THE JOURNAL OF BIOLOGICAL CHEMISTRY
© 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
Vol. 276, No. 10, Issue of March 9, pp. 6945–6949, 2001
Printed in U.S.A.
The Modulation of Oxygen Radical Production by Nitric Oxide in
Mitochondria*
Received for publication, August 21, 2000, and in revised form, November 9, 2000
Published, JBC Papers in Press, December 5, 2000, DOI 10.1074/jbc.M007625200
Theresa M. Sarkela, Jessica Berthiaume‡, Sarah Elfering§, Anna A. Gybina¶, and Cecilia Giulivi储
From the Department of Chemistry, University of Minnesota, Duluth, Minnesota 55812
Biological systems of diverse complexity, when exposed to
NO䡠 1 or stimulated to produce NO䡠, usually present changes in
the rate of oxygen free radical production (Ref. 1 and references
therein). Decreases in the rate of oxygen free radical production
are often attributed to the diffusion-controlled reaction between O2. and NO䡠, which yields peroxynitrite. Increases in
reactive oxygen species (ROS) production are usually associated with the damage and/or inactivation of mitochondrial
* This work was supported by Grant MCB 9724060 from the National
Science Foundation and by a grant from the United Mitochondrial
Disease Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must
therefore be hereby marked “advertisement” in accordance with 18
U.S.C. Section 1734 solely to indicate this fact.
‡ Recipient of the Undergraduate Research Opportunity Program
fellowships from the University of Minnesota.
§ Recipient of the Margaret Mitchell Award from the National Cancer
Institute.
¶ Supported by the Swenson Research Summer Program and the
Research Corporation, Cottrell College Science Awards (9906909).
储 To whom correspondence and reprint requests should be addressed:
Dept. of Chemistry, University of Minnesota, 10 University Dr., Duluth, MN 55812. E-mail: [email protected].
1
The abbreviations used are: NO䡠, nitric oxide; ROS, reactive oxygen
species; O2. , superoxide anion; NMMA, NG-monomethyl-L-arginine;
NOS, nitric-oxide synthase; GSNO, S-nitrosoglutathione; HPLC, high
pressure liquid chromatography.
This paper is available on line at http://www.jbc.org
components by peroxynitrite or peroxynitrite-like species.
Considering that under physiological conditions mitochondria constitute the main intracellular source of oxygen free
radicals (2, 3) and that these organelles can produce NO䡠 (4 – 8),
it becomes of interest to examine whether the production of
mitochondrial ROS is affected by endogenous NO䡠. The accurate documentation of the rates of production of nitrogen and
oxygen radicals is an essential step to understanding the role of
these interactions in different, more complex pathophysiological situations and underlines the relevance of studying the
mechanisms behind these processes in relatively simpler models. Furthermore, transient changes in ROS production have
become an area of intense research given the growing interest
in the role of ROS as mediators of signal transduction pathways, organ preconditioning (9), and apoptotic processes (10).
In this study, we examined the rates of O2 free radical
production by intact mitochondria under various conditions of
endogenous NO䡠 production; different mechanisms underlying
the NO䡠-O2. interactions were quantitatively investigated.
Herein, we demonstrated that mitochondria could generate
O2. and NO䡠; however, unlike other biological systems, no irreversible damage to mitochondrial components was observed.
The enhanced ROS production detected under NO䡠-producing
conditions was attributed to an increase in the steady-state
level of reduced components of the respiratory chain, whereas
a small contribution was attributed to the aerobic decay of
S-nitrosoglutathione (GSNO) in the presence of GSH. Finally,
the implications of these results were discussed in terms of the
ability of NO䡠 to modulate the rates of oxygen free radical
production and/or consumption and the pathophysiological consequences associated with this process.
EXPERIMENTAL PROCEDURES
Chemicals and Biochemicals
EDTA, EGTA, sodium succinate, sodium malate, sodium glutamate,
mannitol, sucrose, HEPES, bovine serum albumin (fatty acid free),
L-arginine, antimycin, 2⫻ crystallized scopoletin, and desferrioxamine
mesylate were purchased from Sigma. Catalase and horseradish peroxidase (Grade I) were obtained from Roche Molecular Biochemicals.
Oxymyoglobin was obtained as described before (5, 6). All other reagents were of analytical grade.
