Characterization of the Magnitude and Kinetics of Xanthine
Oxidase-catalyzed Nitrite Reduction
EVALUATION OF ITS ROLE IN NITRIC OXIDE GENERATION IN ANOXIC
TISSUES*
Haitao
Li,
Alexandre
Samouilov,
Xiaoping
Liu, and
Jay L.
Zweier
From the Molecular and Cellular Biophysics Laboratories,
Department of Medicine, Division of Cardiology and the Electron
Paramagnetic Resonance Center, The Johns Hopkins University School of
Medicine, Baltimore, Maryland 21224
Received for publication, December 26, 2000, and in revised form, April 16, 2001
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ABSTRACT |
Xanthine oxidase (XO)-catalyzed
nitrite reduction with nitric oxide (NO) production has been reported
to occur under anaerobic conditions, but questions remain regarding the
magnitude, kinetics, and biological importance of this process. To
characterize this mechanism and its quantitative importance in
biological systems, electron paramagnetic resonance spectroscopy,
chemiluminescence NO analyzer, and NO electrode studies were performed.
The XO reducing substrates xanthine, NADH, and
2,3-dihydroxybenz-aldehyde triggered nitrite reduction to NO, and the
molybdenum-binding XO inhibitor oxypurinol inhibited this NO formation,
indicating that nitrite reduction occurs at the molybdenum site.
However, at higher xanthine concentrations, partial inhibition was
seen, suggesting the formation of a substrate-bound reduced enzyme
complex with xanthine blocking the molybdenum site. Studies of the pH
dependence of NO formation indicated that XO-mediated nitrite
reduction occurred via an acid-catalyzed mechanism. Nitrite and
reducing substrate concentrations were important regulators of
XO-catalyzed NO generation. The substrate dependence of anaerobic
XO-catalyzed nitrite reduction followed Michaelis-Menten kinetics,
enabling prediction of the magnitude of NO formation and delineation of
the quantitative importance of this process in biological systems. It
was determined that under conditions occurring during no-flow ischemia,
myocardial XO and nitrite levels are sufficient to generate NO levels
comparable to those produced from nitric oxide synthase. Thus,
XO-catalyzed nitrite reduction can be an important source of NO
generation under ischemic conditions.
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INTRODUCTION |
Nitric oxide is an important regulator of a variety of biological
functions, and it also plays important roles in the pathogenesis of
cellular injury. It has been generally accepted that NO is solely
generated in biological tissues by specific nitric- oxide synthases
(NOSs)1 that metabolize arginine to citrulline
with the formation of NO. However,
previous studies have also demonstrated that NOS-independent generation
of NO from nitrite occurs in ischemic tissues, such as the heart,
demonstrating that nitrite can be a source rather than a product of NO,
particularly under acidic conditions (1-3). Although it is
clear that NO formation occurs secondary to nitrite reduction (3),
questions remain regarding the source of the reducing equivalents
required and the role of non-NOS enzymes in this process (1).
Xanthine oxidase (XO) is a ubiquitous enzyme in mammalian cells that
plays important roles in both physiological and pathological conditions. It is involved in the catabolism of purine and pyrimidines, oxidizing hypoxanthine to xanthine and xanthine to uric acid. XO also
reduces oxygen to superoxide and hydrogen peroxide and is one of the
key enzymes responsible for superoxide-mediated cellular injury (4). It
has a central role in the process of injury that occurs upon
reoxygenation of hypoxic cells and tissues (5-7). XO contains critical
flavin, iron sulfur, and molybdenum sites and has some structural
similarity with microbial nitrite reductase (8). Recently, it has been
reported that XO catalyzes reduction of nitrite to NO under hypoxic
conditions (8-10), but a number of important questions have remained
regarding the mechanism, substrate specificity, and magnitude of this process.
XO exhibits broad specificity, accepting a variety of reducing
substrates (11). It was first reported that NADH, but not xanthine, can
act as an electron donor to XO and catalyze nitrite reduction (8, 9).
Xanthine or hypoxanthine was found to inhibit this NO formation from XO
(9). However, more recently, it was reported that xanthine can serve as
a reducing substrate to stimulate nitrite reduction (10). In contrast
to this, other investigators reported that XO in the presence of
xanthine does not reduce nitrite to NO (12). In addition, in these
studies there are major differences regarding the rates of NO formation and Km values of XO for nitrite or the requisite
reducing substrate. Thus, questions remain regarding the substrate
source required as well as the rate of this XO-catalyzed process of NO formation as a function of a given substrate concentration.
Considering the important physiological roles of NO in blood pressure
regulation, vascular tone, neural signaling, and immune function
(13-15) and the functions of the derivatives of NO and superoxide in
biological systems, it is important to determine the mechanism,
magnitude, and role of XO-catalyzed NO generation.
