In biological systems, ONOO^- production depends on production rates of NO and O₂^-, and on the reactions of these two free radicals with other biological components, which limit the local concentrations of NO and O₂^-. In mitochondria, O₂^- is generated through the auto oxidation of semiquinones at Complexes I and III, and it may suffer the SOD-catalyzed dismutation reaction to produce H₂O₂ or react with NO in a classical termination reaction between free radicals. These diffusion-controlled reactions kinetically compete for O₂^- degradation. Results from our laboratory have shown that even in physiopathological situations in which NO production is reduced, such as the mitochondrial dysfunction associated to stunned heart, mitochondrial ONOO^- production rate may be slightly increased if the steady-state concentration of O₂^- is augmented. The enhancement in O₂^- concentration leads to an increase in its degradation by reaction with NO, decreasing NO bioavailability and increasing ONOO^- production rate. Therefore, mitochondrial ONOO^- generation is mainly driven by O₂^- rather than by NO steady-state concentrations. In this scenario, the switch from NO-signaling pathways to oxidative damage takes place. The modification of crucial biomolecules by nitration or oxidation can lead to the bioenergetics failure that underlies physiopathological conditions such as neurodegenerative diseases, ischemia-reperfusion, Diabetes, endotoxic shock and aging.

The mitochondrial matrix is a metabolically differentiated intracellular space concerning superoxide (O₂^-), nitric oxide (NO) and peroxynitrite (ONOO^- ) metabolism because of the impermeability of the mitochondrial inner membrane to O₂^-, H⁺ and ONOO^- and the specific presence of relatively high concentrations of Mn-superoxide dismutase (Mn-SOD), about 3-10 mM.¹ Superoxide anion (O₂^-) is formed through the auto oxidation of ubisemiquinone² at Complex III and of the flavin semiquinone of NADH dehydrogenises.³ In the mitochondrial matrix, O₂^- is consumed through two diffusion-controlled reactions (Figure 1): the disproportionate reaction catalyzed by Mn‑SOD (k=2.3´10⁹ M^-1 s^-1)^4,5 that produces O₂ and hydrogen peroxide (H₂O₂);^6‒9 and the reaction with nitric oxide (NO) to yield peroxynitrite (ONOO^-; k=1.9´10¹⁰ M^-1 s^-1).¹⁰ Thus, from the kinetic point of view, these reactions compete for O₂^- degradation.

As a result of O₂^- disproportionation, isolated respiring mitochondria produce H₂O₂ at rates that depend on the redox state of the components of the respiratory chain and, consequently, on the mitochondrial metabolic state.^6,11 The rates of H₂O₂ production of mitochondria isolated from mammalian organs are in the range of 0.4-0.9 nmol H₂O₂´min^-1´mg protein^-1 in state 4 and 0.05-0.15 nmol H₂O₂´min^-1´mg protein^-1 in state 3,^1,7,11 being H₂O₂ generation in state 4 about 4-16 times higher than in state 3.^1,9 Hydrogen peroxide in the mitochondrial cristae space originates largely from mitochondrial Complex III, whereas mitochondrial Complex I contribute to mitochondrial matrix H₂O₂.¹² In the aristae subspace, “redox nanodomains” have been described which are induced by and control calcium signaling at the endoplasmic reticulum-mitochondrial interface.¹³ Hydrogen peroxide transients sensitize calcium ion release to maintain calcium oscillation. In this scenario, H₂O₂ is considered the major redox metabolite operative in redox sensing, signaling and redox regulation.¹⁴ While 1-10 nM H₂O₂ are considered physiological concentrations that play a role in redox signaling pathways under normal conditions, higher H₂O₂ concentrations lead to adaptive responses and supra physiological concentrations (>100nM) lead to damage of biomolecules.¹⁴

