhibition, with H O serving to limit HOCl synthesis under
2
2
such circumstances. MPO contains two heme prosthetic groups while CAT is a tetramer with
four heme groups, which allows the CAT heme groups to be more susceptible to HOCl attacks
[
51, 52]. However, the ability of CAT Compound I to utilize H O as a substrate could favor
2 2
protein modification and enzyme inactivation rather than CAT heme destruction, especially at
low HOCl concentrations, consistent with other reports [39, 42]. The loss of catalytic activity of
both enzymes by HOCl mediated heme destruction and/or protein modification is also supported
by evaluating different MPO inactivation curves that display the same heme content (e.g., 2:1
CAT:MPO (Fig 2A) and 3:1 MPO:CAT (Fig. 2B) but display different degrees of protection.
Our UV-visible studies showed that higher H O concentration was required to inactivate a
2
2
correspondingly higher concentration of MPO and CAT (Fig. 3A), a process that could be
prevented in the presence of MPO inhibitors and/or HOCl scavengers. Taken together, the ability
of HOCl to mediate MPO/CAT inhibition through multiple pathways may provide new insight
into biological roles, particularly in organs that experience exposure to wide variations of
MPO/CAT levels. The prevention of free iron generation by catalase suppresses reactions
between metals and highly reactive free radicals such as the hydroxyl radical (•OH) produced
through the Fenton reaction, which are extremely damaging to tissues and further perpetuate the
inflammatory environment [53, 54].
The crosstalk between MPO and CAT
CAT is an enzymatic antioxidant, which under normal conditions, serves a signaling function
and plays an important role in protecting the cell from oxidative damage mediated by reactive
oxygen species (ROS) [55, 56]. H O is freely diffusible through membranes, and under
2
2
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6

ACCEPTED MANUSCRIPT
pathological conditions, such as during atherosclerosis, asthma, and other inflammatory
processes, rates of H O production become excessive. CAT deficiency or its inactivation by
2
2
HOCl plays an important role in up-regulating the catalytic activity of MPO that occurs at sites
of inflammation. CAT and MPO crosstalk with each other through the diffusion of H O and
2
2
HOCl, whether the enzymes are in different locations or are co-localized in the same cell [57]. In
our current work, we have shown that HOCl, the final product of MPO, promotes CAT
inactivation and MPO heme destruction resulting in the release of oxidants. Taken together, our
findings support a biologically relevant mechanism, which delineates a novel role for MPO in
the pathogenesis of inflammatory diseases.
Alterations of MPO/CAT ratio in respiratory and cardiovascular pathologies
Specific interrelationships between variants of CAT and MPO and their role in the etiology of
respiratory conditions related to oxidative stress have been infrequently explored. Significantly
increased concentrations of plasma MPO, nitric oxide metabolites, and iron were detected in
asthmatic individuals [58]. It is believed that the loss of SOD and CAT activities is related to
oxidative modifications, while other antioxidant gene polymorphisms are linked to the
susceptibility to develop asthma. In support of this, CAT activity was lower in the red blood cells
of asthmatic children and in bronchoalveolar lavage specimens of asthmatic lungs, as compared
to healthy individuals [59]. In addition to asthma, the crosstalk between CAT and MPO also
plays an important role in cystic fibrosis (CF). Although there are no significant differences in
the concentrations or activities of H O in breath condensate of CF patients compared to healthy
2
2
individuals, sputum specimens from CF patients were found to contain large amounts of active
MPO (5.93±4.8 μM) and CAT (0.31±0.18 μM) [1]. Epithelial lining fluid and plasma in CF
1
7

