Free radical chemistry in biological systems

LAURA B VALDEZ, SILVIA LORES ARNAIZ, JUANITA BUSTAMANTE, SILVIA
ALVAREZ, LIDIA E COSTA, ALBERTO BOVERIS

Laboratory of Free Radical Biology, School of Pharmacy and Biochemistry
University of Buenos Aires

Abstract

Mitochondria are an active source of the free radical superoxide (O₂^-) and nitric oxide (NO), whose production accounts for about 2% and 0.5% respectively, of mitochondrial O₂ uptake under physiological conditions. Superoxide is produced by the auto-oxidation of the semiquinones of ubiquinol and the NADH dehydrogenase flavin and NO by the enzymatic action of the nitric oxide synthase of the inner mitochondrial membrane (mtNOS). Nitric oxide reversibly inhibits cytochrome oxidase activity in competition with O₂. The balance between NO production and its utilization results in a NO intramitochondrial steady-state concentration of 20-50 nM, which regulates mitochondrial O₂ uptake and energy supply. The regulation of cellular respiration and energy production by NO and its ability to switch the pathway of cell death from apoptosis to necrosis in physiological and pathological conditions could take place primarily through the inhibition of mitochondrial ATP production. Nitric oxide reacts with O₂^- in a termination reaction in the mitochondrial matrix, yielding peroxynitrite (ONOO^-), which is a strong oxidizing and nitrating species. This reaction accounts for approximately 85% of the rate of mitochondrial NO utilization in aerobic conditions. Mitochondrial aging by oxyradical- and peroxynitrite-induced damage would occur through selective mtDNA damage and protein inactivation, leading to dysfunctional mitochondria unable to keep membrane potential and ATP synthesis.

Key terms: Superoxide radical, nitric oxide, peroxynitrite, mitochondria, apoptosis, aging

INTRODUCTION

Free radicals are chemical species with an unpaired electron in the outer valence orbitals. Free radicals that are normal metabolites in aerobic biological systems have varied reactivities, ranging from the high reactivity of hydroxyl radical (t1/2 = 1 nsec) to the low reactivity of melanins (t_1/2 = days), with the intermediate reactivity of nitric oxide (t_1/2 = 1-10 sec) and ubisemiquinone (t_1/2 = 10 msec).

There is now evidence that two free radicals of low molecular mass, superoxide anion (O2-; 32 Da) and nitric oxide (NO; 30 Da), are continuously produced in aerobic cells in the specialized mitochondrial membranes that also produce ATP for cellular energy needs. The interaction between the two free radicals appears to play a role in the regulation of cell respiration through the inhibitory effect of NO on cytochrome oxidase activity. Over-production of these species leads mitochondria to a dysfunctional state, which is understood as a step to apoptosis and tissue aging.

1. Mitochondrial production of oxygen free radicals

During animal respiration, the tetravalent reduction of oxygen is carried out by mitochondrial cytochrome oxidase in a process coupled to energy generation and ATP synthesis. The products of the univalent reduction of oxygen are also generated in mitochondria; a small fraction of the total electron transfer is used to univalently reduce oxygen to superoxide anion (O₂^-) by ubisemiquinone and the flavin semiquinone of NADH dehydrogenase of the mitochondrial electron transfer chain (Boveris and Cadenas, 1997). Superoxide anion dismutates to hydrogen peroxide (H₂O₂) by the enzymatic action of Mn-superoxide dismutase (Mn-SOD) specifically located in the mitochondrial matrix. It was recently estimated that O₂^- and H2O2 account for approximately 2% and 1% respectively, of the O₂ uptake by liver and heart mitochondria under physiological conditions (Boveris et al., 1999a). The primary production of O₂^- and H₂O₂ is able to initiate the free radical chain reaction due to hydroxyl radical formation (HO"_) by the Fenton- Haber Weiss chemical reactions (Fig. 1).

FIGURE 1. Scheme of cellular hydroperoxide metabolism indicating the subcellular sources of the intermediates of the partial reduction of oxygen and the corresponding detoxification pathways.