In addition, nitric oxide (NO) is produced through the reaction catalyzed by mitochondrial nitric oxide synthase (mtNOS), an isoenzyme of the NOS family located in the mitochondrial inner membrane that requires NADPH, L-arginine, O₂ and Ca²⁺ for its enzymatic activity.^15‒20 Mitochondrial NOS is a highly regulated enzyme.²¹ Several reports have shown that mtNOS is regulated by the O₂ partial pressure in the inspired air,^22‒25 the sympathetic autonomic system,²⁶ the thyroid hormones,²⁷insulin²⁸ and angiotensin II.²⁹ As well as H₂O₂ generation, mtNOS activity is modified by mitochondrial metabolic state:^19,30,31 during the transition from resting to active respiration, NO release decreases about 40-45%. Nitric oxide production depends on mitochondrial membrane potential (Δy), being this dependence more important at physiological Δy range (150-180mV).^30,31 In addition, mtNOS expression and activity are positively regulated in inflammatory processes^32,33 and positively or negatively modulated by pharmacological situations,³⁴ such as haloperidol,³⁵ chlorpromazine³⁶ and enalapril treatments.³⁷ Moreover, changes in mtNOS activity and expression have been associated to the role of NO as signaling molecule involved in mitochondrial biogenesis,^38,39 understood as de novo formation of mitochondria during the cellular life cycle. In turn, the product of mtNOS activity, i.e. NO, is an effective modulator of mitochondrial function⁴⁰ through the inhibition that it exerts over Complex IV ^41‒43 and Complex III^44,45 activities. Mitochondrial NO is produced at rates of 1.0-1.5 nmol NO´min^-1´mg protein^-1 and kept at a steady-state level of about 10^-9-10^-8 M in the mitochondrial matrix.³⁰

When O₂^- and NO are synthesized simultaneously and in close proximity, they will combine spontaneously to form ONOO^-. This diffusion-limited reaction is a key element in deciding the roles of NO in physiology and pathology.^46‒48 Peroxynitrite is a strong oxidant⁴⁹ that can directly react with biomolecules by one or two-electron oxidations.⁵⁰ Peroxynitrite oxidizes the sulfhydryl group of cysteine and glutathione (GSH),⁵¹ the sulfur atom of methionine,⁵² ascorbate,⁵³ and the Purina and pyramiding bases of DNA.⁵⁴ However, the reaction rate constants are relatively slow for these second-order reactions, ranging from 10³ to 10⁶ M^-1s^-1.⁴⁴ Peroxynitrite is also able to start the lipoperoxidation process in biomembranes and liposome’s⁵⁵ and in isolated LDL.⁵⁶ In addition, ONOO^- can promote protein tyrosine and tryptophan nitration, and lipid nitration that serve as important biological markers in vivo.^57,47 Tyrosine nitration affects protein structure and function, resulting in changes in the catalytic activity of enzymes, altered cytoskeletal organization, and impaired cell signal transduction and is thus increasingly considered as a central aspect of peroxynitrite-mediated cytotoxicity.^58,59 Nevertheless, ONOO^- is normally reduced by the mitochondrial reluctant NADH, ubiquinol (UQH₂) and GSH and kept at intramitochondrial steady-state level of about 5-10nM.⁵⁹ When the steady-state concentration of ONOO^- is enhanced at about 20-50nM, tyrosine nitration, protein oxidation and damage to Fe-S centers might take place. Therefore, the switch from signaling pathways of NO to oxidative damage takes place. Peroxynitrite production rate enhancement has been found in a series of clinical conditions such as Parkinson’s disease,⁶⁰ ischemia-reperfusion⁶¹ diabetes,⁶² endotoxicshock,³² and aging.⁶³ In these physiopathological situations, partial inactivation and dysfunction of Complex I (NADH: ubiquinone oxidoreductase) was also observed.⁶⁴ Because of the fact that mitochondrial Complex I is the major entry point for feeding the respiratory chain with the reducing equivalents and that it is one of the H⁺ pumps that generates the mitochondrial DmH⁺ needed for the subsequent ATP synthesis, changes in Complex I activity lead to impairment of mitochondrial capacity to produce ATP and to cellular bioenergetics imbalance which underlies the above cited pathologies. Moreover, the increase in O₂^- production by modified Complex I intensify the enhancement in ONOO^- generation, leading to a positive feedback toward oxidative damage. Figure 2 outlines the metabolism of reactive oxygen and nitrogen species, in the mitochondrial matrix.

