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Biological Research

versión impresa ISSN 0716-9760

Biol. Res. v.33 n.2 Santiago  2000 

Mitochondrial Function and Nitric Oxide Utilization


Laboratory of Oxygen Metabolism, University Hospital, School of Medicine, University of Buenos


In spite of some new criticism on endosymbiosis as the evolutionary process leading to the development of eukariotic cells, it is widely accepted that modern mitochondria originated from ancestral bacteria (Karlin and Campbell, 1994). Considerable evidence has appeared over the past few years that nitric oxide (NO) is the principal intermediate between nitrite and N2O during denitrification (Goretski et al., 1990). Moreover, NO produced by bacterial reductases decreases its respiration by inhibiting cytochrome oxidase, the terminal electron acceptor of the mitochondrial electron transfer chain (Carr and Ferguson, 1990). Although the exact meaning of the presence of steady-state concentrations of NO in bacteria has not yet been fully defined, it appears that NO has profound regulatory effects in prokariotic organisms. First, it controls the respiratory rate with a remarkable effect at low O2 tensions. Second, the regulation of cytochrome oxidase activity represents a striking factor in the availability of nucleotide triphosphate, the building blocks of DNA synthesis, and in bacterial proliferation. Finally, de-energization results in changes in ionic fluxes, adaptive responses and bacterial survival. In this way, the cytotoxic action of activated macrophages on tumor cells or bacteria was thought to be due to inhibitory NO effects on iron-sulfur compounds and iron loss at complexes I-II of the respiratory chain (Adams and Hamilton, 1984; Steinman and North, 1986). Accordingly, during infection, the induction of nitric oxide synthase (iNOS) is thought to contain microbial proliferation, most likely representing an unspecific but strong immune response in different tissues (Fang, 1997).

In recent years, the discovery of NOS and the production of nitric oxide by mammals, and particularly in humans, has been followed by exciting and intense scientific activity devoted to dissecting NO functions in different cells types and tissues. The results have shown that a) in the studied species, almost every tissue has at least one NOS isoform; b) NO synthases are present as constitutive or inducible isoforms, although the levels of gene expression of constitutive isoforms may be under regulation, and conversely, iNOS may function as a "constitutive" enzyme in some cells (Michel and Feron, 1997), c) NO fluxes and effects are vectorially directed either to the extracellular space (e.g. endothelial cells, synaptic terminals) or to the intracellular millieu (mitochondria). Production and diffusion of cytosolic or mitochondrial NO and its catabolism or utilization (Poderoso et al., 1999a) define the different cell steady-state concentrations at which the nitrogen radical evokes physiological responses.

Recently, different laboratories have been interested in the effects of NO on mitochondrial activities (Ghafourifar and Richter, 1997; Giulivi et al., 1998). The primary question was whether the mitochondria of higher organisms, as an evolutionary product of bacteria, were still sensitive to NO. Furthermore, were mitochondria able to produce and catabolize NO? Did NO play a significant role in the regulation of physiological mitochondrial functions? Finally, could an excess of NO affect mitochondrial function and integrity? The responses to these inquiries were affirmative; it appears that the effects of NO on mitochondria are not an evolutionary relict but rather a well-preserved response completely integrated into the cell function of higher organisms.

The effects of NO on mitochondria: an overview

The results of different experiments performed by a significant number of research groups provide evidence that NO acts on different energy-linked and metabolic mitochondrial pathways. A brief analysis is presented in the following items.

Inhibition of cytochrome oxidase

In the last decade, different groups of investigators observed that NO inhibited mitochondrial respiration in a reversible and dose-dependent manner (Cleeter et al., 1994; Brown and Cooper, 1994; Okada et al., 1996) (Fig 1). The most impressive effects were undoubtedly the inhibition of cytochrome oxidase (complex IV) and other reactions involving the intermediates of complexes I and III (Clementi et al., 1998, Poderoso et al., 1996). At estimated steady-state concentrations in the 0.02-0.1 µM range in mammalian tissues (Poderoso et al., 1998), NO was shown to be a high affinity inhibitor of cytochrome oxidase in a competitive way with oxygen (Boveris et al., 1999, Cleeter et al., 1994, Koivisto et al., 1997). The NO concentrations that produce a half-inhibition of the cytochrome oxidase activity or O2 uptake in isolated mitochondria are in the low range of 80-200 nM. The effect was confirmed in mitochondria and submitchondrial particles from rat muscle (Cleeter et al., 1994), rat liver (Takehara et al., 1995) and rat heart (Borutait’e and Brown, 1996; Cassina and Radi, 1996; Poderoso et al., 1996), in tissue preparations like rat brain synaptosomes (Brown and Cooper, 1994) and in the isolated rat heart (Poderoso et al., 1998). Accordingly, an increase in whole body O2 uptake was found in conscious dogs after administration of NOS inhibitors (Shen et al., 1994).

Figure 1 Inhibition of oxygen uptake of liver mitochondria by nitric oxide

The reversible action of NO on mitochondrial complexes provides the basis for a regulatory effect on O2 uptake, depending on NO utilization. For example, NO concentration should depend on the dissociation of cytochrome oxidase-NO complex which occurs as a first-order reaction with k = 0.13 s-1 (Giuffre et al., 1998).

