Regulation of Mitochondrial Respiration by Oxygen and Nitric ... - NYAS

Regulation of Mitochondrial Respiration by Oxygen and Nitric Oxide ALBERTO BOVERIS,a,b LIDIA E. COSTA,c JUAN J. PODEROSO,d MARIA C. CARRERAS,d AND ENRIQUE CADENASe aLaboratory

of Free Radical Biology, School of Pharmacy and Biochemistry of Cardiological Research, and dUniversity Hospital, School of Medicine, University of Buenos Aires, Buenos Aires, Argentina cInstitute

eDepartment

of Molecular Pharmacology and Toxicology, School of Pharmacy, University of Southern California, Los Angeles, California, USA

ABSTRACT: Although the regulation of mitochondrial respiration and energy production in mammalian tissues has been exhaustively studied and extensively reviewed, a clear understanding of the regulation of cellular respiration has not yet been achieved. In particular, the role of tissue pO 2 as a factor regulating cellular respiration remains controversial. The concept of a complex and multisite regulation of cellular respiration and energy production signaled by cellular and intercellular messengers has evolved in the last few years and is still being researched. A recent concept that regulation of cellular respiration is regulated by ADP, O 2 and NO preserves the notion that energy demands drive respiration but places the kinetic control of both respiration and energy supply in the availability of ADP to F 1-ATPase and of O2 and NO to cytochrome oxidase. In addition, recent research indicates that NO participates in redox reactions in the mitochondrial matrix that regulate the intramitochondrial steady state concentration of NO itself and other reactive species such as superoxide radical (O2−) and peroxynitrite (ONOO −). In this way, NO acquires an essential role as a mitochondrial regulatory metabolite. NO exhibits a rich biochemistry and a high reactivity and plays an important role as intercellular messenger in diverse physiological processes, such as regulation of blood flow, neurotransmission, platelet aggregation and immune cytotoxic response.

INTRODUCTION The regulation of mitochondrial respiration and energy production in mammalian tissues has been exhaustively studied and extensively reviewed. However, a clear understanding of the regulation of cellular respiration is not yet complete. In particular, the role of tissue pO2 as a factor regulating cellular respiration is a matter of controversy. It was considered that maximal rates of mitochondrial respiration could be maintained in a wide range of tissue pO2, from the usual 5 to 30 µM O2 to about 0.8 µM O21–3 based on the [O2]0.5 values (the [O2] that sustains half maximal respiratory rate) of 0.02 to 0.3 µM O2 that were measured with isolated mitochondria and cells.4–5 Considering this high affinity of mitochondrial respiration for O2, the rates bAuthor

to whom correspondence should be addressed. 121

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at which mitochondria perform oxidative phosphorylation were supposed to be rather independent of tissue pO2 and limitation of respiration by [O2] was thought to occur only under severe hypoxia. However, recent [O2]0.5 values carefully determined turned out to be higher than the ones previously measured, especially for active (state 3) respiration.6–10 The [O2]0.5 determined by Costa et al.10 using high-resolution respirometry in liver and heart mitochondria were 0.30–0.40 µM in state 4 and 1.57– 1.69 µM in state 3,10 which implies that the intracellular [O2] prevailing in some tissues, e.g., 3–8 µM in the heart,2–3 would be regulatory under normoxia with respiration slowed below its maximal rate. Defining a critical rate (Vc) as 80% of Vmax9 and considering a classical Michaelis-Menten kinetics it follows that the critical [O2] will be reached at 6 µM O2 (Vc = Vmax [O2]/([O2]0.5 + [O2]) and that limitation of active mitochondrial respiration may occur at physiological normoxia in the heart; higher values of pO2, in the range of 22 to 32 µM O2 and well above the critical [O2], have been reported for skeletal muscle and liver, however.1,2,4,11 Cytochrome oxidase was not usually considered a regulatory enzyme, yet evidence has been obtained that non-catalytic subunits alter enzyme kinetics.12 Direct spectral analysis have indicated that a substantial fraction of cytochrome oxidase is reduced in intact tissues,13 even though this reduction is not observed in isolated mitochondria. It has been suggested that this difference is due to regulatory mechanisms that are lost in vitro.14 It is worth noting that the reduced cytochrome oxidaseNO complex is spectroscopically similar to reduced cytochrome oxidase.15–16 The concept of a complex and multisite regulation of cellular respiration and energy production signaled by cellular and intercellular messengers has evolved in the last few years and is still under development. The classical and elegant concept of the regulation of cellular oxygen uptake by ADP put forward by Lardy and Wellman,17 Chance and Williams,18 and Estabrook19 considers that energy needs drive respiration and that availability of ADP to mitochondrial F1-ATPase exerts the kinetic control of respiration and energy production over a wide range of O2 concentration that certainly includes the physiological conditions (FIG . 1). The new concept of regulation of cellular respiration by ADP, O2 and NO keeps the idea that energy