Mitochondrial ONOO^- generation depends on NO and O₂^- production rates and on the reactions of these two free radicals with other biological components, which limit the local concentrations of NO and O₂^-. Results from our laboratory have shown that even in physiopathological situations in which NO production is reduced, mitochondrial ONOO^- production rate may be slightly increased if the steady-state concentration of O₂^- is augmented, with the consequent oxidation of biomolecules or modification of proteins by nitration.^61,65 We have observed that in myocardial stunning^61,65 and in streptozotocin (STZ)-induced diabetes,^62,39 the cardiac mitochondrial dysfunction implied the reduction in state 3 O₂ consumption sustained by glutamate-malate, the decrease in mitochondrial Complex I-III activity, and the enhancement in H₂O₂ production rate (Table 1), among others. However, while in the mitochondrial dysfunction produced as a consequence of hyperglycemia, an increase in NO production rate (23%) and in mtNOS expression (132%)³⁹ were observed together with a reduction (50%) in Mn-SOD activity, in the mitochondrial impairment that accompanied the initial phase of stunned heart (15 min of ischemia and 30 min of reperfusion), a reduction in NO production rate (28%), without changes in mtNOS expression and SOD activity⁶¹ were detected, as expected for an acute stress model. Strikingly, an increase in tyrosine nitration (about 60-80%) of heart mitochondrial proteins was observed in both experimental models (Table 1).

Taking into account the experimental values of H₂O₂ and NO production rates in both physiopathological situations and the Mn-SOD concentration in the mitochondrial matrix calculated from Mn-SOD activity, the steady-state concentrations of O₂^- and NO and the ONOO^- production rate were estimated (Table 1) .

Superoxide anion production rate was calculated from the experimental H₂O₂ production values and considering the 2:1 stoichiometry of the disproportionation reaction of O₂^- to H₂O₂,

- d [O₂^-]/dt = 2 d[H₂O₂]/dt

In the steady-state, the production rate is equal to the consumption rate of a chemical species. In the case of O₂^-,

- d[O₂^-]/dt = d[O₂^-]/dt

Superoxide is consumed by the reaction with NO (reaction I) and the disproportionation reaction (reaction II) catalyzed by SOD:

$O_{{}_2}{}^{{}^{-} }+ NO \stackrel{k_1}{\to } ONO{O}^{-}$

$2 O_{{}_2}{}^{{}_{{}_{{}^{-}}}} + 2 H^{{}^{+}} \stackrel{k_2}{\to } H_{{}_2}{O}_{{}_2} + O_{{}_2}$

Mitochondrial NO is produced by mtNOS and released into the mitochondrial matrix where NO reacts with O₂^- (reaction I), ubiquinol (UQH₂) (reaction III) and cytochrome oxidase (cyt aa_3-e³⁺) (reaction IV):

$O_{{}_2}{}^{{}^{-}} + NO \stackrel{k_1}{\to } ONO{O}^{-}$

$NO + UQ{H}_{{}_2} \stackrel{k_3}{\to } UQ{H}^{{}^{•} }+ H^{{}^{+}} + N{O}^{{}^{-}}$

$NO + cyt a{a}_{{}_{{}_3}}-F{e}^{{{}^2}^{+}} \stackrel{k_4}{\to } cyt a{a}_{{}_3}-F{e}^{{{}^3}^{+}} + N{O}^{{}^{-}}$

Thus, NO production rate was expressed as:

d[NO]/dt =-d[NO]/dt = k₁ [NO] [O₂^-]+k₃ [NO] [UQH₂]+k₄ [NO] [cyt aa₃-Fe²⁺]

Superoxide and NO steady-state concentrations ([O₂^-]_ss and [NO]_ss) were calculated from the equations 1 and 2, respectively, by mathematical iteration, and using the following rate constants: k₁=1.9´10¹⁰ M^-1 s^-1;¹⁰ k₂=2.3´10⁹ M^-1 s^-1;⁴ k₃=1.5´10⁴ M^-1 s^-1;⁶⁷ k₄=4.0´10⁷ M^-1s^-1.⁶⁸

[O₂^-]_ss=2d[H₂O₂]/dt /(k₁[NO]+k₂[Mn-SOD]) (Eq. 1)

[NO]_ss=d[NO]/dt/(k₁[O₂^-]+k₃[UQH₂]+k₄[cyt aa₃-Fe²⁺])  (Eq. 2)