Biological Materials
Liver mitochondria were isolated from adult Wistar rats by differential centrifugation and purified through Percoll centrifugation (5). This
procedure yielded intact mitochondria minimally contaminated with
other subcellular compartments (5). Essentially, male rats (body weight
of 180 –220 g) were anesthetized using a CO2 chamber. The livers were
quickly removed; washed with 0.22 M mannitol, 70 mM sucrose, 0.5 mM
EGTA, 2 mM Hepes, and 0.1% defatted bovine serum albumin, pH 7.4
(Buffer A); and homogenized in a 10:1 buffer to liver w/w ratio. Large
cell debris and nuclei were pelleted by centrifuging at 600 ⫻ g for 5 min
in a Sorvall SS34 rotor. This supernatant was filtered through two
layers of cheesecloth to remove fat. Mitochondria were pelleted by
centrifuging the supernatant for 10 min at 10,300 ⫻ g in the same rotor.
After suspending the pellet in 5 ml of the previous buffer, the mitochon-
6945
Downloaded from www.jbc.org by guest, on October 4, 2010
Biological systems that produce or are exposed to nitric oxide (NO䡠) exhibit changes in the rate of oxygen
free radical production. Considering that mitochondria
are the main intracellular source of oxygen radicals,
and based on the recently documented production of
NO䡠 by intact mitochondria, we investigated whether
NO䡠, produced by the mitochondrial nitric-oxide synthase, could affect the generation of oxygen radicals.
Toward this end, changes in H2O2 production by rat
liver mitochondria were monitored at different rates of
endogenous NO䡠 production. The observed changes in
H2O2 production indicated that NO䡠 affected the rate of
oxygen radical production by modulating the rate of O2
consumption at the cytochrome oxidase level. This
mechanism was supported by these three experimental
proofs: 1) the reciprocal correlation between H2O2 production and respiratory rates under different conditions of NO䡠 production; 2) the pattern of oxidized/reduced carriers in the presence of NO䡠, which pointed to
cytochrome oxidase as the crossover point; and 3) the
reversibility of these effects, evidenced in the presence
of oxymyoglobin, which excluded a significant role for
other NO䡠-derived species such as peroxynitrite. Other
sources of H2O2 investigated, such as the aerobic formation of nitrosoglutathione and the GSH-mediated decay
of nitrosoglutathione, were found quantitatively negligible compared with the total rate of H2O2 production.
6946
Oxygen and Nitrogen Radicals in Mitochondria
drial fraction was loaded on 20 ml of 30% (v/v) Percoll, 0.225 M mannitol, 1 mM EGTA, 25 mM Hepes, and 0.1% defatted bovine serum albumin, pH 7.4, and spun for 30 min at 95,000 ⫻ g in a Beckman 60Ti rotor.
Mitochondria, collected from the lowest band, were washed once with
Buffer A and twice with 0.15 M KCl. Mitochondrial pellets were gently
suspended in a small volume of ice-cold Buffer A. The respiratory
control and phosphate to oxygen ratios of these purified mitochondria
were 6.5 ⫾ 0.2 and 2.8 ⫾ 0.1, respectively, assessed in 0.225 M sucrose,
5 mM MgCl2, 20 mM KCl, 10 mM potassium phosphate, and 20 mM
Hepes/KOH, pH 7.4 (reaction medium) supplemented with 10 mM succinate and 0.25 mM ADP.
Biochemical Analyses
FIG. 1. Nitric oxide production by L-arginine-supplemented
mitochondria. The rate of NO䡠 production was followed by the oxidation of oxymyoglobin or by the production of L-citrulline (not shown), as
described under “Experimental Procedures.” A, the velocity of NO䡠
production versus L-Arg concentration with no (closed circles), 30 ␮M
(closed triangles), and 75 ␮M (open circles) NMMA. B, from the slopes of
reciprocal plots of velocity versus [L-Arg], the apparent Km was calculated at each concentration of NMMA. These values were plotted versus
the [NMMA].
centrations were confirmed spectrophotometrically at 334 nm.
Protein Determination—Protein concentration was determined by
biuret assay (15) with the modifications introduced by Yonetani (16),
using bovine serum albumin as the standard.
Statistics—Data were expressed as mean ⫾ S.E. and were evaluated
by one-way analysis of variance (ANOVA).
RESULTS AND DISCUSSION
Endogenous Nitric Oxide Modulates Hydrogen Peroxide Production by Mitochondria—The addition of increasing concentrations of L-Arg to intact, coupled mitochondria in State 4
(using either glutamate-malate or succinate) stimulated the
production of NO䡠 (Fig. 1A). The initial velocity plotted versus
L-Arg concentration represented a right rectangular hyperbola;
the Vmax and Km values were 1.5 nmol of NO䡠 ⫻ (min 䡠 mg
protein)⫺1 and 5 ␮M, respectively. Comparable results were
obtained by measuring L-citrulline, cosynthesized with NO䡠 by
NOS (Vmax ⫽ 1.2 ⫾ 0.2 nmol of L-citrulline/min ⫻ mg of protein
and Km ⫽ 8 ␮M). The rate of NO䡠 production was negligible at
L-Arg concentrations of 50 –100 ⫻ Km. This effect was accompanied by an increase in the rate of oxygen consumption (3–5
times), found to be resistant to ADP stimulation, and comparable with the increase obtained in the presence of carbonyl
cyanide p-trifluoromethoxyphenylhydrazone. These experimental observations indicated that the lack of NO䡠 production
at high concentrations of L-Arg was attributed to the uncoupling of mitochondria by this amino acid.