To characterize this XO-catalyzed pathway of NO production and its
quantitative importance in biological systems, electron paramagnetic
resonance (EPR) spectroscopy, chemiluminescence NO analyzer, and NO
electrode studies were performed. The mechanism of NO formation was
shown to occur due to nitrite reduction at the molybdenum site, with
either NADH or xanthine serving as reducing substrates. The kinetic
parameters for nitrite, NADH, and xanthine were determined, enabling
prediction of the magnitude of NO formation and delineation of the
quantitative importance of this process in biological systems.
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EXPERIMENTAL PROCEDURES |
Materials--
Xanthine oxidase from buttermilk (xanthine:
oxygen oxidoreductase) (EC 1.1.3.22), xanthine, oxypurinol,
diphenyleneiodonium chloride (DPI), sodium nitrite,
-NADH, and
2,3-dihydroxybenz-aldehyde were obtained from Sigma.
N-Methyl-D-glucamine dithiocarbamate (MGD) was
synthesized using carbon disulfide and
N-methyl-D-glucamine, as described (16). Ferrous
ammonium sulfate was purchased from Aldrich (99.997%). Dulbecco's
phosphate-buffered saline (PBS) was obtained from Life Technologies Inc.
EPR Spectroscopy--
EPR measurements were performed using a
Bruker-IBM ER 300 spectrometer operating at X-band. Measurements were
performed using a TM110 microwave cavity at ambient
temperature with a modulation frequency of 100 kHz, modulation
amplitude of 2.5 G, and microwave power of 20 mW. NO formation was
measured by spin trapping using the ferrous iron complex of MGD,
Fe-MGD. Solid ferrous ammonium sulfate and MGD (molar ratio, 1:5) were
added to the deoxygenated (argon-purged) PBS buffer with a final
concentration 2 mM in iron. Experiments were performed
under anaerobic conditions achieved by argon purging in a glass-purging
vessel followed by transfer under argon to a quartz flat cell that was
then sealed. Quantitation of NO trapping was determined from the
intensity of NO adduct signal recorded after mixing of Fe-MGD with
aqueous solutions equilibrated with NO gas of known concentration
(3).
Chemiluminescence Measurements--
The rate of the NO
production was measured using a Sievers 270B nitric oxide analyzer
interfaced through a DT2821 A to D board to PC. In the analyzer, NO is
reacted with ozone forming excited-state NO2, which emits
light. Mixing of reagents and separation of NO from the reaction
mixture were done at a controlled temperature of 37 °C in a
glass-purging vessel equipped with heating jacket. An ice-water cooling
condenser was attached to the top of the vessel to reduce the outflow
of vapors during purging. Additionally, an ice-cooled chemical trap
filled with 1.0 M NaOH was placed between the purging
vessel and NO analyzer. The release of NO was quantified by analysis of
the digitally recorded signal from the photomultiplier tube using
specially designed data acquisition and analysis software developed in
our laboratory. Calibration of the magnitude of NO production was
determined from the integral of the signal over time compared with that
from nitrite concentration standards added to acetic acid containing
1% potassium iodide (3). To enhance sensitivity for
measurements of low levels of NO formation, a high quality, low noise
photomultiplier tube was installed in the analyzer.
Electrochemical Measurements--
Electrochemical measurements
of NO generation by XO were carried out at 37 °C using a CHI 832 electrochemical detector with a Faraday cage (CH Instruments, Inc.,
Cordova, IN) and WPI NO electrode (Word Precision Instruments,
Sarasota, FL). The electrochemical detector continuously recorded the
current through the working electrode, which is proportional to the NO
concentration in the solution. The sensor was calibrated before and
after experiments with known concentrations of NO, using NO
gas-equilibrated solutions. The electrode was placed in a closed,
water-jacketed glass electrochemical cell with ports for gas purging.
All measurements were performed at 37 °C with prepurging of argon
gas into the solution for 20 min before measurements, and then
continuous flow of argon above the solution was maintained to assure
anaerobic conditions throughout the measurements.
Statistical Analysis and Kinetic Fitting--
Values are
expressed as mean ± S.D. of at least three repeated measurements,
and the statistical significance of difference was evaluated by
Student's t test. A p value of 0.05 or less was considered to indicate statistical significance. Kinetic fits to the
experimental data were performed on a PC using Table Curve 2D v4
(Jandel Scientific).
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RESULTS |
Role of Reducing Substrates in XO-mediated NO Generation--
NO
is paramagnetic and binds with high affinity to the water-soluble spin
trap Fe2+-MGD, forming a mononitrosyl iron complex with
characteristic triplet spectrum at g = 2.04 with
hyperfine splitting aN = 12.8. From the
intensity of the observed spectrum, quantitative measurement of NO
generation can be performed (17-19). This technique was applied to
measure nitrite-mediated NO generation under anaerobic conditions. In
the absence of nitrite, mixtures of XO (0.1 mg/ml) and its reducing
substrate xanthine (20 µM) gave rise to no signal (Fig. 1A). In the absence of
XO but the presence of nitrite (1 mM), only a trace signal
was seen (Fig. 1B). Whereas xanthine (20 µM)
and nitrite only gave rise to a trace signal (Fig. 1C), upon
addition of the XO reducing substrates NADH (1 mM),
2,3-dithydroxybenz-aldehyde (DBA) (200 µM), or xanthine
(20 µM), a marked increase in the NO signal was seen
(Fig. 1, D-F). Thus, all of the typical types of XO
reducing substrates, including NADH, 2,3-dithydroxybenz-aldehyde, and
xanthine, acted as electron donors for XO-catalyzed nitrite reduction
and triggered large amounts of NO generation under anaerobic conditions.