FIGURE 1. Classical view and new concept of the regulation of cellular O 2 uptake.

BOVERIS et al.: REGULATION OF MITOCHONDRIAL RESPIRATION

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demands drive respiration but places the kinetic control of both respiration and energy supply in the availability of ADP to F1-ATPase and of O2 and NO to cytochrome oxidase (FIG . 1). In addition, recent reports by Poderoso et al.20–22 and by Boveris et al.23 indicate that NO participates in redox reactions in the mitochondrial matrix that regulate the intramitochondrial steady state concentration of NO itself and other reactive species such as superoxide radical (O2−) and peroxynitrite (ONOO−). In this way, NO acquires an essential role as a mitochondrial regulatory metabolite. Nitric oxide, a diatomic gas with an unpaired electron in its external orbitals, exhibits a rich biochemistry and a high reactivity and plays important roles as intercellular messenger in diverse physiological processes, such as regulation of blood flow, neurotransmission, platelet aggregation and immune cytotoxic response.24 Endothelium produced NO stimulates the guanylate cyclase activity of the underlying vascular smooth muscle cells and the increased level of cGMP produces muscle relaxation and vasodilation.25–26 This action slows down blood flow and increases the equilibration time of HbO2 with tissue O2 resulting in better tissue oxygenation. The coupling of the endothelial production of NO and blood flow to changes in tissue pO2 may be thought as dependent on cellular metabolites that are produced when mitochondrial metabolism becomes O2 limited and that reach endothelial cells. Since Granger and Lehninger27 early recognized macrophage cytotoxicity as exerted partly on mitochondrial respiration, the recognition of NO production in activated macrophages and neutrophils28–29 prompted the assay of NO effects on mitochondrial function. Two British research groups, Cleeter et al.30 and Brown and Cooper,31 simultaneously reported the inhibitory effects of NO on cytochrome oxidase activity using skeletal muscle mitochondria30 and brain synaptosomes.31 The inhibition is reversible and competitive with O231 suggesting that tissue NO may raise the [O2]0.5 and therefore become the first known physiological regulator to act directly on the mitochondrial respiratory chain. Cytosolic Ca2+ has been frequently suggested as a regulator of mitochondrial function, likely as part of a role as intracellular second messenger that signals activation of diverse and specialized cell responses, many of which require increased energy production.14 However, cytosolic Ca2+, as a cation able to discharge mitochondrial membrane potential, seems to act as antagonist for the coordinated activation of mitochondrial respiration and energy supply.32

OXYGEN DEPENDENCE OF MITOCHONDRIAL RESPIRATION The dependence of the respiratory rate of isolated mitochondria and cells on [O2] is hyperbolic; therefore it is conveniently described by [O2]0.5, the oxygen concentration that provides half-maximal respiration rate. The [O2]0.5 values are also sometimes referred to as K0.5 and KmO2; however, since, owing to their operational nature they are different with mitochondria in state 4 or in state 3 and depend on [NO], it is convenient to use only the [O2]0.5 notation and concept. The use of high resolution respirometers is advisable to precisely measure the rates of O2 uptake that occur in the 0.01 to 5 µM O2 range which is essential to determine [O2]0.5 values. The [O2]0.5 of active (state 3) and resting (state 4) respiration were recently reexamined in tightly coupled mitochondria isolated from rat liver and