The rates of NO䡠 production in mitochondria, maintained in
State 4, were decreased significantly when NMMA was included
in the reaction mixture (Fig. 1A). The addition of this compound
did not affect the Vmax but increased the apparent Km values (15
and 30 ␮M, respectively). These results indicated that this compound is acting as a competitive inhibitor of mitochondrial NOS,
as has been shown by other reports (17–19). The KI was calculated from the slope of a plot of Km(app) versus [NMMA] (Fig. 1B),
resulting in a value (15 ␮M) similar to that reported for mouseinducible NOS (13 ␮M) (18). Even at high concentrations of
NMMA (0.5 mM), a slow rate of NO䡠 was detectable (0.02 nmol of
NO䡠 ⫻ (min 䡠 mg protein)⫺1) not accompanied by L-citrulline
production. This slow rate has been attributed to the nonenzymatic release of NO䡠 from GSNO in the presence of reduced
glutathione and superoxide dismutase.2
To examine whether endogenous NO䡠 affected the mitochondrial production of oxygen free radicals, the production of H2O2
2
M. Steffen, T. Sarkela, A. A. Gybina, T. W. Steele, N. J. Traaseth, D.
Kuehl, and C. Giulivi, Biochem. J., in press.
Downloaded from www.jbc.org by guest, on October 4, 2010
Oxygen Consumption—The oxygen uptake of mitochondria was
measured using a Clark-type O2 electrode from Hansatech (King’s
Lynn, UK) at 30 °C (5, 7). Intact, purified mitochondria (0.5–2 mg of
protein/ml) were maintained in 1 ml of reaction medium in the presence
of 10 mM succinate as substrate and, where indicated, different
amounts of L-Arg or NG-monomethyl-L-arginine (NMMA).
NO䡠 Detection—The production of NO䡠 by mitochondria (1–2 mg/ml)
was evaluated as the change in the absorbance at 581–592 nm at 22 °C
using a dual wavelength, double beam SLM-Aminco DW-2C UV-visible
spectrophotometer. The sample cuvette contained 2 ml of reaction medium, 0.1– 0.3 mM oxymyoglobin, 10 mM succinate, and 0.1 mM Larginine under constant stirring. When NMMA was used in the incubation, the protein concentration was 3–5 mg/ml. Ten ␮M catalase and
superoxide dismutase were also present in the reaction medium to
avoid unspecific side reactions (namely, the reaction of NO䡠 with the
superoxide anion or of oxymyoglobin with peroxynitrite/hydrogen peroxide); however, no significant changes were found with or without
these enzymes. The rates of NO䡠 production were taken during the first
4 –5 min when the ratios of myoglobin:catalase (in heme):NO䡠 were
28:6:1, thereby avoiding a significant, if any, inhibition of catalase by
NO䡠. The L-citrulline production was monitored by a modified colorimetric technique from Prescott and Jones (11).
Hydrogen Peroxide Production—The rate of H2O2 production by intact
mitochondria (0.12– 0.15 mg of protein/ml) was measured in reaction
medium with 5 ␮M horseradish peroxidase and 1 mM scopoletin (12). This
high concentration of scopoletin was required to compete with GSH for the
horseradish peroxidase-H2O2 complex (data not shown). The recovery of
H2O2 was 80 –90% assayed with glucose/glucose oxidase.