Nitric oxide diffusion to and from cytosol was not included in Eq. 2. Ubiquinol and cytochrome aa₃ contents were taken as 277mM and 5.6mM, respectively, considering a mitochondrial matrix volume of 7.0ml´mg protein^-1. ^66,69

Once the O₂^- and NO steady-state concentrations were calculated, the ONOO^- production rate was estimated from Eq. 3, taking into account the second-order rate constant k₁:

d[ONOO^-]/dt=k₁[O₂^-] [NO] (Eq. 3)

In the physiological conditions assessed (Table 1, control data), the mitochondrial O₂^- steady-state concentrations calculated were about of 0.05nM, because of its short half-life (50-100 ms) and its degradation by the reaction catalyzed by Mn-SOD. In these experimental situations, NO steady-state concentrations have resulted ~9 nM, more than 100-fold higher than O₂^- concentration. Therefore, the second-order reaction rate between O₂^- and NO to produce ONOO^- is converted in a pseudo-first order reaction respect to O₂^-, being O₂^- concentration the driving factor to generate ONOO^- in a given time. Consequently, under normal mitochondrial conditions, ONOO^- production and concentration are relatively low. Moreover, taking into account that the rate constant of the ONOO^- formation reaction is approximately 8 times higher than the rate constant of the O₂^- dismutation reaction, physiological concentrations of SOD (in mM range) can effectively compete with NO concentration (nM) for O₂^- consumption (Figure 1). It is known that in physiological situations, only about 15-20% of the O₂^- generated in mitochondria is catabolized through its reaction with NO; but this pathway consumes about 80% of mitochondrial NO. On the other hand, Table I shows that although the mitochondrial NO steady-state concentration is reduced (28%) in the stunned heart, the ONOO^- generation is slightly increased (20%), mainly because of an increase (70%) in O₂^- steady-state concentration, leading to an increase (~80%) in nitration of tyrosine residues of mitochondrial proteins. Therefore, mitochondrial ONOO^- generation is mainly driven by O₂^- steady-state concentration rather than by NO steady-state concentration. In pathological situations in which the O₂^- steady-state concentration is increased and Mn‑SOD expression is not modified, as it is the case in the stunned heart, the metabolic pathway of O₂^- degradation through its reaction with NO is exacerbated, leading to an enhancement in ONOO^- generation (Figure 2). In addition, in experimental diabetes, an increase in the NO steady-state (28%) was observed together with a large increment in O₂^- steady-state concentration (3.5 fold), this latter as a consequent of not only the enhancement of O₂^- production but also the reduction in SOD active concentration. This physiopathological situation caused a 5-fold increase in ONOO^- production rate in heart mitochondria from diabetic in comparison with control animals. Accordingly, when O₂^- formation is stimulated more than two-fold the rate of NO synthesis and Mn-SOD concentration is reduced, NO is quantitatively converted to ONOO^- acquiring fundamental importance the ONOO^--derived chemical reactions (Figure 3). The conversion from reversible inhibition of cellular respiration by NO to pathological inhibition of mitochondrial function by the NO-derived ONOO^- has been observed in many physiopathological conditions, and it seems to be controlled by O₂^- steady-state concentration rather than by NO concentration. Mitochondrial dysfunction accompanied by ONOO^- generation increase is a hallmark of heart hypoxia-reperfusion injury,⁶⁰ sepsis,³² diabetes,⁶¹ among others. Moreover, these results agree with the modulation of the NO bioavailability in the vascular endothelium, another interesting microenvironment. In this case, an increased O₂^- production by NADPH oxidase (NOX 1 and NOS 2 isoforms) compromises the NO bioavailability, this latter fact associated.⁷⁰ In a high-blood pressure experimental model, it has been observed that the flavanol(-)-epicatechin regulates NO bioavailability not only through the modulation of NOS activity but also by regulating O₂^- production and NOX expression, suggesting that the reaction between O₂^- and NO is a key pathway in the endothelium-dependent vasorelaxation process.⁷⁰

Figure 1 Mitochondrial O2- is consumed by two competitive diffusion-controlled reactions: the dismutation reaction catalyzed by SOD and the free radical termination reaction with NO. NO and O2- steady-state concentrations and SOD concentration in the mitochondrial matrix are shown between brackets.