Changes in mitochondrial membrane potential

In addition to the inhibition of O2 uptake, NO produces a decrease in the membrane potential of mitochondria (Takehara et al., 1995) (Figs 2 and 3). The half maximal effect of NO on membrane potential is observed in liver mitochondria at approximately 0.25 µM NO, an effect that is slightly less sensitive than the inhibition of O2 uptake. Nitric oxide or its by-product peroxynitrite and associated changes in membrane potential could affect calcium fluxes across the mitochondrial membrane and modify the permeability transition pore, a multichannel that operates calcium outflow. This topic is relevant since calcium concentration in the mitochondrial matrix is partially responsible for mtNOS activation; mtNOS shares immunological aspects with iNOS but appears to be a calcium-dependent variant or isoform.

Figure 2 Nitric oxide-induced changes in membrane potential of liver mitochondria. The data are expressed as a percentage of the respective depolarization induced by FCCPF
Figure 3 The rate of recovery of mitochondrial membrane potential depends on the utilized NO concentrations

Mitochondrial NO steady-state concentration and the regulation of O2 uptake

The inhibition of O2 uptake is also observed in perfused organs where the steady-state intracellular NO concentration reached upon infusion depends on NO concentration in the infusion fluid, perfusate flow, NO tissue uptake, and NO binding to cellular and mitochondrial components, such as myoglobin and cytochrome-oxidase (Poderoso et al., 1998).

On the other hand, the biological effects of NO are related to simultaneous O2 concentrations; lowering the pO2 amplifies NO effects (Takehara et al., 1996; Cassina and Radi, 1996). There is, in fact, a true competition between O2 and NO for the cytochrome oxidase Cu2+ reaction center (Boveris et al., 1999; Cassina and Radi, 1996; Zhang et al., 1996). The inhibition of the O2 uptake by NO in isolated rat liver and heart mitochondria is consistent with NO’s affinity for cytochrome oxidase, which is 150 times higher than the corresponding O2 affinity (Boveris et al., 1999). This fact is relevant because O2 concentrations in myocardial cells are as low as 3 to 8 µM O2 (Gayeski and Honig et al., 1991), a condition in which a NO steady-state concentration of 50 nM would effectively compete with O2, producing a cytochrome oxidase inhibition of appriximately 30% (Poderoso et al., 1998). It was also recently confirmed that NO inhibition of the mitochondrial electron transfer chain is followed by a decrease in ATP synthesis (Brookes et al., 1999).

The NO-inhibitory effects on cytochrome oxidase activity and oxygen metabolism have been considered to be a regulatory and adaptative physiological mechanism in response to changes in O2 availability (Poderoso et al., 1998). This inhibition has been further implicated in inflammatory, ischemic, or neurodegenerative diseases (Brown, 1997). It is likely that the two roles express both the reversible effect of NO on cytochrome oxidase and its participation in the generation of peroxynitrite (ONOO-). Furthermore, both effects depend on the ability of NO to react with O2- to produce ONOO-. The utilization of NO by the reaction of NO with O2- determines the intramitochondrial NO steady-state concentrations and the O2/NO ratio, which regulates cytochrome oxidase activity. The formation of the powerful oxidant ONOO- was repeatedly associated with mitochondrial and cellular toxic effects. In this way, physiology or toxicity depend on the amplification of a normal control pathway based on the intramitochondrial NO steady-state concentrations.

The NO-induced mitochondrial production of oxygen active species

Superoxide anion (O2-) and hydrogen peroxide (H2O2) are reactive oxygen species normally produced as by-products of mitochondrial respiration involving the univalent and bivalent reduction of oxygen. (Valdez et al., 2000). Moreover, it was observed that both O2- and H2O2 are produced in mitochondria and in the cytosol in specific ways. There is a strict compartmentalization for O2- that does not diffuse outside mitochondria. Accordingly, manganese-superoxide dismutase (Mn-SOD) is strictly localized in the mitochondrial matrix. In contrast, H2O2 is freely diffusible across mitochondrial membranes and therefore it provides signals for nuclear or cytosolic processes that are regulated by oxidants and represents the presence of NO and NO-derived O2- active species within mitochondria. The concentration of O2- is similar in mitochondria and in bacteria, in the 10-10 M range (Boveris and Cadenas, 1997; Imlay and Fridovich, 1991). The production of non-charged H2O2 by stoichiometric dismutation of O2- is catalyzed by SOD with diffusion-controlled rate reactions (k=2.3 x 109 M-1 s-1 ) hydrogen peroxide is partially utilized by glutathione peroxidase and by peroxisomal catalase.

The exposure of mitochondria or submitochondrial particles to NO initiates or increases the mitochondrial production of both O2- and H2O2 (Poderoso et al., 1996; Packer et al., 1996). At 0.2-1 µM NO, mitochondria isolated from almost all rat tissues have NO concentration-dependent H2O2 production rates between 0.05 and 0.3 nmol/ protein.