Detection of GSNO—Sample preparation proceeded as follows. Aliquots of the reactions were withdrawn at different time points. The
aliquots were treated to avoid thiol oxidation or thiol-disulfide exchange,
to terminate any metabolic process, and to prevent any artifactual GSNO
production. To satisfy these criteria, the samples were kept on ice protected from light during the duration of the procedure and promptly
precipitated with perchloric acid in the presence of a metal chelator,
desferrioxamine mesylate. The samples (0.8 ml) were treated with 20 ␮l of
10 mM desferrioxamine mesylate and 36 ␮l of 70% perchloric acid, kept on
ice for 10 min, and centrifuged at 14,000 rpm for 10 min at 4 °C. The
supernatants were treated with 1 volume of 0.15 M K2HPO4, 10 mM
sodium citrate, and 5 mM EDTA (phosphate-citrate-EDTA). The samples
were kept on ice for 30 min and centrifuged at 14,000 rpm for 15 min at
4 °C. The supernatants were filtered through a 2-␮m filter and kept at
⫺80 °C until HPLC analysis, usually performed during the same day. The
samples were analyzed by HPLC using a modified method from Stamler
and Feelisch (13). The separation and detection of GSNO were performed
by using a C18 Aqua HPLC column (column specifications: length and
inner diameter, 250 mm ⫻ 4.6; particle size, 5 ␮m) (Phenomenex™,
Torrance, CA) connected with a SecurityGuard™ column from the same
manufacturer (4 ⫻ 3 mm C18 cartridge). The different components were
eluted with an isocratic gradient of 99% 0.1 M monochloroacetic acid,
0.125 mM EDTA, 1.5 mM sodium octyl sulfate, pH 2.8, and 1% acetonitrile.
The mobile phase was purged with helium, and the flow rate was set at
0.8 ml/min. The chromatography equipment consisted of a Class-VP Shimadzu HPLC system equipped with two high pressure LC10ADvp pumps
and an automatic injector Sil-10ADvp. The chromatograms were analyzed
using the software provided with the system (version 5.03). The peaks
were monitored by a photodiode array detector (215 and 336 nm) and an
electrochemical detector (L-ECD-6A from Shimadzu set at ⫺300 mV)
connected in series. The identification of the derivatives was performed by
comparison with synthetic GSNO. This latter compound was prepared
according to Hart (14) by acid-catalyzed nitrosation of GSH (Elemental
analysis: Found, C, 34.5; H, 4.9; N, 15.2; S, 9.0; H2O, 0.94; C10H16N4O7S䡠
H2O requires C, 34; H, 5.1; N, 15.8; S, 9.1; H2O, 5.1%). Fresh solutions of
GSNO were prepared immediately before each experiment, and the con-
Oxygen and Nitrogen Radicals in Mitochondria
6947
FIG. 2. Effect of L-arginine on O2 uptake and H2O2 production by intact mitochondria. The H2O2 production (A) and O2 uptake (B) of
intact, purified mitochondria were measured in the presence of 5 mM glutamate/0.5 mM malate (closed circles) or 10 mM succinate (open circles).
The O2 uptake was measured using 1 mg of protein/ml suspended in reaction medium as described under “Experimental Procedures.” The rate of
H2O2 production was measured using 0.12 mg of protein/ml in reaction medium using the horseradish peroxidase-scopoletin method. Other
experimental conditions were described under “Experimental Procedures.”
effects for the NO䡠 production, respiratory rates, and H2O2
production (Figs. 1–3) indicated that these events were biochemically linked, acting at a common site. The involvement of
NO䡠 in these effects was supported by the reversibility of the
effects observed with NMMA (upon the addition of L-Arg) and
L-Arg (upon the addition of oxymyoglobin). These results also
precluded a significant role for peroxynitrite because this species causes irreversible damage of respiratory chain components (24). Furthermore, the fact that oxymyoglobin reversed
the modulation of the O2 uptake and ROS production not only
excluded ONOO⫺ but also the involvement of other NO䡠-derived species, such as nitrate, also produced in the presence of
oxymyoglobin (23).
Although the association between respiratory rates, hydrogen peroxide production, and NO䡠 generation was apparent
from these results, no quantitative correlation was demonstrated. Moreover, given the fact that mitochondria are endowed with a pool of GSNO2 and that its synthesis (25) and
decay may produce oxygen radicals (26) at rates that may be
significant, we sought to explore these processes using quantitative strategies.
Role of S-Nitrosoglutathione Metabolism in the Hydrogen
Peroxide Generation by Mitochondria—The major mitochondrial thiol is constituted by glutathione (about 10 mM) (27).
Glutathione is synthesized in the cytosol, imported to the mitochondria, and involved in a protective role against oxidative
stress (28). Thiols react with NO䡠 and/or its metabolites, resulting in the formation of stable NO䡠 donors or nitrosothiols (29).
Recent reports suggest that the formation of GSNO in vivo may
include the production of O2. via the oxidation of the intermediate GSNO . (25) (Equations 1 and 2).
GS⫺ ⫹ NO䡠 ¡ [GSNO . ]
(Eq. 1)
关GSNO . ] ⫹ O2 ¡ GSNO ⫹ O2.