Figure 2 Metabolism of reactive oxygen and nitrogen species in mitochondrial matrix. The NO produced by mtNOS can readily interact with Complexes III and IV. O2- is formed at Complexes I and III and undergoes a very fast reaction with NO to form ONOO- or it is catabolized by Mn-SOD to form H2O2. NO and H2O2 are considered mitochondrion-cytosol signaling molecules.

Figure 3 schematic representations showing the changes in the steady-state concentrations of reactive oxygen and nitrogen species, in two physio pathological situations in which heart mitochondrial dysfunction has been observed. Mitochondrial NO production decreased in the myocardial stunning while NO generation increased in STZ-induced diabetes. However, mitochondrial ONOO- production was increased in both physio pathological situations due to the enhancement in the O2- steady-state concentration.

To conclude, in physiopathological conditions, ONOO^- generation is mainly driven by O₂^- steady-state concentration. The enhancement in O₂^- concentration increases its degradation by reaction with NO, declining NO bioavailability and increasing ONOO^- concentration. This way, the switch from NO-signaling pathways to oxidative damage takes place, with protein tyrosine nitration, protein oxidation and damage to Fe-S centers. Among them, changes in Complex I structure and function can exacerbate ONOO^- generation-secondary to O₂^- production rate enhancement-producing to a positive feedback toward oxidative distress, bioenergetics failure and the subsequent cell death.

This work was supported by research grants from the University of Buenos Aires (UBACYT 200-201-301-00731 and 200-201-601-00132), Agencies National de Promotion Scientific y Technological (ANPCYT, PICT 2014-0964), and Conseco National de Investigations Scientifics y Technical (CONICET; PIP 112-201-101-00444).

The author declares no conflict of interest.