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FIGURE 2. Oxygen concentration ([O 2]) and its first derivative (rate of O 2 uptake; d[O2]/dt) during the respiratory activity of rat liver mitochondria (0.35 mg prot/ml) suspended in 0.25 M sucrose, 0.5 mM EGTA, 5 mM MgCl 2, 1.5% BSA, 10 mM HEPES, and 6 mM phosphate buffer (pH 7.35). ADP pulse: 0.3 mM. A: initial rate of O 2 uptake, ∆[O2]/ ∆t, used to produce one data point in F IGURE 3. B: time course of the differential rates of O 2 uptake, d[O2]/dt), that generate the data points of F IGURE 4. Modified from Costa et al.10

TABLE 1. Oxygen dependence of active (state 3) mitochondrial respiration determined by four approaches Aerobic mitochondria

Anaerobic mitochondria

ADP pulses

O2 pulses

Initial rates

Differential rates

Air-saturated medium

H 2O 2

Vmax (nmol O2/sec ⋅ mg prot)

1.80 ± 0.02

1.78 ± 0.09

1.84 ± 0.07

1.82 ± 0.08

[O2]0.5 (µM)

2.45 ± 0.32

1.69 ± 0.09

1.04 ± 0.02

1.09 ± 0.04

Vmax (nmol O2/sec ⋅ mg prot)

1.47 ± 0.10

1.55 ± 0.09

1.51 ± 0.09

1.53 ± 0.10

[O2]0.5 (µM)

3.02 ± 0.30

1.45 ± 0.10

1.07 ± 0.04

1.12 ± 0.10

Liver mitochondria

Heart mitochondria


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heart by Costa et al.10 The [O2]0.5 values corresponding to state 4 respiration (0.30– 0.40 µM) were not different from previously reported values obtained using advanced instrumentation6–9 and are given, as well as methodological details, elsewhere.10,23 The new [O2]0.5 values corresponding to state 3 respiration were determined using four different experimental approaches and opened the way to the new concept of respiratory regulation by pO2.10,23 In the first approach, termed “initial rates,” ADP pulses were added to mitochondria in the resting state 4, at a different [O2] in different runs, collecting one data point in each run, i.e. the maximal rate, ∆O2/∆t, usually constant during several sec after ADP addition (FIG . 2). Hyperbolic fitting of the individual data points gave the [O2]0.5 (FIG . 3 and TABLE 1, initial rates), which tend to be overestimated because only a few points could be obtained in the range of limiting [O2]. In the second approach, termed “differential rates,” ADP pulses were also added to mitochondria in resting state 4, with excess ADP to exhaust O2 in the reaction medium and taking advantage of the 0.2–1 sec period in which dO2/dt can be measured in high resolution respirometers (FIG . 2). The dO2/dt values were fitted to a single hyperbola as a function of [O2] and gave the highest possible accuracy for the determination of [O2]0.5 (FIG . 4 and TABLE 1, differential rates). The third approach, termed “O2 pulses,” consisted of the injection of air-saturated reaction medium to mitochondria incubated in anaerobiosis for 3–5 min in the presence of non-limiting [ADP] (FIG . 5). In order to reach easily anaerobiosis without accumulation of metabolic by-products, mitochondria were suspended in a reaction medium in which O2

FIGURE 3. Oxygen dependence of the state 3 respiration of rat liver mitochondria showing the hyperbolic fitting of the initial rates (∆[O2]/∆t). For details see text and FIGURE 2. The triangle indicates the mean value of the O 2 uptake rate measured in air-saturated buffer.

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FIGURE 4. Oxygen dependence of the state 3 respiration of rat liver mitochondria. The dotted line is the hyperbolic fitting of the data points (d[O2]/dt) collected at 1 sec intervals after an ADP pulse. For details see text and F IGURE 2. Different symbols indicate different runs. The calculated [O 2]0.5 is 1.7 µM. Modified from Costa et al.10

FIGURE 5. Oxygen concentration ([O 2]) and its first derivative (d[O2]/dt) during the respiration of rat liver mitochondria (0.30 mg prot/ml) that were incubated in anaerobiosis for 3–5 min and added with an O 2 pulse. For details see text. Experimental conditions as in FIGURE 2.