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Fig. 1.
EPR measurement of NO generation from XO and
nitrite. Spectra are shown of the
(MGD)2-Fe2+-NO adduct formed in solutions of 2 mM (MGD)2-Fe2+ complex in PBS
buffer (pH 7.4) with 0.1 mg/ml XO, 20 µM xanthine
(A); 1.0 mM nitrite (B); 1.0 mM nitrite, 20 µM xanthine (C);
1.0 mM nitrite, 0.1 mg/ml XO, and 1.0 mM NADH
(D); 1.0 mM nitrite, 0.1 mg/ml XO, and 200 µM 2,3-dithdroxybenz-aldehyde (E); or 1 mM nitrite, 0.1 mg/ml XO, and 20 µM xanthine
(F). Spectra were obtained after incubation for 30 min at
37 °C under anaerobic conditions.
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To further confirm the existence of the XO-catalyzed mechanism of NO
generation in the absence of any possible effects or perturbation by
the spin trap, studies were performed measuring NO generation using a
specific electrochemical NO sensor. Prior to the addition of XO, no
detectable NO generation was seen from nitrite (1 mM) in
the presence of xanthine (10 µM) or NADH (1 mM). However, after addition of XO (0.015 mg/ml), prominent
NO generation was triggered from xanthine or NADH (Fig.
2, traces A and B).
The magnitude and rate of NO generation in the presence of xanthine
(Fig. 2, trace A) was considerably higher than that with NADH (Fig. 2, trace B), whereas with xanthine and
nitrite in the absence of XO, no NO generation was observed (Fig. 2,
trace C). Of note, the substrate DBA also stimulated NO
generation from nitrite in the presence of XO, with magnitude and rate
approaching that seen from xanthine (data not shown).

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Fig. 2.
Electrochemical measurement of NO generation
from XO and nitrite. The time course of NO generation was measured
using an electrochemical NO sensor under anaerobic conditions at
37 °C in PBS, pH 7.4. The arrow shows the time at which
XO (0.015 mg/ml) was added to A and B. Tracing A shows the data for nitrite (1.0 mM) in
the presence of xanthine (10 µM), B shows
nitrite (1.0 mM) in the presence of NADH (1.0 mM), and C shows nitrite (1.0 mM)
and xanthine (10 µM) without XO added.
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Thus, both EPR spin trapping and NO electrode studies demonstrate that
XO can generate NO production in the presence of substrates that reduce
the enzyme. To confirm that XO-mediated NO generation occurs and to
directly measure the rate of this NO generation, further studies were
performed using a chemiluminescence NO analyzer. Mixing of reagents was
performed at 37 °C in the glass-purging vessel. NO was purged from
the solution with argon gas and then reacted with ozone in the analyzer
to form excited-state NO2, which emits light. This method
provides direct measurement of the rate of NO generation as a function
of time (3). With nitrite (1 mM) alone or nitrite in the
presence of xanthine (5 µM) or NADH (1 mM),
no measurable rate of NO formation was observed (Fig. 3, trace C). However, with the
addition of XO (0.02 mg/ml) in the presence of xanthine or NADH,
prominent NO generation was triggered (Fig. 3, traces A and
B). Additional control experiments performed with XO in the
presence of xanthine or NADH but in the absence of nitrite confirmed
that nitrite was required for NO generation. Again, a higher rate of
XO-mediated NO generation was seen with xanthine than with NADH. As was
seen with the electrode measurements, the substrate DBA (40 µM) also stimulated NO generation from nitrite in the
presence of XO with a rate approaching that seen with xanthine (data
not shown).

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Fig. 3.
Measurement of the rate of NO generation from
XO-catalyzed nitrite reduction. Measurements were performed using
a chemiluminescence NO analyzer under anaerobic conditions at 37 °C
in PBS, pH 7.4. The arrows show the time at which XO (0.02 mg/ml) was added to A and B. Tracing A
shows the data for 1.0 mM nitrite and 5 µM
xanthine, B shows 1.0 mM nitrite in the presence
of 1.0 mM NADH, and C shows 1.0 mM
nitrite without XO.