1. Boveris A, Cadenas E. Cellular sources and steady-state levels of reactive oxygen species. In: Clerch LB, Massaro DJ, editors. Oxygen, gene expression and cellular function. New York: Marcel Deckker: Springer; 1997: p. 1‒25.
2. Boveris A, Cadenas E. Mitochondrial production of superoxide anions and its relationship to the antimycin insensitive respiration. FEBS Lett. 1975;54(3):311‒314.
3. Turrens JF, Boveris A. Generation of superoxide anion by the NADH dehydrogenase of bovine heart mitochondria. Biochem J. 1980;191(2):421‒427.
4. Mc Cord JM, Fridovich I. Superoxide dismutase. An enzymic function for erythrocuprein (hemocuprein) J Biol Chem. 1969;244(22):6049‒6055.
5. Klug D, Rabani J, Fridovich I. A direct demonstration of the catalytic action of superoxide dismutase through the use of pulse radiolysis. J Biol Chem. 1972;247:4839‒4842.
6. Boveris A, Chance B. The mitochondrial generation of hydrogen peroxide. Biochem J. 1973;134(3):707‒716.
7. Boveris A, Cadenas E, Stoppani AOM. Role of ubiquinone in the mitochondrial generation of hydrogen peroxide. Biochem J. 1976;156(2):435‒444.
8. Cadenas E, Boveris A, Ragan CI, et al. Production of superoxide radicals and hydrogen peroxide by NADH-ubiquinone reductase and ubiquinol-cytochrome c reductase from beef-heart mitochondria. Arch Biochem Biophys. 1977;180(2):248‒257.
9. Chance B, Sies H, Boveris A. Hydroperoxide metabolism in mammalian organs. Physiol Rev. 1979;59(3):527‒605.
10. Kissner R, Nauser T, Bugnon P, et al. Formation and properties of peroxynitrite as studied by laser flash photolysis, high-pressure stopped-flow technique and pulse radiolysis. Chem Res Toxicol. 1997;10(11):1285‒1292.
11. Boveris A, Valdez LB, Zaobornyj T, et al. Mitochondrial metabolic states regulate nitric oxide and hydrogen peroxide diffusion to the cytosol. Biochim Biophys Acta. 2006;1757(5-6):535–542.
12. Bleier L, Wittig I, Heide H, et al. Generator-specific targets of mitochondrial reactive oxygen species. Free Radic Biol Med. 2015;78:1‒10.
13. Booth DM, Enyedi B, Geiszt M, et al. Redox nanodomains are induced by and control Calcium signaling at the ER-mitochondrial interface. Mol Cell. 2016;63(2):240‒248.
14. Sies H. Hydrogen peroxide as a central redox signaling molecule in physiological oxidative stress: Oxidative eustress. Redox Biol. 2017;11:613‒619.
15. Ghafourifar P, Richter C. Nitric oxide synthase activity in mitochondria. FEBS Lett. 1997;418(3):291–296.
16. Giulivi C, Poderoso JJ, Boveris A. Production of nitric oxide by mitochondria. J Biol Chem. 1998;273:11038–11043.
17. Tatoyan A, Giulivi C. Purification and characterization of a nitric-oxide synthase from rat liver mitochondria. J Biol Chem. 1998;273(18):11044–11048.
18. Elfering SL, Sarkela TM, Giulivi C. Biochemistry of mitochondrial nitric-oxide synthase. J Biol Chem. 2002;277(41):38079‒38086.
19. Traaseth N, Elfering S, Solien J, et al. Role of calcium signaling in the activation of mitochondrial nitric oxide synthase and citric acid cycle. Biochim Biophys Acta. 2004;1658(1-2):64‒71.
20. Ghafourifar P, Cadenas E. Mitochondrial nitric oxide synthase. Trends Pharmacol Sci. 2005;26(4):190‒195.  
21. Valdez LB, Zaobornyj T, Bombicino S, et al. Physiological regulation of heart mitochondrial nitric oxide synthase. In: Alvarez S, Evelson P, editors. Free Radical Pathophysiology. India: Transword Research Network; 2008: p. 177‒190.
22. Alvarez S, Valdez LB, Zaobornyj T, et al. Oxygen dependence of mitochondrial nitric oxide synthase activity. Biochem Biophys Res Commun. 2003;305(3):771–775.
23. Valdez LB, Zaobornyj T, Alvarez S, et al. Heart mitochondrial nitric oxide synthase. Effects of hypoxia and aging. Mol Aspects Med. 2004;25(1-2):49‒59.
24. Gonzales GF, Chung A, Miranda S, et al. Heart mitochondrial nitric oxide synthase is upregulated in male rats exposed to high altitude (4,340 m). Am J Physiol Heart Circ Physiol. 2005;288(6):2568‒2573.
25. Zaobornyj T, Valdez LB, La Padula P, et al. Effect of sustained hypobaric hypoxia during maturation and aging on rat myocardium. II. mtNOS activity. J Appl Physiol. 2005;98(6):2370‒2375.
