From the Department of Pathology and Center for Free Radical Biology, University of Alabama at Birmingham, Birmingham, Alabama 35294
Received for publication, April 21, 2003 , and in revised form, May 1, 2003.
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ABSTRACT |
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INTRODUCTION |
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Besides the ubiquitous cellular defenses against oxidative stress such as superoxide dismutase and glutathione (4, 5), CcOX has recently been proposed to contribute to the antioxidant defenses of mitochondria by directly scavenging ONOO (68). Studies performed on purified CcOX have shown that the enzyme in turnover may represent a significant sink for ONOO (68). The detailed mechanisms are not entirely clear with one study indicating a catalytic production of NO from the interaction of ONOO with the enzyme (6) and other studies suggesting a peroxynitrite reductase activity in CcOX with the product being nitrite (7, 8). Furthermore, it has been suggested that NO can be oxidized to nitrite by CcOX directly (9, 10) or by oxygen in a reaction that is accelerated in the lipid bilayer of mitochondrial membranes (11, 12). The suggestion that CcOX can metabolize ONOO is in agreement with other studies showing that some hemeproteins and synthetic iron-porphyrin complexes can scavenge this highly reactive species (see Refs. 13 and 14 for recent reports).
Despite these observations, the potential interaction of ONOO with the high concentrations of reductants used to assess purified CcOX activity has not been defined (68). Specifically ascorbate, a ubiquitous antioxidant, is used in some of these studies at millimolar concentrations. This is potentially important because ascorbate has been shown to increase NO bioavailability in blood vessels, an effect which may be mediated by enhanced synthesis of NO and/or by prevention of its breakdown (15). In the mitochondrion the role of ascorbate is unknown, but its concentration in the mitochondrial matrix has been estimated in the low millimolar range.2 Moreover, Li et al. (16) have recently proposed that the mitochondrial respiratory chain can recycle ascorbate from its oxidized form (DHA) to the reduced form. In this study, we investigated the possible contribution of ascorbate to the release of NO from ONOO in the presence and absence of CcOX using both the purified enzyme and isolated mitochondria.
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MATERIALS AND METHODS |
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Polarographic measurements (21) were performed in a thermostated chamber equipped with electrodes for oxygen and NO (Instech, Plymouth Meeting, PA and WPI, Sarasota, FL, respectively). The NO electrode was calibrated using the acidified nitrite/potassium iodide method as recommended by the supplier (World Precision Instruments, Inc., Sarasota, FL). In experimental traces, a known amount of authentic NO (from the calibrated stock solution) was always added at the end to quantify the amount of NO generated in the experiment. Data were digitally recorded and analyzed with Windaq software (Dataq Instruments, Inc., Akron, OH). Unless otherwise stated, all isolated CcOX incubations were performed at 20 °C in 25 mM K+ phosphate buffer, pH 7.3, with 0.1% w/v lauryl maltoside. Isolated mitochondrial incubations were performed in respiration medium comprising KCl (100 mM), sucrose (25 mM), HEPES (10 mM), MgCl2 (5 mM), KH2PO4 (5 mM), EGTA (1 mM), pH 7.3, at 37 °C. Errors are expressed as standard deviations of at least three independent experiments unless otherwise specified. Statistical significance was assessed with an unpaired Student's t test and a p value < 0.05.
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RESULTS |
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To assess the role of CcOX in the production of NO from ONOO, we added ONOO to ascorbate, TMPD, or cytochrome c2+ alone under the same conditions as described previously. As Fig. 1C shows, addition of ONOO to ascorbate produced a significant amount of NO comparable with that observed when ONOO was added to CcOX in turnover (2.0 ± 0.02 versus 2.4 ± 0.13 µM; quantified in Fig. 1D). This suggests that ascorbate, not CcOX, is mainly responsible for the NO generation seen in Fig. 1A. The concentration of ascorbate in the cytosol (22, 23) and the reported ascorbate levels in the mitochondrial matrix (see the Introduction) suggest that this mechanism of NO generation from ONOO may be relevant to mitochondrial physiology.
However, these data do not preclude the possibility that CcOX alone may contribute to the NO generation from ONOO. To test this, we examined isolated mitochondria respiring in the presence of glutamate, malate, and ADP (Fig. 1E). In this system, electrons are delivered to CcOX through the respiratory chain (complex I ubiquinone complex III cytochrome c complex IV), thus reducing substrates such as ascorbate are not required. Addition of ONOO to respiring mitochondria resulted in neither NO generation nor inhibition of respiration. However, in the presence of 1 mM ascorbate NO was produced and consequently respiration was inhibited. The effect of authentic NO on mitochondrial respiration is also shown further suggesting that the inhibition of respiration caused by ONOO is due to NO.