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FIGURE 6. Oxygen dependence of the state 3 respiration of rat liver mitochondria that were pulsed with ADP in aerobiosis () or with O2 after anaerobiosis (●, ▲). The data points (d[O2]/dt) were collected at 1 sec intervals. For details see text and F IGURES 2 and 5. The calculated [O 2]0.5 are 1.60 µM O2 (ADP pulse) and 1.05 µM O2 (O2 pulse).

was diminished to 10–20 µM O2 by flushing N2. The non-linear regression fitting of dO2/dt as a function of [O2] from two consecutive O2 pulses and from an ADP pulse show that the [O2]0.5 was significantly lower in the case of the O2 pulses than with the ADP pulse (FIG . 6, TABLE 1). Because of the relatively low [O2] in air-saturated reaction medium and the fast rate of state 3 respiration, a fourth approach delivering more O2 to the reaction medium and termed “H2O2 pulse” was introduced. In this condition 10 µM O2, enough to reach the Vmax, is easily added to anaerobic catalasesupplemented mitochondria by the injection of a few µl of a H2O2 solution. The [O2]0.5 values obtained by this approach were similar to the ones obtained by the O2 pulses (TABLE 1). The lower [O2]0.5 values obtained after anaerobiosis suggest the existence of an O2-dependent system that is able to modulate mitochondrial respiration at low [O2] and that the regulator is competitive with O2, since [O2]0.5 is increased in its presence while Vmax is not changed. The production of NO by mitochondrial nitric oxide synthase (mtNOS)33–34 during active state 3 respiration seems to afford the referred regulation.35

THE EFFECTS OF NO ON THE MITOCHONDRIAL RESPIRATORY CHAIN The rich biochemical properties of NO make possible its multiple effects on the mitochondrial respiratory chain: (a) NO inhibits cytochrome oxidase activity competitively with oxygen; (b) NO inhibits electron transfer between cytochromes b and

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c and increases the mitochondrial production of O2−; and (c) NO inhibits electron transfer and NADH-dehydrogenase function in Complex I. Cytochrome Oxidase (Complex IV) Concerning the inhibition of cytochrome oxidase activity, NO binds to the enzyme in its reduced and oxidized forms; in the reduced form NO binds to the binuclear reaction center formed by cytochrome a3 heme and CuB. The cytochrome oxidase activity of the isolated enzyme15,31 and of rat heart submitochondrial particles,21 as well as the active respiration of mitochondria isolated from rat muscle ,30 liver,36 heart21 and brown adipose tissue37 and of rat brain synaptosomes31 are effectively inhibited by 0.05–2 µM NO (see FIG . 7). Binding and inhibition are reversible and removable by washing21,30 or by addition of excess myoglobin or hemoglobin.21,36 The degree of inhibition of cytochrome oxidase activity by NO depends on the O2 concentration in the reaction medium 31,36,37; NO and O2 compete for the binding site at the reaction center of cytochrome oxidase. Then, the inhibition of mitochondrial respiration by NO can be expressed as a function of the ratio [O2]/ [NO]; half-maximal inhibition of state 3 respiration is reached at a ratio of 150 O2/ NO (FIG . 7, inset) which clearly indicates the very high affinity of NO for cytochrome oxidase. Ratios of 400–500 O2/NO and 500–1000 O2/NO have been reported to inhibit 50% the respiration of rat brain synaptosomes31 and of rat brown adipose tissue,37 respectively.

FIGURE 7. Inhibition of cytochrome oxidase activity and active respiration by NO. – The inhibition of O 2 uptake, normalized for the different experiments, is plotted against the ratio of O2 and NO concentrations. Rat liver mitochondria in the presence of: , ADP (state 3), and , 20 µM dinitrophenol (state 3u); data from Takahara et al.36 Rat heart mitochondria, ∆, in the presence of ADP; data from Poderoso et al.20