26. Fellet AL, Balaszczuk AM, Arranz C, et al. Autonomic regulation of pacemaker activity: role of heart nitric oxide synthases. Am J Physiol Heart Circ Physiol. 2006;291(3):1246‒1254.
27. Carreras MC, Peralta JG, Converso DP, et al. Modulation of liver mitochondrial NOS is implicated in thyroid-dependent regulation of O2 uptake. Am J Physiol Heart Circ Physiol. 2001;281(6):2282‒2288.
28. Finocchietto P, Barreyro F, Holod S, Peralta et al. Control of muscle mitochondria by insulin entails activation of Akt2-mtNOS pathway: Implications for the metabolic syndrome. PLoS One. 2008;3:e1749.  
29. Costa LE, La Padula P, Lores Arnaiz S, et al. Long-term angiotensin II inhibition increases mitochondrial nitric oxide synthase and not antioxidant enzyme activities in rat heart. J Hypertens. 2002;20(12):2487‒2494.
30. Valdez LB, Zaobornyj T, Boveris A. Mitochondrial metabolic states and membrane potential modulate mtNOS activity. Biochim Biophys Acta. 2006;1757(3):166–172.  
31. Valdez LB, Boveris A. Mitochondrial nitric oxide synthase, a voltage-dependent enzyme, is responsible for nitric oxide diffusion to cytosol. Front Biosci. 2007;12:1210–1219.
32. Alvarez S, Boveris A. Mitochondrial nitric oxide metabolism in rat muscle during endotoxemia. Free Radic Biol Med. 2004;37(9):1472‒1478.
33. Vanasco V, Magnani ND, Cimolai MC, et al. Endotoxemia impairs heart mitochondrial function by decreasing electron transfer, ATP synthesis and ATP content without affecting membrane potential. J Bioenerg Biomembr. 2012;44(2):243–252.
34. Boveris A, Lores Arnaiz S, Bustamante J, et al. Pharmacological regulation of mitochondrial nitric oxide synthase. Meth Enzymol. 2002;359:328‒339.
35. Lores Arnaiz S, Coronel MF, Boveris A. Nitric oxide, superoxide, and hydrogen peroxide production in brain mitochondria after haloperidol treatment. Nitric oxide. 1999;3(3):235‒243.
36. Lores Arnaiz S, Czerniczyniec A, Bustamante J, et al. Brain mitochondrial nitric oxide synthase: in vitro and in vivo inhibition by chlorpromazine. Arch Biochem Biophys. 2004;430(2):170‒177.
37. Boveris A, D' Amico G, Lores Arnaiz S, et al. Enalapril increases mitochondrial nitric oxide synthase activity in heart and liver. Antioxid Redox Signal. 2003;5(6):691‒697.
38. Navarro A, Torrejon R, Bandez MJ, et al. Mitochondrial function and mitochondria-induced apoptosis in an overstimulated rat ovarian cycle. Am J Physiol Endocrinol Metab. 2005;289(6):1101‒1109.
39. Bombicino SS, Iglesias DE, Rukavina Mikusic IA, et al. Hydrogen peroxide, nitric oxide and ATP are molecules involved in cardiac mitochondrial biogenesis in Diabetes. Free Radic Biol Med. 2017;112:267‒276.  
40. Boveris A, Costa LE, Cadenas E, et al. Regulation of mitochondrial respiration by adenosine diphosphate, oxygen, and nitric oxide. Meth Enzymol. 1999;301:188‒198.
41. Cleeter MW, Cooper JM, Darley Usmar VM, et al. Reversible inhibition of cytochrome c oxidase, the terminal enzyme of the mitochondrial respiratory chain, by nitric oxide. Implications for neurodegenerative diseases. FEBS Lett. 1994;345(1):50‒54.  
42. Brown GC, Cooper CE. Nanomolar concentrations of nitric oxide reversibly inhibit synaptosomal respiration by competing with oxygen at cytochrome oxidase. FEBS Lett. 1994;356(2-3):295‒298.  
43. Takehara Y, Nakahara H, Inai Y, et al. Oxygen-dependent reversible inhibition of mitochondrial respiration by nitric oxide. Cell Struct and Funct. 1996;21(4):251‒258.
44. Poderoso JJ, Carreras MC, Lisdero C, et al. Nitric oxide inhibits electron transfer and increases superoxide radical production in rat heart mitochondria and submitochondrial particles. Arch Biochem Biophys. 1996;328(1):85‒92.
45. Iglesias DE, Bombicino SS, Valdez LB, et al. Nitric oxide interacts with mitochondrial complex III producing antimycin-like effects. Free Radic Biol Med. 2015;89:602‒613.  
46. Pacher P, Beckman JS, Liaudet L. Nitric oxide and peroxynitrite in health and disease. Physiol Rev. 2007;87(1):315‒424.
47. Bartesaghi S, Radi R. Fundamentals on the biochemistry of peroxynitrite and protein tyrosine nitration. Redox Biol. 2018;14:618‒625.