(Eq. 2)
If this mechanism had been active in mitochondria, assuming
oxygen is the only suitable electron acceptor for Equation 2,
increases in NO䡠 production would have been followed by increases in the rates of O2. and GSNO production. Studies were
undertaken to explore the occurrence of this mechanism in
mitochondria and to explore whether the increase in oxygen
radical production observed in NO䡠-producing mitochondria
could be associated with the formation of GSNO. In addition,
the decay of GSNO, which includes a complex chemistry of
homolytic and heterolytic mechanisms (26, 30), could also result in the production of hydrogen peroxide. Toward this end,
the rates of the production and decay of GSNO were evaluated
in terms of their potential to contribute to the rate of hydrogen
peroxide produced by mitochondria.The concentration of GSNO
from intact mitochondria (analyzed and quantified by HPLC as
Downloaded from www.jbc.org by guest, on October 4, 2010
and the oxygen consumption of intact mitochondria were assessed under different conditions of NO䡠 generation. Mitochondria were supplemented with either malate-glutamate or succinate during the experiments to ensure a nonlimiting pool of
NADPH, required for the activity of NOS.
The addition of L-Arg to intact, coupled substrate-supplemented mitochondria increased the rate of H2O2 production in a
dose-response manner (Fig. 2A). The concentrations of L-Arg
required for the half-maximal effect were 29.6 ␮M (glutamatemalate) and 34 ␮M (succinate). At saturating concentrations of
L-Arg, the rate of H2O2 production increased by 42% (glutamatemalate) and 58% (succinate), whereas the respiratory rates decreased 33% (glutamate-malate) and 27% (succinate) (Fig. 2B).
The concentrations of L-Arg required for the 50% inhibition of the
respiratory rates were 24.7 ␮M (glutamate-malate) and 31.5 ␮M
(succinate). The effects observed on the respiratory rates and
H2O2 production were completely reversed by adding 0.2–1 mM
oxymyoglobin to the reaction mixture (not shown). These latter
experiments might wrongly indicate that the rate of H2O2 production was decreased by the reaction of H2O2 with oxymyoglobin via its pseudocatalatic/peroxidatic activity, without the participation of NO䡠. This possibility seemed unlikely based on the
following evidence. First, the rate constants for the reaction of
oxymyoglobin or oxyhemoglobin with H2O2 in their peroxidatic
and catalatic activities are 4 –5 orders of magnitude smaller than
those corresponding to the reaction of oxymyoglobin with NO䡠
(21–23), indicating that this molecule will preferentially react
with oxymyoglobin rather than with H2O2. Second, the addition
of NMMA, which blocks the production of NO䡠 by mitochondrial
NOS (independently of the presence of oxymyoglobin) decreased
the rate of H2O2 production, excluding the role of oxymyoglobin
in the modulation of H2O2 production by NO䡠-producing mitochondria (see below).
The addition of NMMA to L-Arg- and succinate-supplemented mitochondria decreased the rate of H2O2 production
and increased the respiratory rates in a dose-response manner
(Fig. 3, A and B, respectively). The concentrations of NMMA
required for the half-maximal rate of H2O2 production were
13.9 (glutamate-malate) and 13.8 ␮M (succinate); at saturating
concentrations of NMMA, the rate of H2O2 production was
decreased 42% (malate-glutamate) and 37% (succinate). The
concentrations of NMMA required for the half-maximal respiratory rates were 15.7 (glutamate-malate) and 10.2 ␮M (succinate); at saturating concentrations of NMMA, the respiratory
rates were increased by 85% (malate-glutamate) and 39% (succinate). The effects observed on the respiratory rates and H2O2
production mediated by NMMA were completely reversed by
adding increasing amounts of L-Arg (not shown).
The reciprocal association between ROS and NO䡠 production
with the O2 uptake (Figs. 1–3, Table I) and the similar concentrations of NMMA and L-Arg required for the half-maximal
6948
Oxygen and Nitrogen Radicals in Mitochondria
FIG. 3. Effect of NMMA on O2 uptake and H2O2 production by intact
mitochondria. The H2O2 production (A)
and O2 uptake (B) of intact, purified mitochondria supplemented with 0.1 mM LArg were measured in the presence of 5
mM glutamate/0.5 mM malate (closed circles) or 10 mM succinate (open circles).
Other experimental conditions were described in the legend to Fig. 1 and under
“Experimental Procedures.”
TABLE I
Oxygen consumption, H2O2 production, and NO䡠 generation in intact
mitochondria
The rates of O2 consumption, H2O2 production, and NO䡠 generation
were measured using intact, purified mitochondria in reaction medium
with 10 mM succinate and at saturating concentrations of either L-Arg
or NMMA. Results are expressed as mean ⫾ S.E. (n ⫽ 7) from three
separate mitochondrial preparations.
