The effect of nitric oxide on mitochondrial respiration

doi:10.1016/j.niox.2019.04.005

Nitric Oxide

Volume 88, 1 July 2019, Pages 61-72

https://doi.org/10.1016/j.niox.2019.04.005 Get rights and content

Highlights

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Nitric oxide, at low concentrations, causes a reversible inhibition of cytochrome c oxidase.
•
Peroxynitrite oxidizes most of the mitochondrial components, causing severe damage.
•
The mechanisms by which nitric oxide kills or protects cells are still far from clear.
•
Peroxynitrite is most likely the responsible agent of nitric oxide-induced cytotoxicity.

Abstract

This article reviews the interactions between nitric oxide (NO) and mitochondrial respiration. Mitochondrial ATP synthesis is responsible for virtually all energy production in mammals, and every other process in living organisms ultimately depends on that energy production. Furthermore, both necrosis and apoptosis, that summarize the main forms of cell death, are intimately linked to mitochondrial integrity.

Endogenous and exogenous •NO inhibits mitochondrial respiration by different well-studied mechanisms and several nitrogen derivatives. Instantaneously, low concentrations of •NO, specifically and reversibly inhibit cytochrome c oxidase in competition with oxygen, in several tissues and cells in culture. Higher concentrations of •NO and its derivatives (peroxynitrite, nitrogen dioxide or nitrosothiols) can cause irreversible inhibition of the respiratory chain, uncoupling, permeability transition, and/or cell death. Peroxynitrite can cause opening of the permeability transition pore and opening of this pore causes loss of cytochrome c, which in turn might contribute to peroxynitrite-induced inhibition of respiration. Therefore, the inhibition of cytochrome c oxidase by •NO may be involved in the physiological and/or pathological regulation of respiration rate, and its affinity for oxygen, which depend on reactive nitrogen species formation, pH, proton motriz force and oxygen supply to tissues.

Introduction

Mitochondrial function integrity is key to cell life. Alterations in mitochondrial function have been widely shown to lead to the disruption of cell function, such as tissue or organism disease or even death. Although traditionally regarded as the powerhouse of the cell, the metabolic functions of mitochondria go far beyond bioenergetics. Mitochondria catabolize nutrients for energy, generate macromolecule biosynthetic precursors, compartmentalize metabolites for redox homeostasis maintenance and work as hubs for metabolic waste management. Mitochondrial demands and their complex integration into cell biology outweigh the provision of ATP, a notion which changes our perception of mitochondria and puts organelles in the limelight of cell biology and medicine [1].

Mitochondrial calcium uptake plays a central role in cell signaling and the regulation of mitochondrial function, whereas excessive mitochondrial calcium accumulation has been widely associated with pathological scenarios. Mitochondrial function and dysfunction are thus considered key players in metabolic disease, cancer cell metabolism, neurodegeneration and aging. Mitochondria produce free radical species, possibly also nitric oxide (•NO), and are major targets of oxidative damage. In this sense, several reports have focused on the mechanisms of mitochondrial radical generation and the potential regulatory role of uncoupling proteins, as well as oxidative injury targets [2]. Particularly •NO and reactive •NO species (RNS) have multidimensional effects on mitochondrial function, some of them discussed in this review.

Joseph Priestley discovered both •NO (‘nitrous air’) and oxygen (‘dephlogisticated air’) in the early 1770's and performed the reaction between these two gases, producing the soluble brown gas nitrogen dioxide to quantify the amount of oxygen (O₂) in normal air [3,4]. Priestley applied this method to show that mice consumed the O₂ contained in air and thus developed the first accurate measurement of respiration. •NO was subsequently used as a chemical tool, but it was not until the late 1980's that this gas was proven to be produced and utilized by animals, both as a physiological regulator and a cytotoxic agent [[5], [6], [7], [8]].

The paradox of •NO acting as both a physiological regulator and a cytotoxic agent was apparently resolved when •NO was found to react with superoxide to produce peroxynitrite (ONOO⁻), which is a potent oxidant and harmful agent. Therefore, it was suggested that •NO was mostly responsible for the physiological regulation, while peroxynitrite was responsible for the cytotoxic properties [9]. However, this appealing dichotomy may not be as clear-cut as once appeared.