48. Ferrer Sueta G, Campolo N, Trujillo M, et al. Biochemistry of peroxynitrite and protein tyrosine nitration. Chem Rev. 2018;118(3):1338‒1408.
49. Koppenol WH. The basic chemistry of nitrogen monoxide and peroxynitrite. Free Radic Biol Med. 1998;25(4-5):385‒391.
50. Radi R, Peluffo G, Alvarez MN, et al. Unraveling peroxynitrite formation in biological systems. Free Radic Biol Med. 2001;30(5):463‒488.
51. Radi R, Beckman JS, Bush KM, et al. Peroxynitrite oxidation of sulfhydryls: the cytotoxic potential of superoxide and nitric oxide. J Biol Chem. 1991;266(7):4244–4250.
52. Pryor WA, Jin X, Squadrito GL. One- and two-electron oxidations of methionine by peroxynitrite. Proc Natl Acad Sci. U S A. 1994; 91(23):11173–11177.
53. Bartlett K, Church DF, Bounds PL, et al. The kinetics of the oxidation of L‑ascorbic acid by peroxynitrite. Free Radic Biology and Medicine. 1995;18(1):85‒92.
54. King PA, Anderson VE, Edwards JO, et al. A stable solid that generates hydroxyl radical upon dissolution in aqueous solution: reactions with proteins and nucleic acid. J Am Chem Soc. 1992;114(13):5430–5432.  
55. Radi R, Beckman JS, Bush KM, et al. Peroxynitrite-induced membrane lipid peroxidation: the cytotoxic potential of superoxide and nitric oxide. Arch Biochem Biophys. 1994;288(2):481–487.
56. Darley Usmar VM, Hogg N, O Leary VJ, et al. The simultaneous generation of superoxide and nitric oxide can initiate lipid peroxidation in human low density lipoprotein. Free Radic Res Commun. 1992;17(1):9–20.
57. Radi R. Protein tyrosine nitration: biochemical mechanisms and structural basis of functional effects. Acc Chem Res. 2013;46(2):550‒559
58. Souza JM, Peluffo G, Radi R. Protein tyrosine nitration–functional alteration or just a biomarker? Free Radic Biol Med. 2008;45(4):357–366.
59. Valdez LB, Alvarez S, Arnaiz SL, et al. Reactions of peroxynitrite in the mitochondrial matrix. Free Radic Biol Med. 2000;29(3-4):349‒356.
60. Valdez LB, Bandez MJ, Navarro A, Boveris A. Brain mitochondrial dysfunction and complex I syndrome in Parkinson’s disease. In: Ed. Ranna AQ, editor. Etiology and Pathophysiology of Parkinson’s disease. Croatia: InTech; p. 317–328.
61. Valdez LB, Zaobornyj T, Bombicino S, et al. Complex I syndrome in myocardial stunning and the effect of adenosine. Free Radic Biol Med. 2011;51(6):1203–1212.
62. Bombicino SS, Iglesias DE, Rukavina Mikusic IA, et al. Diabetes impairs heart mitochondrial function without changes in resting cardiac performance. Int J Biochem Cell Biol. 2016;81(Pt B):335–345.
63. Navarro A, Boveris A. Rat brain and liver mitochondria develop oxidative stress and lose enzymatic activities on aging. Am J Physiol Regul Integr Comp Physiol. 2004;287(5):1244–1249.  
64. Boveris A, Carreras MC, Poderoso JJ. The regulation of cell energetics and mitochondrial signaling by nitric oxide. In: Ignarro L. editor. Nitric Oxide: Biology and Pathobiology. 2nd ed. London: Elsevier Academic Press; 2010:441–482.
65. Valdez L, Bombicino S, Iglesias D, et al. Mitochondrial complex I inactivation after ischemia-reperfusion in the stunned heart. In: Gelpi R, Boveris A, Poderoso JJ, editors.Biochemistry of Oxidative Stress: Physiopathology and Clinical Aspects. Advances in Biochemistry in Health and Disease. USA: Springer; 2016:245–257.
66. Costa LE, Boveris A, Koch OR, et al. Liver and Heart mitochondria in rats submitted to chronic hypobaric hypoxia. Am J Physiol. 1988;255(Pt 1):123‒129.
67. Poderoso JJ, Carreras MC, Schöpfer F, et al. The reaction of nitric oxide with ubiquinol: kinetic properties and biological significance. Free Radic Biol Med. 1999;26(7-8):925‒935.
68. Antunes F, Boveris A, Cadenas E. On the mechanism and biology of cytochrome oxidase inhibition by nitric oxide. Proc Natl Acad Sci U S A. 2004;101(48):16774‒16779.
69. Boveris A, Stoppani AO. Inhibition of electron and energy transfer in mitochondria by 19-nor-ethynyltestosterone acetate. Arch Biochem Biophys. 1970;141(2):641‒655.
70. Litterio MC, Vazquez Prieto MA, Adamo AM, et al. (-)-Epicatechin reduces blood pressure increase in high-fructose-fed rats: effects on the determinants of nitric oxide bioavailability. J Nutr Biochem. 2015;26(7):745‒751.