Taken together, the data in Fig.
1 show that at 50100 µM
ONOO, CcOX does not catalyze the reduction of
ONOO to NO, whereas ascorbate does. However, the
concentrations of ascorbate used in the experiments described above are either
nonphysiological (20 mM) or in the high intracellular physiological
range (1 mM) (22,
23). To further investigate
the ability of ascorbate to release NO from ONOO, we
measured NO production at different concentrations of ascorbate and
ONOO. Fig.
2A shows that ONOO addition to
phosphate buffer at neutral pH did not result in significant release of
NO. Even upon subsequent addition of ascorbate, NO was not
produced, thus ruling out the possibility of NO generation from a
reaction between ascorbate and the products of ONOO
decomposition, and
(24). However, when
ONOO was added again to the chamber in the presence of
ascorbate (50 µM), a transient production of NO was
observed. The height of the NO peak was proportional to the
concentration of ONOO in the range 10100
µM and to the concentration of ascorbate up to 1 mM
(Fig. 2, B and
C). However, above 1 mM ascorbate, the
generation of NO reaches a plateau. The saturation kinetics in the
supra-millimolar range suggest that the production of NO does not occur
through a direct reaction between ONOO and ascorbate but
through a more complex mechanism.
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To provide further insight to the NO production by
ONOO and ascorbate, we studied the yield of NO under
different conditions (Fig. 3).
Because the reactivity of both ascorbate and ONOO might be
affected by even trace catalytic amounts of transition metals, we performed
the same experiment described in Fig.
2A, in the presence of the metal chelators, neocuproine,
EDTA, or DTPA (Fig. 3, A and
B). The peak height of NO generated was not
decreased significantly by neocuproine or DTPA and only slightly by EDTA
(10% less NO versus control, p = 0.003).
Interestingly, DTPA increased NO stability, as measured by the slope of
the decomposition curve for NO, by
5-fold compared with control
(p = 0.04).
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To determine whether the generation of NO from ascorbate plus
ONOO requires the reduced form of ascorbate, we studied the
interaction of ONOO with DHA, the oxidized form where one of
the hydroxyl groups of ascorbate is replaced by a carbonyl.
Fig. 3C shows that
although a small amount of NO is still generated (5% of control),
the reaction yield is significantly reduced suggesting that ascorbate must act
as a reductant for this reaction to occur.
The effects of NaHCO3 and superoxide dismutase are also shown in
Fig. 3C. These data
show that generation of NO from ascorbate plus ONOO is
abrogated by the presence of NaHCO3. In contrast, superoxide
dismutase enhanced the generation of NO by a small but significant
amount (p = 0.043). Because the stability of ONOO
is affected by several factors, including the equilibrium with its protonated
form (ONOOH, pKa 6.8), we also studied the
effect of pH on the amount of NO produced
(Fig. 3D). A decrease
in the yield of the reaction was observed with decreasing pH.
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DISCUSSION |
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In considering a possible mechanism for the formation of NO from
ascorbate plus ONOO, the data in
Fig. 3 are of particular
importance. First, the observation that less NO is made at acidic pH
suggests that the source of NO is ONOO rather than
ONOOH (pKa 6.8). This is consistent with
the observation that NaHCO3 abolished NO formation in this
system, because it is known that CO2 can react with
ONOO but not ONOOH
(26).
Several mechanisms may exist for the generation of NO from
ONOO. For example, it is possible that ascorbate or one of
its derivatives (e.g.
) may
react with ONOO to form an intermediate that is an NO
donor, as previously shown, for low-molecular weight compounds such as HEPES
and glutathione (5,
25). However, the rapid
appearance of NO in the medium following ONOO addition
(see Figs. 1,
2,
3) suggests that any such
intermediate would have a very short half-life so that both are made and
degraded within the response time of the NO electrode. In addition,
because the decomposition of certain NO donors is accelerated in the
presence of metals, the lack of an effect of metal chelators on NO
generation (Fig. 3A)
suggests this is not a significant pathway.
Another possible mechanism for NO generation from
ONOO could be the scavenging of
by the ascorbate system (ascorbate plus
).