ACCEPTED MANUSCRIPT
patients were characterized by a marked deficiency of non-enzymatic antioxidants such as GSH,
taurine, and methionine compared to controls [1, 60]. An abnormal accumulation of total iron,
and iron-regulatory cytokines such as interleukin-1 beta and tumor necrosis factor alpha were
found in the sputum of the patients with chronic bronchitis and, to a greater extent, in patients
with CF [61, 62]. The involvement of HOCl in mediating the generation of free iron through
heme destruction in disease pathology might highlight its involvement in the production of •OH
in the presence of H O . Accumulation of free iron may promote oxidative injury and enhance
2
2
bacterial growth [63]. Additionally, overload of free iron is also associated with many
inflammatory disorders such as arthritis, diabetes, psychiatric illness, and liver disease, and is
also a risk factor for cancer and heart diseases [4, 5, 7, 64, 65].
Furthermore, MPO and its oxidant products, which predict increased cardiovascular risk, have
been implicated in the pathogenesis of atherosclerosis [66]. MPO mediates lipid peroxidation
and conversion of LDL to an atherogenic form [66]. Recently, it has been shown that MPO-
derived oxidants can also modify HDL, causing an impairment of cholesterol efflux [66]. Thus,
MPO has been considered a useful biomarker for cardiovascular disease [67]. CAT, among other
antioxidant enzymes, has been shown to protect the endothelium of human aorta against
apoptosis associated with oxidized forms of LDL [68]. Such results highlighted the key role of
H O in atherogenesis as well as CAT and other related antioxidant enzymes’ role in insulating
2
2
against oxidative stress of the cardiovascular system by impacting H O -mediated signaling and
2
2
HOCl-production [69, 70]. Therefore, blocking the MPO/HOCl chlorination machinery might be
a useful therapeutic approach in vascular diseases and other inflammatory conditions, where iron
overload is a significant etiopathological factor.
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Conclusion
In conclusion, it is conjectured that CAT and MPO crosstalk with each other, independent of
their localization, through the diffusion of the substrate (H O ) and product (HOCl) in a common
2
2
pro-oxidant mechanism. Therefore, HOCl overproduction, which occurs in many inflammatory
diseases, could lead to the imbalance of MPO and CAT through protein modification and/or
heme destruction. Inactivation of both enzymes leads to H O accumulation, which, when
2
2
combined with free iron, causes further oxidative damage to the host by generation of •OH.
Thus, we describe a HOCl-based regulatory mechanism that enables hemoprotein instant release
of free iron and protein modification that can be attenuated by CAT. This may provide new
insights into biological roles for MPO and CAT, particularly in organs that experience elevated
MPO/HOCl levels. The crosstalk between MPO and CAT is remarkable and suggests that there
are biological roles for their interaction which remain to be considered. These are under current
investigation in our laboratories
Acknowledgements: Dr. Iyad Ali was a Fulbright Senior Research Scholar in Dr. Abu-Soud
laboratory.
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Figure legends
Figure 1. MPO inactivation by self-generated HOCl and its prevention by CAT. (A) A typical
recording by an H O -selective electrode demonstrating the dramatic MPO feedback inhibition
2
2
mediated by self-generated HOCl after incremental consecutive addition of H O (10 µ M, 3 µL
2
2
in 3 ml reaction mixture) (denoted by the arrows) to a continuously stirred phosphate buffer (200
−
mM, pH 7.4) containing 40 nM MPO and 100 mM Cl , at 25°C. Identical experiment
-
performed (B) By replacing MPO/Cl with 40 nM CAT. (C) In the presence of combination of
-
4
0 nm MPO, 40 nM CAT and 100 mM Cl . The data shown are representative of three
independent experiments.
Figure 2. Area under the curves of the H O selective-electrode traces reflecting the maximum
2
2
signal intensity to time of H O consumption by both MPO and CAT, were determined and
2
2
plotted as a function of H O added to the same solution mixture. A) When an incremental H O
2
2
2
2
solution (10 µM, 3 µL) was added to a continuously stirred phosphate buffer (200 mM, pH 7.4)
supplemented with fixed amount of MPO (40 nM) and increasing concentration of CAT (0, 40,
−
5
0,120, and 160 nm) in the presence of fixed amount of Cl (100) mM, at 25°C. B) When an
incremental H O solution (10 µM, 3 µL) was added to a continuously stirred phosphate buffer
2
2
(200 mM, pH 7.4) supplemented with fixed amount of CAT (40 nM) and increasing
−
concentration of MPO (0, 40, 50,120, and 160 nm) in the presence of fixed amount of Cl (100)
mM, at 25ºC. The data shown are representative of three independent experiments.
Figure 3. Melatonin inhibits the MPO chlorinating activity and prevents MPO/CAT inactivation
mediated by HOCl. (A) A typical recording by an H O -selective electrode demonstrating the
2
2
dramatic effect of CAT in protecting MPO from HOCl insults after incremental consecutive
addition of equal amounts of H O (10 µM, 3 µL in 3 ml reaction mixture) (denoted by the
2
2
2
0

ACCEPTED MANUSCRIPT
arrows) to a continuously stirred phosphate buffer (200 mM, pH 7.4) containing 40 nM MPO, 40
−
nM CAT, and 100 mM Cl , at 25°C. (B) An identical experiment performed in the presence of
1
00 µM melatonin. The data shown are representative of three independent experiments.
Figure 4. Absorbance spectra of MPO (0.6 µM) (dashed line), CAT (0.6 µM) (dotted line), and
combination of both (sold line) recorded in 200 mM phosphate buffer, (pH 7.4) from 350-700
nm, at 25ºC. The data shown are representative of three independent experiments.
Figure 5. Effects of methionine in inhibiting MPO/CAT heme destruction mediated by MPO-
generated HOCl. A cuvette containing phosphate buffer supplemented with CAT (~0.6 µM) and
-
1
00 mM Cl was mixed well and used to zero the absorbance reading from 300-500 nm (dotted
line), at 25ºC. The dashed line is the prominent Soret absorbance centered at 430 nm after adding
of MPO (~0.6 µM) to the solution mixture, followed by sequential incremental addition of H O
2
2
2
(20 µM, 3 µl) to the solution mixture. The spectra were recorded immediately after each H O
2
addition (A). Spectra collected 10 min. after each H O addition (B). The same experiments in
2
2
the presence of methionine (100 µM) (C). The data shown are representative of three
independent experiments.
Figure 6. The susceptibility of MPO versus CAT heme destruction by HOCl. A typical
recording by an H O -selective electrode when three consecutive addition of exogenous HOCl
2
2
(
10 µM)/H O (10 µM) to a continuously stirred phosphate buffer (200 mM, pH 7.4)
2 2
supplemented with 40 nM MPO and 40 nM CAT at 25°C to insure MPO/CAT inactivation.
-
The reaction mixture then received 100 mM Cl followed by two consecutive H O additions
2
2
(denoted by the arrows). The data shown are representative of three independent experiments.
Figure 7. Working kinetic model for the H O and HOCl interaction with both MPO and CAT.
2
2
2
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Figure 1
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Figure 2
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Figure 3
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