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The Ubiquinol-Cytochrome b-Cytochrome c Space (Complex III) Rat heart submitochondrial particles added with NO show a marked inhibition of their succinate-cytochrome c activity with half maximal effect at about 0.7 mM NO (FIG . 8 and ref. 22) with a NO-induced reduction of cytochrome b.21,22 This second effect of NO on the mitochondrial respiratory chain results in increased O2− production in submitochondrial particles and H2O2 generation in whole mitochondria, being about 0.5 mM NO the concentration required for half maximal effect (FIG . 8 and ref. 21). The interaction of NO with the NO-reactive component of the ubiquinonecytochrome b area of the mitochondrial respiratory chain, likely an iron-sulfur center, is also reversible but is not affected by the O2/NO ratio. The time course of the inhibition of cytochrome oxidase produced by a 1 mM NO pulse as a function of [O2] shows a marked O2 dependence (a steeper slope) when ascorbate-TMPD, reductants of cytochrome oxidase, were added, and a lower O2 dependence (a less marked slope) when succinate and antimycin, reductants of the ubiquinonecytochrome b isopotential pool, were supplied (FIG . 8, inset). In the first case, the marked O2 dependence indicates the competition of NO and O2 for cytochrome oxidase. In the latter case, the lower O2 dependence of the inhibition of the ubiquinolcytochrome c electron transfer reflects the O2 dependence of O2− production by ubisemiquinone autoxidation.38,39

FIGURE 8. Inhibition of succinate–cytochrome c reductase activity and increase in the generation of O 2− produced by NO in rat heart submitochondrial particles. Modified from Poderoso et al.20 Inset: Effect of [O 2] on the NO-dependent inhibition of cytochrome oxidase activity.

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NADH-Dehydrogenase (Complex I) Prolonged exposure of cells to NO results in a persistent inhibition of complex I activity 26,40 simultaneously with a decrease in the cellular content of reduced glutathione. The inhibition is reversible by exposing the cells to high intensity light and appears to result from S-nitrosylation of thiol groups in the enzyme.40 It has been claimed that S-nitrosylation of complex I may play a role in neurodegenerative diseases.40 MITOCHONDRIAL PRODUCTION OF NO AND INHIBITION OF CYTOCHROME OXIDASE ACTIVITY Nitric oxide is produced during the oxidation of L -arginine (Arg) to citrulline catalyzed by nitric oxide synthase (NOS). The recent finding of a mitochondrial enzyme (mtNOS) in the inner mitochondrial membrane by Giulivi et al.33,34 and by Ghadoufar and Richter41 supports the idea of a physiological role for NO in mitochondrial respiration. The production of NO has been measured in whole mitochondria and in mitochondrial membranes isolated from a few rat and mouse organs (FIG . 9 and TABLE 2). Giulivi35 assayed the effects of the endogenous NO production on the rates of mitochondrial respiration and cytochrome oxidase activity by supplementing mitochondria with either the substrate Arg or the inhibitor of NOS N(G)monomethyl-L -arginine (NMMA). The [O2]0.5 of the respiration of tightly coupled mitochondria oxidizing succinate in the presence of ADP ranged from 2 to 3 µM in agreement with those obtained by the ADP pulses (TABLE 1, initial rates). In the presence of Arg the rates of O2 uptake decreased significantly at all measured O2

FIGURE 9. Mitochondrial production of NO. Rat thymus mitochondrial membranes (0.25 mg protein/ml) were supplemented with 0.1 mM NADPH, 1 mM arginine, 1 mM Cl 2Ca, 1 µM superoxide dismutase, 0.5 µM catalase and 10 µM oxyhemoglobin in 50 mM phosphate buffer (pH 7.4). a: complete reaction mixture; b: as in a plus NMMA.