It is well established that •NO causes rapid and reversible inhibition of cytochrome c oxidase at low levels, so that •NO is potentially a physiological regulator of respiration. •NO is produced by different isoforms of •NO synthase in different locations within and outside cells, and •NO is consumed (particularly by hemoglobin and myoglobin) in different locations [10]. Interestingly, •NO is a major regulator of O₂ supply via vasodilation of vascular smooth muscle and inhibits the main consumer of O₂ by inhibiting cytochrome c oxidase. The fine balance between O₂ delivery to cells and O₂ consumption by the electron transport chain may be due to the gradients of •NO and O₂ which interact in several ways. In this regard, it has been reported that O₂ is a substrate of all •NO synthases; the Km of •NO synthases for O₂ is potentially within the physiological range in the heart. Hemoglobin and myoglobin produce •NO at low O₂ and consume •NO at high O₂, and •NO oxidation of the globins inactivates their ability to transport O₂ (and •NO). Combining the actions of •NO in different tissues and its regulation of blood flow contributes to better substrate delivery and utilization by different tissues, including muscles and myocardium, in basal metabolism or exercise [11]. Therefore, low physiological levels of •NO (1–200 nM •NO) can cause substantial inhibition of respiration, and potentially make tissue respiration highly sensitive to oxygen tension.

In a variety of tissues, organisms and conditions respiration can become very sensitive to the O₂ level, and the competition between •NO and O₂ at cytochrome c oxidase might play a role in this sensitivity. Nevertheless, there is no direct evidence for this in vivo, the apparent Km of •NO synthase for O₂ is considerably higher than Km of cytochrome c oxidase for O₂ (in the absence of •NO), so that at moderately low O₂ levels •NO synthase might be unable to produce sufficient •NO to inhibit cytochrome c oxidase [12].

•NO becomes an inhibitor of the enzyme, in competition with O₂. Evidence suggests that at low [O₂] •NO will activate the soluble guanylate cyclase to produce vasodilatation and therefore increase the local supply of O₂. This effect has been suggested to be a significant contributing factor in hypoxic vasodilatation [13]. •NO therefore behaves as a rheostat for respiration and eventually acts to ration the consumption of the O₂ available. Then, elevated •NO from its decreased inactivation by cytochrome c oxidase at low [O₂], is a protective mechanism against tissue hypoxia. It could be considered as a useful scenario for the response of tissues to this deleterious condition [14].

Even before •NO was discovered as the responsible agent, it was known that activated macrophages produced a compound which was cytotoxic to other cells by irreversibly inhibiting their mitochondrial respiration [[15], [16], [17]]. •NO and its derivatives peroxynitrite and nitrogen dioxide can indeed irreversibly inhibit mitochondrial respiration; however, a radically different effect of •NO on mitochondrial respiration was reported in 1994 [18]. Very low levels of •NO caused a completely reversible inhibition of mitochondrial respiration at cytochrome c oxidase in competition with O₂. In fact, the Ki was estimated to be 270 nM •NO when the oxygen concentration was between 125 and 165 μM O₂, and 60 nM •NO when the oxygen concentration was 18–38 μM O₂, as determined in synaptosomes [19]. This raised the exciting question whether •NO was a physiological regulator of mitochondrial respiration, in fact the only direct regulator of this metabolic pathway known so far. In this context, we will review the effects of •NO on cytochrome c oxidase, the impact of •NO derivatives and other radicals on mitochondria, and tisular and cellular •NO measurements in vivo, all of which ultimately lead to the modulation of mitochondrial respiration.

Section snippets

Cytochrome c oxidase

Cytochrome c oxidase (cytochrome aa3, complex IV) is the terminal complex of the mitochondrial respiratory chain, responsible for about 90% of O₂ consumption in mammals, and essential for virtually all energy production in cells [20,21]. It is located in the mitochondrial inner membrane, and catalyzes the oxidation of cytochrome c²⁺ to cytochrome c³⁺ and the reduction of O₂ to water, which is coupled to the pumping of protons into the intermembrane space of the mitochondria. This oxidase

Reversible •NO inhibition of cytochrome c oxidase

•NO binds to the O₂ binding site of cytochrome c oxidase, as first reported in 1955 by Wainio [26]. This study found that the addition of •NO to the reduced form of isolated cytochrome c oxidase induced a shift in the optical spectrum of cytochromes aa3 similar to the shift induced by carbon monoxide, which suggested that •NO binds to the same site of O₂ on the enzymatic complex, i.e. the reduced form of heme a3. This raised the possibility that •NO could be an inhibitor of cytochrome c oxidase

Tissue •NO measurements in vivo

The measurements of •NO levels in vivo have been previously described and performed by different techniques like Malinski's •NO-selective electrode, microsensors for •NO and carbon fiber microelectrodes in living tissues [55]. Abundant literature has presented detailed studies of •NO concentration and its changes in dynamics in vivo. Relevant evidence demonstrates heterogeneous •NO concentration dynamics in the hippocampal subregions, functionally dependent on the stimulation of the NMDA

•NO produced in vivo by cells and tissues and effects on mitochondrial respiration

Research into whether •NO can regulate in vivo cellular and tissue respiration is crucial but complicated for several reasons such as: (a) •NO can react with cellular components to produce peroxynitrite, nitrosothiols and other derivatives, which may affect respiration generally by an irreversible inhibition; (b) •NO is a potent vasodilator which increases O₂ supply; (c) •NO may affect ATP consumption indirectly modulating cellular O₂ consumption; (d) •NO can affect muscle contraction by