Additions
⫺d[O2]/dt
24.2 ⫾ 0.4
L-Arg
12.7 ⫾ 0.2
nmol ⫻ (min 䡠 mg protein)
⫺1
0.11 ⫾ 0.03
0.02 ⫾ 0.01a
0.24 ⫾ 0.02
1.50 ⫾ 0.05a
Not detectable. Significantly different (p ⬍ 0.01) by one-way analysis of variance (ANOVA).
a
described under “Experimental Procedures”) was 0.37 ⫾ 0.03
nmol/mg of protein, and its rate of production (under optimal
conditions for NO䡠 production) was 10 –20 pmol of GSNO (min
mg 䡠 protein)⫺1. According to the mechanism outlined before, if
this rate were equivalent to that of O2. production (see Equation
2), it would have represented a small fraction (3–5%) of the rate
of hydrogen peroxide obtained with NO䡠-producing mitochondria (Table I), constituting a minor contribution to the total
rate of H2O2 production.
The decay of GSNO may result in H2O2 production if the
pathway indicated in Fig.4 is operative. To evaluate the contribution to the rate of H2O2 by GSNO decay, the rate of H2O2
generation was evaluated in a control (mitochondria treated
with NMMA for 30 min) and in GSNO-depleted mitochondria
(mitochondria incubated with NMMA at 25°C for 3– 4 h until
the concentration of GSNO was not detectable by HPLC). No
significant difference was found in the rates of H2O2 production
between GSNO-depleted and control mitochondria (98 ⫾ 30
and 137 ⫾ 30 ␮M/min, respectively; mean ⫾ S.E.; n⫽3), indicating that the decay of GSNO did not contribute significantly
to the H2O2 generation by mitochondria; however, it did not
preclude in vivo situations in which higher concentrations of
GSNO may be achieved, and its decay may have a substantial
contribution to the rate of H2O2 production.
Interaction of Nitric Oxide and Respiratory Chain Carriers—We
investigated whether the modulation of ROS by NO䡠-producing
mitochondria was the result of the interaction between NO䡠 and
certain electron carriers of the respiratory chain. This hypothesis is
based on the high affinity of NO䡠 for metallo-containing proteins
(31, 32) and on the fact that certain inhibitors of the respiratory
chain (those that increase the steady-state reduction level of electron carriers at the NADH dehydrogenase and ubiquinol-cytochrome c reductase segments) enhance the production of ROS.
Toward this end, the steady-state reduction of the carriers of
the respiratory chain was evaluated in NO䡠-producing mitochondria by visible spectroscopy (Fig. 5). Under these conditions, 77% of cytochromes b and c was reduced, whereas 50% of
cytochrome oxidase was reduced. The steady-state reduction of
cytochrome oxidase (in percentage) was found within the experimental range of the inhibition of the oxygen consumption
under identical conditions. This result agreed with those pre-
FIG. 4. Synthesis and decay of GSNO. GS䡠, glutathionyl radical;
GS⫺, glutathionyl anion; SOD, superoxide dismutase.
FIG. 5. Steady-state reduction of components of the respiratory chain under different conditions. Rat liver mitochondria were
isolated as described under “Experimental Procedures.” The respiratory
control ratio and phosphate to oxygen ratio number of these preparations were 4.7 and 2.1, respectively, assayed in the presence of 10 mM
succinate and 0.45 mM ADP in reaction buffer (0.225 M sucrose, 10 mM
potassium phosphate, 5 mM MgCl2, 20 mM Hepes, pH 7.4). The absorption spectra were recorded using an SLM-Aminco DW-2C UV-visible
spectrophotometer as described previously (33). The difference spectra
of samples, which contained 1 mg of mitochondrial protein in reaction
buffer, were recorded under the following conditions: 20 mM succinate
(State 4; striped bar), succinate plus 0.5 mM ADP (State 3; white bar),
succinate and 50 nM antimycin (State 4 plus antimycin; gray bar); and
succinate and 0.15 mM L-arginine (State 4 plus L-arginine; black bar).
The concentrations of each carrier were calculated using the following
extinction coefficients: cytochromes c-c1, ⑀550 –540 ⫽ 19 mM⫺1 cm⫺1; cytochromes bK-T, ⑀564 –575 ⫽ 20 mM⫺1 cm⫺1; cytochromes aa3, ⑀605– 630 ⫽ 16
mM⫺1 cm⫺1.
viously obtained in which changes in the respiratory rates
correlated with those in cytochrome oxidase activity under
identical conditions of NO䡠 production (7).