The dependence of the H2O2 production rate in rat liver mitochondria on NO concentration is shown in Figure 4. The addition of NO has a marked effect which under these conditions, expresses that the main source of O2- is the reaction of NO with membrane ubiquinol to release NO and ubisemiquinone, which is in turn autoxidized giving O2- and the respective ubiquinone (reactions 1-2) (Poderoso et al., 1999b).

Figure 4 Nitric oxide utilization and NO-induced mitochondrial production of H2O2 are related to the concentration of electron transfer chain intermediaries. a: control; b, c and d: 0.1, 0.2 and 0.3 mg mitochondrial protein/ml


NO + UQH2 ® NO- + UQH• + H+
UQH• + O2 ® UQ + O2- + H+

Production of O2 active species determines NO utilization and modulates NO effects on O2 uptake

Previous experiments in our laboratory confirmed that the rates of NO utilization by mitochondria under aerobic physiological conditions depend on the ability of NO itself to generate a sustained production of O2 active species. In this way, the formed O2- reacts quickly with NO to form peroxynitrite (ONOO-) with a diffusion-controlled rate constant (1.9 x 10-10, ref Koppenol, 1998) (reaction 3).


NO + O2- ® ONOO-

Considerable evidence supports the notion that NO decay or utilization is based upon the mitochondrial production of O2 active species elicited by NO itself through reactions 1-3. First, the amperometric decay of a single 0.1-2 µM NO pulse is decreased by the addition of SOD (Poderoso et al., 1996) and increased by the addition of exogenous soluble ubiquinone (Poderoso et al., 1999a). Second, extraction of membrane-bound ubiquinone almost completely prevents mitochondrial NO utilization and consequently prolongs NO effects, which are reverted by reconstitution of the membranes. Third, the degree of NO inhibition of cytochrome oxidase is linearly related to the concentration of mitochondrial ubiquinone (Poderoso et al., 1999a).

The formation of peroxynitrite in mitochondria: an undesirable consequence of NO metabolism

As mentioned above, the aerobic metabolism of NO necessarily involves the formation of peroxynitrite, which is able to nitrate and/or oxidize the side chain of constitutive aminoacids of mitochondrial proteins. Sulfhydrils of cysteine and methionine are particularly sensitive to ONOO- effects. The addition of ONOO- to mitochondria results in the inhibition of Complexes I and II (Cassina and Radi, 1996), aconitase (Castro et al., 1994) and ATPase (Radi et al., 1994).

The potential damage induced by ONOO- depends on the interception by matrix scavengers such as reduced glutathione or by the activity of some mitochondrial enzymes such as gluthatione peroxidase (Sies et al., 1997). However, it seems clear that as O2- production is stoichiometrically related to NO, the final ONOO- mitochondrial concentration for a given condition should depend on the corresponding NO concentration. This fact acquires relevance under those conditions of increased NO, such as after NOS induction or after activation of mitochondrial NOS (Ghafourifar and Richter, 1997; Poderoso et al., 1999a). Accordingly, we previously reported the spontaneous intramitochondrial ONOO- formation during endotoxemia in the rat (Boczkowski et al., 1999). This finding was accompanied by an increase of steady-state concentrations of both NO and H2O2 in the diaphragm, the organ under study. The relative contribution of O2- to the formation of ONOO- through reaction 3 or its dismutation to freely diffusible H2O2 by Mn-SOD-catalyzed reaction depends on the respective constant reaction rates and on NO concentrations. It is apparent that at low 50-100 nM NO, most of the formed O2- should be dismutated to H2O2; in contrast, at 0.5 µM NO, the net formation of ONOO- is clearly favored. Moreover, ONOO- or ONOO--derived radicals are able to react with ubiquinol to form additional O2- (Poderoso et al., 1999a) and thus this reaction will modify the final ONOO-/H2O2 ratio at any NO mitochondrial level. Ubiquinol-centered reactions detoxify mitochondria from prolonged NO-effects finally producing nitrite, nitrate, and H2O2.


Nitric oxide has profound effects on the mitochondrial function. It first appeared to be as an integral mitochondrial regulator of O2 uptake, ATP synthesis (Brookes et al., 1999) and the production of oxygen active species. The inhibitory NO effects on cytochrome oxidase depend on the matrix NO steady-state concentrations and consequently, on NO utilization. In aerobic conditions, nitric oxide decays primarily through a sequence of ubiquinol-centered reactions, ultimately producing peroxynitrite and hydrogen peroxide (Fig 4). Considering the high affinity of NO for cytochrome oxidase, these pathways are important to avoid a sustained inhibition of the mitochondrial transfer chain. Additionally, the mechanism will become a dangerous one, whether or not NO concentration increases in the mitochondrial matrix. The consequence will be the oxidation of proteins and lipids, mitochondrial damage and alterations in the life cycle, as judged by mitochondrial influence on cell apoptosis. (Valdez et al., 2000).

Figure 5 . Physiological and pathological effects of NO reactions in mitochondria.

Corresponding Author: JJ Poderoso. Laboratory of Oxygen Metabolism, Cordoba 2351 - 1120, Buenos Aires, Argentina. email: Fax: +541145083983

Received: February 24, 2000. Accepted: February 24, 2000


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