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Kinetics of XO-catalyzed Nitrite Reduction--
The rate of NO
formation derived from XO-catalyzed nitrite reduction was measured
under anaerobic conditions using the NO analyzer. The concentration
dependence of each reducing substrate was first determined in the
presence of a fixed nitrite concentration of 1 mM. Each of
the typical reducing substrates, xanthine, DBA, and NADH, acted as
electron donors to support XO-catalyzed nitrite reduction, and each of
these reactions followed Michaelis-Menten kinetics (Fig.
4, A-C). For each reducing
substrate, the apparent values of Km,
Kcat, and Vmax were
determined by fitting the data to the Michaelis-Menten equation, and
the values for each reducing substrate are shown inside each curve. For
each of these reducing substrates, the rate of XO-mediated NO formation was also determined as a function of nitrite concentration (Fig. 4,
D-F). Again, typical Michaelis-Menten kinetics were
observed as a function of nitrite concentration, and the apparent
Km, Kcat, and
Vmax values are shown inside each curve. From
these kinetic data, it is possible to predict the magnitude of
XO-catalyzed NO formation as a function of nitrite and reducing
substrate concentration and to determine the quantitative importance of
this mechanism of NO generation in a given biological system where
these substrate levels are known.

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Fig. 4.
Kinetics of NO generation from XO as a
function of reducing substrate or nitrite concentration. Initial
rates of NO generation were measured by chemiluminescence NO analyzer
as described in Fig. 3. A shows the effect of [NADH] on
the rate of NO generation from 0.04 mg/ml XO and 1.0 mM
nitrite. B shows the effect of [xanthine] on the rate of
NO generation from 0.02 mg/ml XO and 1.0 mM nitrite.
C shows the effect of [2,3-dithdroxybenz-aldehyde] on the
rate of NO generation from 0.02 mg/ml XO and 1.0 mM
nitrite. D shows the rate of NO generation by 0.04 mg/ml XO
and 1.0 mM NADH in the presence of 0.2-4 mM
nitrite. E shows the rate of NO generation by 0.02 mg/ml XO,
5 µM xanthine in the presence of 5 µM-2.5
mM nitrite. F shows the rate of NO generation by
0.02 mg/ml XO, 40 µM 2,3-dithdroxybenz-aldehyde in the
presence of 0.5 mM-5 mM nitrite. For each of
these graphs, the corresponding fits (solid lines)
Km, Vmax, and
Kcat data were obtained using the
Michaelis-Menten equation.
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Determination of the Mechanism and Reaction Site of Nitrite
Reduction--
The effects of site-specific inhibitors of XO were
studied to investigate the reaction sites involved in the process of
the XO-catalyzed nitrite reaction observed with different reducing substrates. Oxypurinol binds to the molybdenum site of XO. It was observed that oxypurinol inhibited XO-catalyzed nitrite reduction regardless of the type of reducing substrate present. Near total inhibition of NO generation was seen in the presence of either xanthine
or NADH (Fig. 5). Because oxypurinol
inhibits substrates binding at the molybdenum site of the enzyme, this
suggests that nitrite binds to the reduced molybdenum site. DPI,
which acts at the FAD site, inhibited XO-dependent nitrite
reduction only when NADH was used as the reducing substrate, and it did
not inhibit NO generation when xanthine was used (Fig. 5). This
suggests that NADH donates electrons to FAD, and then electrons are
transported back to reduce the molybdenum that in turn reduces nitrite
to NO. When xanthine or aldehydes are the electron donors, both XO reduction (by xanthine or aldehydes) and oxidation (by nitrite) takes
place at the molybdenum site, so that only oxypurinol could inhibit
XO-dependent NO formation.

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Fig. 5.
Effect of site-specific inhibitors on
XO-mediated NO formation. Rates of NO generation were measured by
chemiluminescence NO analyzer as described in Fig. 3. The inhibitive
effect of oxypurinol, which binds to the molybdenum site, and DPI,
which modifies the flavin, were determined for xanthine (X)-
or NADH-mediated NO generation. For the left set of bars,
experiments were performed with 0.5 mM nitrite, 5 µM xanthine, and 0.02 mg/ml XO, and for the right
set of bars, experiments were performed with 1.0 mM
nitrite, 1.0 mM NADH, and 0.04 mg/ml XO. Control, without
inhibitor; DPI, 20 µM; oxypurinol, 20 µM.
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Inhibitive Effect of High Xanthine Levels on NO Generation--
It
has been reported that excess xanthine (>100 µM) can
exert inhibition of XO catalytic function due to binding of xanthine to
reduced forms of the enzyme generated in the process of XO-catalyzed oxygen reduction. This kind of substrate-bound reduced XO complex inhibits intramolecular electron transport from the molybdenum center
to FAD (20, 21). In view of the questions regarding the role and
potency of xanthine as a reducing substrate for the process of
XO-mediated NO formation, NO analyzer and EPR spin trapping studies
were performed to detect XO-catalyzed NO generation under anaerobic
conditions in the presence of high concentrations of xanthine (10-100
µM). Although XO-catalyzed NO generation followed classic
Michaelis-Menten kinetics with low concentrations of xanthine (<20
µM, Fig. 4B), higher concentrations of
xanthine showed significant concentration-dependent
inhibition of NO formation (Fig. 6). From fitting of the data in Fig. 6A, Ki was
calculated to be 55 µM. These results suggest that a
xanthine-reduced XO complex is formed and inhibits the maximum rate of
NO generation from XO-catalyzed nitrite reduction. It is likely that
excessive xanthine inhibits XO-catalyzed nitrite reduction by binding
to reduced forms of the enzyme, in turn blocking the binding of nitrite
to the molybdenum site.