Experimental evidence for the association between the increased reduced state of the respiratory chain carriers and H2O2
Downloaded from www.jbc.org by guest, on October 4, 2010
NMMA
⫹d[NO䡠]/dt
⫹d[H2O2]/dt
Oxygen and Nitrogen Radicals in Mitochondria
production in NO䡠-producing mitochondria was provided by evaluating the steady-state reduction of the respiratory chain carriers and the rate of ROS production under a variety of conditions,
including those that entailed changes in NO䡠 production (Fig. 6).
A significant correlation was found between the reduction level of
cytochrome b (Fig. 6) or other components of the respiratory chain
(not shown) and the production of H2O2 (Fig. 6) (r ⫽ 0.958), indicating that an increased reduction of these carriers under a variety
of conditions (including those in which NO䡠 is produced) leads to an
increased rate of ROS production.
Based on our results, it seemed that the modulation of ROS
production was accomplished through the reversible inhibition of
the mitochondrial respiratory rate by NO䡠 at the cytochrome oxidase level. The transient decrease (or inhibition, depending on the
concentration of NO䡠 and oxygen) in the respiratory rate leads to an
increased steady-state reduction of the respiratory chain carriers,
which in turn by reacting with oxygen resulted in the production of
ROS. It is likely that cytochrome oxidase represents the crossover
point in NO䡠-producing mitochondria (Fig. 5), excluding a significant interaction of NO䡠 with other available components of the
respiratory chain (cytochromes c or b) under these experimental
conditions. However, if cytochrome oxidase were the crossover
point in this process, it would have been fully reduced. This apparent discrepancy can be bridged by considering that the interaction
of NO䡠 with cytochrome oxidase is complex, constituted by the
reversible binding of NO䡠 to the heme a3 of cytochrome oxidase and
by the reduction of NO䡠 to N2O by this oxidase (7).
Considering the results presented in this study, it could be
wrongly surmised that the effect of NO䡠 on the respiratory chain
is not different from that of other xenobiotics used to enhance
ROS production in mitochondria (e.g. NADH-supplemented mitochondria plus rotenone or FADH2-supplemented mitochondria
plus antimycin). Nitric oxide has two distinctive properties from
the aforementioned inhibitors; it is endogenously produced (4 –
8), and it has a transient effect (5, 7). This latter property, based
on its cytochrome c oxidase-mediated catabolism, allows a slow
but continuous flow of electrons through the chain, even when a
complete suppression of the O2 consumption has been accomplished. This type of modulation of the respiratory chain has the
advantage of avoiding a complete reduction of the components of
the respiratory chain and the subsequent burst of ROS when
lower ratios of [NO䡠]/[O2] are encountered.
Conclusions—The mitochondrial production of ROS has been
considered as a side process of the normal oxidative metabolism, its rate ranging between two levels determined by the
mitochondrial metabolic states, namely States 4 (maximum)
and 3 (minimum) (2). This study demonstrated that the mitochondrial production of ROS is not limited to these two values
and may exhibit a degree of values modulated by endogenous
NO䡠. Cellular conditions that would affect the availability of
either L-Arg or other cofactors required for NOS activity would
generate variable amounts of NO䡠 and O2. . Although further
studies need to be performed to fully understand the implications of these observations, two different groups of reports may
support the importance of our observations. First, some studies
support the important role of manganese superoxide dismutase
in a system that produces both O2. and NO䡠 in preventing the
formation of the powerful oxidant peroxynitrite and the deleterious effect that the inactivation/lack of this enzyme may
cause (20). Second, some studies examine the role of mitochondrial ROS, and that of endogenous agents that modulate their
concentrations, as activators of intracellular signaling cascades
involved in a variety of responses.
Acknowledgments—We thank T. Githu and M. Raveendranathan for
technical assistance.