This would serve to drive the equilibrium (NO +
ONOO) to the left. Literature evidence to date has
concentrated on the diffusion-limited forward reaction between NO and
to yield ONOO. However, a significant back-reaction does
exist (k = 0.02 s1 at 25 °C) and
could be enhanced in the presence of a sink for
(27). In this regard, Jackson
et al. (28) have
shown that ascorbate can effectively scavenge
and compete with NO for reaction with
,
although only at concentrations in the high millimolar range
(28). Notably, the reaction
between
and
is
1000x faster than that between ascorbate and
(k = 2.3 x 108 M1
s1 versus 3 x 105
M1 s1, respectively)
(29,
30). Thus, in a system where
ascorbate oxidation is enhanced (e.g. CcOX in turnover),
scavenging may be enhanced because of greater formation of
(see above). Such enhancement may decrease into the physiological range the
effective concentration of ascorbate required for such
scavenging. In support of a role for
scavenging by ascorbate, Fig. 3
shows that superoxide dismutase can mildly enhance NO formation in this
system. However this effect appears small, because this experiment was
performed at 5 mM ascorbate at which point
scavenging may have already been saturated (see concentration dependence,
Fig. 2B).
Additional mechanisms for the ascorbate-driven generation of NO from
ONOO may be hypothesized including reactions between
ascorbate/
and other known decomposition products of ONOO
(e.g.
,
OH,
(27,
31)). However, it is not yet
clear whether any of these reactions would yield NO.
Figs. 1,
2,
3 suggest an 2% yield of
NO from ONOO. In a physiologic context, where
ONOO must have come from NO originally, this raises
the question whether this reaction is a significant pathway for the
regeneration of NO. However, given that soluble guanylate cyclase and
cytochrome c oxidase are sensitive to low nanomolar concentrations of
NO and that cell signaling is amplified at these enzymes, biological
responses are plausible. Moreover, it does appear that ascorbate can act as an
antioxidant for the prevention of ONOO formation
(28), thus increasing NO
availability.
In the context of the mitochondrion, the organelle has been proposed as a
potential source of ONOO
(32). A significant store of
1 mM ascorbate is proposed to exist in the matrix (see
Introduction), and thus ascorbate-driven ONOO decomposition
may occur in this organelle. Whereas the data in
Fig. 1 do not show NO
generation from ONOO in mitochondria, it is unclear whether
ascorbate survived the mitochondrial isolation procedure and, if so, what its
redox state was, because DHA does not catalyze NO release from
ONOO (Fig.
3C). Overall, water-soluble antioxidants such as
glutathione (5) and ascorbate
may comprise the primary mitochondrial defense against ONOO.
This raises the possibility that only ONOO made in
situ may be damaging to the organelle. Furthermore, the ability of
CO2 to modulate ascorbate-driven ONOO degradation
(Fig. 3) is unique from a
mitochondrial perspective, because the organelle is a major cellular source of
CO2. Protein tyrosine nitration mediated by ONOO
is enhanced by CO2, thus raising the possibility of modulation of
this post-translational modification by ascorbate of mitochondrial
proteins.
Whereas it is unlikely that the ascorbate plus ONOO reaction represents a significant source of NO, it is the balance between these reactive nitrogen species that is important for cellular function. Therefore, these results have implications for the role of mitochondria and reactive nitrogen species in processes such as apoptosis, because it has been shown that NO can prevent cytochrome c release from mitochondria, whereas ONOO enhances it (33, 34). Thus, ascorbate may act as a modulator of mitochondrial apoptotic signaling as has previously been proposed for glutathione (31).
In summary, we have shown that the generation of NO from
ONOO by CcOX in turnover is most likely because of
ascorbate, and it appears that, whereas CcOX does possess a
ONOO reductase activity
(7), this is not the primary
source of NO in this system. Further studies are required to elucidate
the precise roles of
and the
scavenging of
in this system.
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FOOTNOTES |
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Supported by the Ministero dell'Istruzione, dell'Universitá e della
Ricerca of Italy (Programma di Ricerca Scientifica Interuniversitario
Nazionale "Bioenergetica: aspetti genetici, biochimici e
fisiopatologici"). Present address: Dept. of Biochemical Sciences,
University of Rome "La Sapienza," Rome I-00185, Italy.
Supported by the American Heart Association and the University of Alabama,
Birmingham, Clinical Nutrition Research Center. To whom correspondence should
be addressed: Dept. of Pathology, University of Alabama, Rm. 329, BMR-2, 901
19th St. South, Birmingham, AL 35294. Tel.: 205-975-9507; Fax: 205-934-7447;
E-mail:
brookes{at}uab.edu.
1 The abbreviations used are: NO, nitric oxide; ONOO,
peroxynitrite;
,
ascorbyl radical; CcOX, cytochrome c oxidase; DHA, dehydroascorbate;
DTPA, diethylenetriaminepentaacetic acid;
,
superoxide anion; TMPD,
N,N,N',N'-tetramethyl-p-phenylenediamine.
2 T. M. Hagen, personal communication.
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ACKNOWLEDGMENTS |
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REFERENCES |
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