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Fig. 6.
Inhibition of XO-mediated NO generation by
high xanthine concentrations. A, rates of NO generation
were measured by chemiluminescence NO analyzer as described in Fig. 3
from 1.0 mM nitrite in the presence of different xanthine
concentrations (1-100 µM), with XO (0.02 mg/ml) under
anaerobic conditions, pH 7.4, 37 °C. The points show the
measured experimental values ± S.D., and the line
shows a least squares fit of the data to the equation with competitive
inhibition (Equation 9). A good fit was obtained with a correlation
coefficient r2 > 0.98. B, NO
generation measured by EPR spin trapping with
(MGD)2-Fe2+ after 30 min in anaerobic
conditions, pH 7.4, 37 °C. Production of NO by XO (0.02 mg/ml)-catalyzed nitrite (1.0 mM) reduction was measured as
a function of xanthine concentration (10-100 µM). With
both techniques, significant inhibition was seen with high xanthine
concentrations.
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NO Generation under Conditions Occurring in Ischemic
Tissues--
To determine the magnitude of NO generation that can
occur in biological tissues that typically contain low nitrite
concentrations, measurements of XO-derived NO generation were performed
in the presence of 5-40 µM nitrite, the range of
concentrations observed in myocardial tissue (1-3). A process of
xanthine-stimulated NO generation was seen that was similar to that
observed with higher nitrite levels (Fig.
7). Maximum rates of NO generation of 18, 31, 56, and 135 pmol/s/mg were observed for 5, 10, 20, and 40 µM nitrite, respectively, at xanthine concentrations
ranging from 1 to 10 µM. With further increase in
xanthine levels, progressive inhibition was seen, as was observed with
1 mM nitrite (Fig. 6).

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Fig. 7.
XO-mediated NO formation with cellular
nitrite levels. Initial rates of NO generation as a function of
xanthine concentration were measured by chemiluminescence NO analyzer
with 5, 10, 20, or 40 µM nitrite and 0.02 mg/ml of XO,
with measurements performed as described in Fig. 3. With low nitrite
concentrations, typical of cellular levels, the process of NO
generation was generally similar to that observed with higher,
mM levels. Maximum rate of NO generation was seen with low
xanthine concentrations followed by progressive inhibition at
high substrate levels. The points show the measured
experimental values with ± S.D., and the line shows a
least squares fit of the data to the rate equation with competitive
inhibition (Equation 9). A good fit was obtained for each curve with
correlation coefficients r2 > 0.97. From these
fits of the data to Equation 9, the Vmax
values were determined to be: 18.9, 33.6, 61.6, and 148.9 pmol/s/mg,
and the Km values were determined to be 0.012, 0.026, 0.053, and 1.0 µM for 5, 10, 20, and 40 µM nitrite, respectively, whereas the
Ki value remained almost constant at 53 µM.
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Effects of pH on XO-catalyzed NO Generation--
Under ischemic
conditions, marked intracellular acidosis occurs, and pH values in
tissues, such as the heart, can fall to levels of 6.0 or below (2). In
order to assess the NO formation under different physiological or
pathological conditions and to further characterize the mechanism of
XO-catalyzed nitrite reduction, experiments were performed to
measure the effect of different pH values on the magnitude of NO
generation. Measurements were performed with 0.02 mg/ml of XO in the
presence of 1 mM nitrite. As shown in Table
I, it was observed for each of the three
substrates xanthine, NADH, and DBA that under acidic conditions
increased XO-catalyzed NO generation occurs. In contrast, under
alkaline conditions, prominent inhibition was seen.
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Table I
Effect of pH on NO generation rate
Measurements were performed with 0.02 mg/ml XO in the presence of 1 mM nitrite.
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DISCUSSION |
In view of the critical role of NO in normal physiology and
disease, it is of crucial importance to understand the biochemical mechanisms of NO formation that occur in biological cells and tissues.
In addition to the formation of NO from specific NOS enzymes, it is
clear that nitrite derived from either NO metabolism or dietary sources
can be an important source of NO formation, particularly under
conditions of limited tissue perfusion and resulting acidosis. Although
NO can be formed by the simple process of nitrite disproportionation
that is accelerated under acidic conditions, it has been previously
observed that in ischemic tissues, nitrite is also reduced to form NO.
Questions have remained regarding the role of non-NOS enzymes in this
process of nitrite reduction (2).