Effects of •NO in tissue mitochondria (apart from cytochrome c oxidase inhibition)

Poderoso et al. reported that •NO inhibited the cytochrome bc1 complex (complex III) in submitochondrial particles. This inhibition was partially reversible and occurred at •NO concentrations somewhat higher than those inhibitingcytochrome c oxidase [33]. However, the inhibition observed may be due to the S-nitrosoglutathione/dithiothreitol system used to generate •NO, or to peroxynitrite generated from •NO and superoxide in this system. Poderoso et al. [72] also demonstrated that •NO can react

Irreversible inhibition of mitochondrial respiration by •NO

In addition to its roles in normal physiology, it is important to appreciate that •NO also has pathophysiological actions. •NO reacts with various O₂ species in the cell to form highly reactive molecules that damage cellular components through various mechanisms.

Cells exposed to •NO (or •NO-producing cells) show immediate but reversible inhibition of respiration at cytochrome c oxidase. However, after several hours of exposure to •NO, irreversible inhibition arises probably due to conversion of

Peroxynitrite-mediated mitochondrial respiration inhibition

•NO reacts rapidly with superoxide (⋅O₂⁻) to produce peroxynitrite (ONOO⁻), which may act as an oxidant itself, isomerize to nitrate, or protonate and dissociate to give nitrogen dioxide (•NO₂) and hydroxyl radical (⋅OH), all of them strong oxidants [79,80]. Peroxynitrite reacts with protein and non-protein thiols, tyrosine residues, unsaturated fatty acids, DNA, •NO and a variety of other molecules. Addition of peroxynitrite to mitochondria causes extensive protein modification and

•NO-induced ROS, RNS and mitochondrial permeability transition

Apart from inhibiting respiration, •NO has two other effects on mitochondria relevant to the induction of cell death: (i) induction of reactive O₂ species (ROS) and RNS production from mitochondria, and (ii) induction of mitochondrial permeability transition (MPT) by RNS.

The mitochondrial respiratory chain can produce superoxide, which dismutates to hydrogen peroxide, while inhibition of the chain may enhance the production of these ROS. At moderate levels, •NO can acutely increase ⋅O₂⁻ and H₂O₂

•NO and peroxynitrite effects on mitochondrial superoxide and hydrogen peroxide production and on the PTP

Poderoso et al. [33] reported that the addition of •NO or •NO donors greatly increased superoxide production by submitochondrial particles, and also greatly increased hydrogen peroxide production by both isolated mitochondria and submitochondrial particles respiring on succinate. The half-maximal stimulation was at 0.3 μM •NO, a concentration which also half-inhibited complex III activity, and it was thus suggested that the inhibition of complex III caused superoxide and hydrogen peroxide

Exogenous or endogenous •NO-induced mitochondrial damage and cellular death

Abundant literature discusses •NO-related mitochondrial inhibition or damage in cells and tissues in relation to pathology. Much of this literature is difficult to interpret in terms of specific mechanisms because (a) agents other than •NO may be involved, (b) •NO may give rise to a variety of chemical products in cells, and (c) a variety of indirect effects may cause mitochondrial damage (e.g. calcium, free radicals, necrosis and apoptosis). However, it is clear that •NO and its products play

Discussion

The effects of •NO and peroxynitrite on mitochondria are clearly distinct and should be distinguished (see Table 1). •NO causes reversible and relatively specific inhibition of cytochrome c oxidase. However, high levels of •NO for long periods can cause other effects which may be mediated by the reversible nitrosylation of protein thiols and probably by the removal of iron from iron-sulfur centers. Peroxynitrite, by contrast, potentially oxidizes most of the components in the mitochondria,

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The effect of nitric oxide on mitochondrial respiration

Highlights

Abstract

Introduction

Section snippets

Cytochrome c oxidase

Reversible •NO inhibition of cytochrome c oxidase

Tissue •NO measurements in vivo

•NO produced in vivo by cells and tissues and effects on mitochondrial respiration

Effects of •NO in tissue mitochondria (apart from cytochrome c oxidase inhibition)

Irreversible inhibition of mitochondrial respiration by •NO

Peroxynitrite-mediated mitochondrial respiration inhibition

•NO-induced ROS, RNS and mitochondrial permeability transition

•NO and peroxynitrite effects on mitochondrial superoxide and hydrogen peroxide production and on the PTP

Exogenous or endogenous •NO-induced mitochondrial damage and cellular death

Discussion

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Neurosci. Biobehav. Rev.

Biochem. Biophys. Res. Commun.

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Biochem. Biophys. Res. Commun.

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Arch. Biochem. Biophys.

J. Biol. Chem.

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