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concentrations. Two concentrations of Arg were used: 5 µM (about the Km of mt-NOS for Arg33) and 0.1 mM. The observed values of [O2]0.5 were 18 and 40 µM at low and high concentrations of Arg, respectively. Conversely, addition of NMMA to intact mitochondria increased the O2 uptake by 40–50% of the control values at low O2 levels and decreased the [O2]0.5 to 1.2 µM. Concerning cytochrome oxidase, the enzyme activity as a function of [O2] was determined in mitochondria supplemented with either Arg or NMMA. The effects of NOS substrate and inhibitor were similar to those observed in mitochondrial state 3 respiration. The [O2]0.5 were 0.9, 5 and 45 µM with NMMA, endogenous and exogenous Arg, respectively. Giulivi plotted the data concerning the effect of Arg and NMMA on state 3 respiration and cytochrome oxidase with the Eadie-Hofstee treatment and found them to fit to a single line indicating that cytochrome oxidase inhibition by endogenous NO entirely accounts for respiratory regulation. The unchanged Vmax and the different [O2]0.5 indicate a competitive inhibition kinetics in which NO inhibited the respiratory chain competing with O2 for cytochrome oxidase.35 Furthermore, the rate of ATP synthesis in intact mitochondria, evaluated by measuring the amount of ATP in aliquots taken at different time points during state 3 respiration, was similarly decreased in the presence of Arg, as well as the respiratory rates in state 4.35 Assuming the classical view of “all or nothing,” where mitochondria are in state 3 or in state 4, and that tissue O2 uptake is accounted by the sum of the O2 uptakes of mitochondria respiring in state 4 and in state 3, the fraction of mitochondria in state 3 and in state 4 under physiological conditions were estimated as 28% and 72%, respectively, for rat heart.42 Alternatively, considering for heart mitochondria an [O2] of 6 µM and a [NO] of 30 nM, the corresponding ratio 200 O2//NO indicates an inhibition of 33% for both state 3 and state 4 respiration (FIG . 7, inset) and the actual respiratory rate can be estimated as 0.67 × maximal respiratory rate measured at saturating [O2]. In such case, with kinetic NO control, the fraction of mitochondria in state 3 (X) and in state 4 (1−X) are estimated as: (X) × (NO-inhibited state 3 O2 uptake) + (1−X) × (NO-inhibited state 4 O2 uptake) = O2 uptake of the perfused organ / content of mitochondria in the tissue. The data used for the calculation are (0.67 × 135 nmol O2/min.mg prot) and (0.67 × 28 nmol O2/min.mg prot) for NOinhibited state 3 and state 4 respiration,42 3.05 µ mol O2/min.g heart21 and 53 mg mitochondrial prot/g heart.43 The fraction of mitochondria in state 3 and state 4 are 54% and 46%, respectively.

TABLE 2. Mitochondrial production of nitric oxide Mitochondria Rat liver mitochondria Rat liver mitochondrial membranes

NO production (nmol/min.mg prot)

Reference

1.4

a

0.9–4.2

a, b, c

Rat thymus mitochondrial membranes

0.12–0.35

c

Mouse brain mitochondrial membranes

1.6

d

aGiulivi et al.33 bGhafourifar and Richter. 41 cJ. Bustamante, personal communication. dS. Lores Arnaiz, personal communication.

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THE INTRAMITOCHONDRIAL STEADY STATE CONCENTRATION OF NITRIC OXIDE The understanding of the NO regulation of mitochondrial respiration requires knowledge of its mitochondrial metabolism and intramitochondrial steady state level. For the following analysis only mitochondrial NO production and utilization are considered disregarding NO production by endothelial NOS and the fast reactions of NO with muscle cytosolic myoglobin and blood hemoglobin that certainly have an effect on mitochondria1 and cellular NO steady state concentrations. Concerning NO and O2− metabolism it is convenient to consider the intramitochondrial space as a specialized intracellular compartment due to the selective permeability of the inner mitochondrial membrane (FIG . 10). The key features are the impermeability of the inner membrane to O2− and H+, the relative impermeability of the same membrane to ONOO−, and the presence of Mn-SOD at a content which is about five times lower than CuZn-SOD in the cytosol. The reactions of O2− with NO (k = 2 × 1010 M−1 s−1) and with Mn-SOD (k = 2.4 × 109 M−1 s−1) are apparently the only ones that occur in the mitochondrial matrix at rates that effectively contribute to O2− utilization. Considering intramitocondrial MnSOD as 3 µM44 and intramitocondrial [NO] as 30 nM,45 it can be calculated that the intramitocondrial production of ONOO− will account for 8% of O2− utilization with the remaining 92% yielding H2O2 as final product. Under conditions of endothelium NOS activation by bradikinin, intramitochondrial NO reaches 100 nM21 and consequently ONOO− formation may account for as much as 27% of O2− utilization. Moreover, in conditions of mtNOS induction, intramitochondrial NO may reach 0.3 µM NO and the production of ONOO− will account for more than 50% of O2− utilization. Besides this oxidative pathway, intramitochondrial NO is metabolized through reductive one-electron transfer reactions from cytochrome oxidase,46 and ubiquinol.22 The two reductive

FIGURE 10. Mitochondrial metabolism of NO and its effect regulating cytochrome oxidase activity.


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