Although it has recently been reported that XO can reduce nitrite to
NO, questions remain regarding the biological importance of this
pathway of NO production, as well as the mechanism, magnitude, and
substrate specificity of this process. It was first reported that NADH,
but not xanthine, can act as an electron donor to XO and catalyze
nitrite reduction (8, 9). Xanthine or hypoxanthine was found to inhibit
this NO formation from XO (9). However, more recently, it was reported
that xanthine can serve as a reducing substrate to stimulate nitrite
reduction (10). In contrast to this, other investigators reported that
XO in the presence of xanthine does not reduce nitrite to NO (12). In
addition, in these studies, there were large differences regarding the
rates of NO formation and Km values of XO for
nitrite or the requisite reducing substrate. In view of these
uncertainties, it has not been possible to ascertain the biological
relevance and importance of this pathway of NO generation. Therefore,
we performed a series of studies using EPR, chemiluminescence NO analyzer, and NO electrode techniques to measure the magnitude and
kinetics of NO formation that arises due to XO-mediated nitrite reduction.
Data obtained using each of these three methods confirmed that XO does
reduce nitrite to NO under anaerobic conditions. It was observed that
each of the typical reducing substrates xanthine, DBA, and NADH could
act as electron donors to support this XO-mediated nitrite reduction
(Figs. 1-3). The results of these studies, along with the inhibition
seen with oxypurinol, suggested that reduced XO was the direct electron
donor to nitrite, with nitrite binding and reduction occurring at the
molybdenum site. Whereas NADH-stimulated NO generation was inhibited by
the flavin modifier DPI, NO generation stimulated by xanthine or
DBA was unaffected. Thus, whereas xanthine or DBA directly reduces the
molybdenum center, NADH initially reduces the flavin, which
subsequently transfers electrons to the molybdenum.
From past studies, questions remain regarding the Km
and Vmax values observed for
XO-mediated NO generation under anaerobic conditions as a function of
nitrite and reducing substrate concentration. Initial studies reported
a Km of 22.9 mM for nitrite reduction in
the presence of NADH (9). However, a subsequent study reported a
Km value of 2.4 mM (8). A more recent
study measuring NADH depletion rather than NO generation reported a
Km of 16 mM (10). In our studies, we
observed that the Km for nitrite was consistently
about 2.3 mM in the presence of 1.0 mM NADH.
This observation agrees closely with the report of Zhang et
al. (8). In contrast with the first two studies that report only
enzyme reduction by NADH and the recent study reporting a
Km of 36 mM for nitrite in the presence
of xanthine, we observed that the Km for nitrite is
2.4 ± 0.2 mM for each of the three types of
substrates studied, NADH (1 mM), xanthine (5 µM), and DBA (40 µM). Possible reasons for
variable results of the prior studies could relate to the conditions
used for NO purging from the solutions, leak of oxygen into the
measurement system, or partial enzyme inactivation or denaturation.
Indeed, in the most recent report, Godber et al. (10)
acknowledge that phase equilibration and gas flow factors led them to
delay their measurements for 2 min after initiation of the reaction. In
their system, they measure the spontaneous liberation of NO into the
gas headspace and follow steady state conditions with equilibration of
the XO in the presence of NO that is generated. Because only a small
fraction of the NO generated is used for detection, this approach
limits the sensitivity that can be obtained, and therefore, these
studies were performed with high nitrite concentrations in the range of
5 to 120 mM, about 1000 times above typical tissue levels.
In contrast, in our study we rapidly purged all the NO into the gas
phase and this enabled efficient NO detection and limited NO-mediated
effects on the enzyme, enabling detection of the initial rate of the
enzyme with physiological/pharmacological nitrite concentrations of 5 µM to 1 mM. Of note, in our study we measured
the NO formation rate within the first 40 s of XO addition to the
purging vessel, and parallel measurements of the rates of NO generation
performed by NO electrode yielded similar values.
Although xanthine was the highest efficiency reducing substrate of
XO-catalyzed nitrite reduction, excessive xanthine exhibited inhibition
of NO production. Previous studies reported that enzyme inactivation
resulted from NO-induced conversion of XO to its relatively inactive
desulfo-form (10). However, this could not explain why these
investigators observed that XO kept its activity (>95%) when NADH
acted as reducing substrate (10). Furthermore, we also observed that
the presence of excess NADH (>10 mM) or DBA (>2
mM) had no inhibitive effect on XO-catalyzed NO generation. In a previous study, we showed that ONOO
markedly
inhibits XO activity in dose-dependent manner, whereas NO
from NO gas in concentrations up to 200 µM had no effect
(22). So inactivation of XO (10) could be caused by ONOO
formation triggered upon exposure to oxygen at the time of
spectrophotometric activity assay. Our results suggest that excessive
xanthine acts to inhibit XO by binding to the molybdenum site of the
reduced enzyme (20, 21), thus blocking the binding of nitrite at this enzyme site. This xanthine-mediated inhibition, which has also been
demonstrated by Godber et al. (10), may explain the
prior failure to detect XO-mediated NO generation from nitrite in
studies in which 150 µM xanthine were used (12).