REFERENCES
1. Beckman, J. (1996) in Nitric Oxide: Principles and Actions (Lancaster, J., Jr.,
ed) pp. 1–71, Academic Press, San Diego, CA
2. Boveris, A., Oshino, N., and Chance, B. (1972) Biochem. J. 128, 617– 630
3. Giulivi, C., Boveris, A., and Cadenas, E. (1999) in Reactive Oxygen Species in
Biological Systems: An Interdisciplinary Approach (Gilbert, D., and Colton,
E., eds) pp. 77–99, Plenum Press, New York
4. Giulivi, C. (1996) Proceedings of the 3rd Annual Meeting of The Oxygen Society,
Abstr. 33, p. 25, Los Angeles, CA
5. Giulivi, C., Poderoso, J. J., and Boveris, A. (1998) J. Biol. Chem. 273,
11038 –11043
6. Tatoyan, A., and Giulivi, C. (1998) J. Biol. Chem. 273, 11044 –11048
7. Giulivi, C. (1998) Biochem. J. 332, 673– 679
8. Ghafourifar, P., and Richter, C. (1997) FEBS Lett. 418, 291–296
9. Van den Hoek, T. L., Becker, L. B., Shao, Z., Li, C., and Schumacker, T. (1998)
J. Biol. Chem. 273, 18092–18098
10. Ghafourifar, P., Klein, S. D., Schucht, O., Schenk, U., Pruschy, M., Rocha, S.,
and Richter, C. (1999) J. Biol. Chem. 274, 6080 – 6084
11. Prescott, L. M., and Jones, M. E. (1969) Anal. Biochem. 32, 408 – 419
12. Boveris, A., Martino, E., and Stoppani, A. O. M. (1977) Anal. Biochem. 80,
145–158
13. Stamler, J. S., and Feelisch, M. (1996) in Methods in Nitric Oxide Research,
(Feelisch, M. and Stamler, J. S., eds) pp. 521–539, John Wiley & Sons, New
York
14. Hart, T. W. (1985) Tetrahedron Lett. 26, 2013–2016
15. Gornall, A. G., Bardawill, C. J., and David, M. M. (1949) J. Biol. Chem. 177,
751–766
16. Yonetani, T. (1961) J. Biol. Chem. 236, 1680 –1686
17. Feldman, P. L., Griffith, O. W., Hong, H., and Stuehr, D. J. (1993) J. Med.
Chem. 36, 491– 496
18. Olken, N. M., and Marletta, M. A. (1993) Biochemistry 32, 9677–9685
19. Olken, N. M., Osawa, Y., and Marletta, M. A. (1994) Biochemistry 33,
14784 –14791
20. Williams, M. D., Van Remmen, H., Conrad, C. C., Huang, T. T., Epstein, C. J.,
and Richardson, A. (1998) J. Biol. Chem. 273, 28510 –28515
21. Giulivi, C., Hochstein, P., and Davies, K. J. A. (1993) Free Radic. Biol. Med. 16,
123–129
22. Saunders, B. C., Holmes-Siedle, A. G., and Stark, B. P. (1964) in Peroxidase:
The Properties and Uses of a Versatile Enzyme and of Some Related Catalysts, pp. 169 –181 Butterworth & Co., Washington, D.C.
23. Doyle, M. P., and Hoekstra, J. W. (1981) J. Inorg. Biochem. 14, 351–356
24. Radi, R., Rodrı́guez, M., Castro, L., and Telleri, R. (1994) Arch. Biochem.
Biophys. 308, 89 –95
25. Gow, A. J., Buerk, D. G., and Ischiropoulos, H. (1997) J. Biol. Chem. 272,
2841–2845
26. Singh, S. P., Wishnok, J. S., Keshive, M., Deen, W. M., and Tannenbaum, S. R.
(1996) Proc. Natl. Acad. Sci. U. S. A. 93, 14428 –14433
27. Giulivi, C., and Cadenas, E. (1998) Biochim. Biophys. Acta 1366, 265–274
28. Meister, A. (1983) Science 220, 470 – 477
29. Stamler, J. S., Simon, D. I., Jaraki, O., Osborne, J. A., Francis, S., Mullins, M.,
Singel, D., and Loscalzo, J. (1992) Proc. Natl. Acad. Sci. U. S. A. 89,
8087– 8091
30. Wong, P. S.-Y., Hyun, J., Fukuto, J. M., Shirota, F. N., DeMaster, E. G.,
Shoeman, D. W., and Nagasawa, H. T. (1998) Biochemistry 37, 5362–5371
31. Cleeter, M. W. J., Cooper, J. M., Darley-Usmar, V. M., Moncada, S., and
Moncada, A. H. V. (1994) FEBS Lett. 345, 50 –54
32. Brown, G. C., and Cooper, C. E. (1994) FEBS Lett. 356, 295–298
33. Martens, M. E., Peterson, P. L., Lee, C. P., Hart, Z., Nigro, M. A., Glasberg, M.,
Hatfield, J. S., and Chang, C. H. (1988) Ann. Neurol. 24, 630 – 636
Downloaded from www.jbc.org by guest, on October 4, 2010
FIG. 6. Rate of H2O2 production by mitochondria and the
steady-state reduction of cytochrome b. The rates of H2O2 production and the steady-state reduction of cytochrome bK-L were evaluated
in mitochondria under State 1, State 4 (succinate or malate-glutamate),
State 3 (succinate or malate-glutamate plus ADP), State 3u (succinate
or malate-glutamate plus carbonyl cyanide p-trifluoromethoxyphenylhydrazone), State 4 plus L-Arg, and State 4 plus NMMA. Other experimental details were described under “Experimental Procedures” and in
the legend to Fig. 5.
6949