It has been reported that purine and aldehyde substrate hydroxylation
takes place via a base-catalyzed mechanism and that substrate must be
protonated for hydroxylation (10). The rate of XO reduction by purine
and aldehydes greatly increases when the pH value is increased from 6.0 to 8.0, and this increased rate of XO reduction will lead to an
increased rate of nitrite reduction. However, our experiments showed
that acidic conditions promote XO-catalyzed nitrite reduction. NO
generation increased as the pH was decreased from 8.0 down to 7.4 or
from 7.4 down to 6.0, suggesting that nitrite reduction takes place via
an acid-catalyzed mechanism, presumably due to nitrite protonation.
HNO2 concentration increases when the pH decreases, and it
could be the direct binding substrate of XO. Although the decrease of
pH would decrease the rate of XO reduction by reducing substrates, it
would greatly increase the speed of XO oxidation by
nitrite/HNO2.
From the studies performed, it is clear that XO can catalyze the
process of NO generation from nitrite under anaerobic or markedly
hypoxic conditions similar to those occurring in ischemic tissues. The
key questions are, what is the magnitude of this process, and whether
the levels of NO produced are likely to have functional significance.
To address these critical questions, a kinetic model can be constructed
that enables prediction of the magnitude of XO-catalyzed NO formation
and understanding the quantitative importance of this mechanism of NO
generation in biological systems. The following equations define the
steps in the reaction mechanism.
|
(Eq. 1)
|
|
(Eq. 2)
|
|
(Eq. 3)
|
where EOX is the fully oxidized
enzyme, Ered is the 2-electron reduced enzyme,
and E'red is the 1-electron reduced enzyme. S refers to the reducing substrate of XO, such as xanthine,
and P is the corresponding product. It should be noted that
for each xanthine oxidized, 2 molecules of nitrite could be reduced to NO. The total enzyme concentration, [Et], can thus
be defined as follows.
|
(Eq. 4)
|
From Equations 1-4, the rate of NO generation can be derived, and
this can be expressed in the form of the Michaelis-Menten equation,
|
(Eq. 5)
|
where terms are defined as follows.
To consider the inhibitive effect of xanthine, two more equations
must be considered.
|
(Eq. 6)
|
|
(Eq. 7)
|
The total enzyme concentration [Et] is
defined as follows.
|
(Eq. 8)
|
The rate of NO generation can be expressed as follows,
|
(Eq. 9)
|
where terms are defined as follows.
It was observed that over a broad range of physiological nitrite
concentrations from 5 to 40 µM nitrite that Equation 9
provided a good fit to the experimental data measuring the rate of NO
generation from XO in the presence of xanthine (Fig. 7). Also, at
higher nitrite levels of 1 mM, a good fit of the
experimental data was obtained (Fig. 6). From Equation 9, it is
predicted that Km, Vmax, and
Ki can vary as a function of nitrite concentration. The equation for Ki expression can be simplified as
follows:
|
(Eq. 10)
|
where a and b are constants determined from
fitting of the experimental data. From the experimental data,
a = 53 µM and b = 2 × 10
3. Thus, the Ki
expression predicts that Ki will remain almost
constant, with a value of 53 µM at low nitrite
concentrations (
1 mM), but would greatly increase at
high nitrite levels of 5 mM and above, consistent with the
results of Godber et al. (10).
Our experiments also showed that nitrite reduction took place via an
acid-catalyzed mechanism; thus, we suggest that protonated nitrite
(HNO2) may directly bind to reduced XO. Decrease of pH increases the HNO2 concentration but will decrease
[Ered] because xanthine and aldehydes donate
electrons to XO via a base-catalyzed mechanism (10). Overall NO
generation depends on the combination of these opposite effects of NO
on XO reduction and nitrite reduction.
It has been previously demonstrated that the activity of XO in the
postischemic rat heart is 16.8 milliunits/g of protein (23), which
corresponds to 0.013 mg of XO/g of protein or ~3.4 µg/g of cell
water. The total XO and xanthine dehydrogenase (XDH) activity, however,
is 10-fold above this value. In the ischemic heart, xanthine levels
rise from near zero to values on the order of 10-100 µM,
and nitrite levels are ~10 µM (1, 2, 23). At normal pH
values of 7.4, the rate of nitrite degradation due to simple chemical
disproportionation is ~ 0.05 pM/s, as previously reported (3), whereas the rate of XO-catalyzed nitrite reduction would
be ~ 100 pM/s. When pH decreases to 6.0, the rate of
nitrite disproportionation will increase to 4 pM/s (3),
whereas the rate of XO-catalyzed nitrite reduction would be estimated
to increase to ~115 pM/s. If XDH catalyzes this process
or is converted to XO, the magnitude of this NO generation would be
increased by up to an order of magnitude. It has been previously
reported in studies from rat heart homogenates that maximally activated
nitric oxide synthase produces 1.5 nM/s NO. Thus xanthine
oxidoreductase-mediated NO generation could approach that of the
maximal NO production from NOS. Under conditions with increased tissue
nitrite concentrations, the magnitude of NO production from this
pathway would be further increased; however, it is also clear that with
marked elevations in xanthine, it would be inhibited. Because under the
acidic and markedly hypoxic conditions occurring during ischemia, NOS
does not function to synthesize NO, this NOS-independent NO generation could be of particular importance. Indeed, it has been shown that the
acidosis occurring during ischemia results in reversible denaturation of NOS, which progresses to irreversible denaturation and enzyme degradation (24). Thus, XO-catalyzed nitrite reduction could be an
important source of NO generation in the ischemic heart. NO derived
from nitrite would accumulate during ischemia. Initially, it could
serve to provide protection via compensatory vasodilatation, whereas
upon reperfusion it would react with superoxide, forming peroxynitrite,
which can result in protein nitration and cellular injury (25, 26).
XDH is the reduced form of xanthine oxidoreductase, and XDH prefers NAD
as an electron acceptor but will use molecular oxygen in the absence of
NAD (27-29). XDH also contains the same active centers as XO, and it
can convert to XO by oxidation of protein cysteine residues to
cystines; this causes a conformational change in the vicinity of the
FAD (27, 30, 31). In rat hearts, XDH concentration is 5-10 times
higher than the concentration of XO (23). It was recently reported that
NO generation also occurs from XDH in a manner similar to that seen
with XO (10). Thus, it is likely that overall production of NO from
xanthine oxidoreductase would be substantially higher than that
estimated for XO alone.
Overall, it is clear that XO-mediated NO generation can potentially be
an important source of NO under ischemic conditions in biological
tissues that contain substantial levels of the enzyme along with
nitrite and reducing substrates. In tissues such as the liver and
gastrointestinal tract, which contain high levels of the enzyme, this
could be even more pronounced than for the example of the heart
considered above (32). Beyond the obligatory need for the enzyme, the
levels of tissue nitrite and enzyme reducing substrates have a critical
role in controlling this process. Nitrite is required, and overall it
is the most limiting substrate, because its Km is
~2.5 mM, whereas typical tissue levels of nitrite are at
least 2 orders of magnitude below this value. A number of factors that
increase tissue nitrite levels, such as prior activation of
constitutive or inducible NOS in inflammatory conditions, dietary
sources, pharmacological sources, or bacterial sources, could all
modulate this pathway of NO generation. This pathway also requires a
reducing substrate, such as NADH or xanthine. Xanthine was the most
effective substrate, triggering NO generation under anaerobic
conditions with a Vmax 4-fold higher than that of NADH (Fig. 4). Although only low xanthine concentrations are required, because its Km value is about 1.5 µM, high levels of xanthine, above 20 µM,
resulted in prominent substrate-mediated inhibition (Fig. 6). If
particularly high levels of xanthine accumulate, this pathway would be
inhibited, and perhaps this may serve a regulatory role to prevent
overproduction of NO.
Thus, XO can be an important source of NOS-independent NO generation.
Under anaerobic conditions, XO reduces nitrite to NO at the molybdenum
site of the enzyme with xanthine, NADH, or aldehyde substrates serving
to provide the requisite reducing equivalents. The
substrate-dependent rate relationship for anaerobic nitrite reduction by XO was determined, and it was demonstrated that under conditions of tissue ischemia, the rate of NO generation is greatly increased above the rate of nitrite disproportionation. This NO production from the enzyme could serve as an alternative source of NO
under ischemic conditions in which NO production from NOS is impaired.
 |
FOOTNOTES |
*
This work was supported in part by National Institutes of
Health Grants HL38324, HL63744, and HL65608 and by an AHA
Scientist Development grant (to X. L.).The costs of publication of this article were defrayed in part by the
payment of page charges. The 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: EPR Center, Johns
Hopkins Asthma and Allergy Center, 5501 Hopkins Bayview Circle, Rm.
LA-14, Baltimore, MD 21224. Tel.: 410-550-0339; Fax: 410-550-2448; E-mail: jzweier@welch.jhu.edu.
Published, JBC Papers in Press, April 18, 2001, DOI 10.1074/jbc.M011648200
 |
ABBREVIATIONS |
The abbreviations used are:
NOS, nitric-oxide
synthase;
XO, xanthine oxidase;
XDH, xanthine dehydrogenase;
NADH, nicotinamide adenine dinucleotide;
DBA, 2,3-dithydroxybenz-aldehyde;
NO, nitric oxide;
MGD, N-methyl-D-glucamine
dithiocarbamate;
EPR, electron paramagnetic resonance;
DPI, diphenyleneiodonium chloride;
FAD, flavin-adenine dinucleotide;
PBS, phosphate-buffered saline..
 |
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