2. Nitric oxide in biological systems

The recognition of the endogenous formation of nitric oxide (NO) by nitric oxide synthases (NOS) opened a new line of thought in free radical biochemistry. Nitric oxide is a free radical in terms of its unpaired electron, but it is a rather non-reactive free radical since it is not able to give initiation reactions; however, it readily reacts with (O₂^- in a termination reaction. Nitric oxide has been shown to be involved in numerous regulatory functions such as vasodilation in the arterial vascular beds (Ignarro, 1989; Moncada et al., 1991), neurotransmission (Dawson and Dawson, 1996) and immunological response (Moncada et al., 1991). Nitric oxide has also been described to participate in apoptosis mediated cell death and in a large number of pathophysiological conditions such as arthritis, atherosclerosis, cancer, diabetes, and neurodegenerative diseases (Wink and Mitchell, 1998).

Nitric oxide is produced in vivo during the oxidation of L-arginine to L-citrulline catalyzed by NOS, in the presence of NADPH and O₂ (Moncada et al., 1991; Griffith and Stuehr, 1995). There are three widely recognized isoforms of this enzyme, two of which are constitutive forms: neuronal NOS (nNOS) and endothelium NOS (eNOS), and the third one is the inducible form (iNOS) (Knowles and Moncada, 1994). The recent discovery of NO production by a mitochondrial NOS (mtNOS) (Ghafourifar and Richter, 1997; Tatoyan and Giulivi, 1998; Giulivi, 1998; Giulivi et al., 1998) added a new isoform of the enzyme and started a revolution in terms of both regulation of tissue oxygen uptake and of free radical toxicity. It has been reported that this NOS isoform is expressed in a constitutive form and is located in the inner membrane of rat liver mitochondria (Tatoyan and Giulivi, 1998; Giulivi, 1998). Recent observations indicate induction phenomena in mtNOS activity in an experimental model of endotoxemia (Boczkowski, et al., 1999).

3. Regulation of mitochondrial oxygen uptake

Mitochondrial respiration is regulated not only by O₂ and ADP concentrations, but also by NO levels (Boveris et al., 1999b). The intracellular oxygen concentration in mammalian organs and tissues in the 5-25 mM O2 range is close and partially overlaps with the critical concentration (2-6 mM), which limits the rate of mitochondrial respiration (Costa et al., 1997). It has been recognized that NO reversibly inhibits the cytochrome oxidase activity of the mitochondrial respiratory chain competitively with O₂ (Cleeter et al., 1994; Poderoso et al, 1996). Half maximal mitochondrial oxygen uptakes are observed at O₂/NO ratios of approximately 150-300. At the physiological NO (50 nM) and O2 (10 mM) concentrations, an inhibition of about 25-35% is expected to occur. At higher NO concentrations (about 0.2 mM), this species also inhibits electron transfer between the ubiquinone-cytochrome b pool and cytochrome c (Poderoso et al., 1996).

4. Mitochondrial utilization pathways of nitric oxide

There are three main reactions that utilize NO in mitochondria and make NO action temporally reversible. The three reactions are those in which NO reacts: a) with superoxide (reaction 1), b) with ubiquinol (reaction 2), and c) with cytochrome oxidase (reaction 3).
 

	k1
NO + O2-	®	ONOO-	(reaction 1)

	k2
NO + UQH2	®	UQH· + H+ + NO-	(reaction 2)

	k3
NO + Cyt a32+	®	Cyt a33+ + NO	(reaction 3)

k1 = 1.9 x 1010 M-1 s-1	(Koppenol, 1998)
k2 = 4 x 103 M-1 s-1	(Poderoso et al., 1999)
k3 = 4 x 107 M-1 s-1	(Giulivi, 1998)

The rapid reaction of NO with O₂^- (diffusion-controlled rate) accounts for approximately 85% of the rate of mitochondrial NO utilization in aerobic conditions. The reaction leads to the formation of peroxynitrite (ONOO-), which is a strong oxidizing and nitrating species. At physiological pH, ONOO- protonates to yield peroxynitrous acid (ONOOH; pKa = 6.8) (Koppenol, 1998). Peroxynitrous acid rearranges to an intermediate [HO·····NO2] that breaks and yields the free radicals HO· and ·NO₂ (Radi et al., 1999) (Fig. 2). The reaction of NO with ubiquinol produces ubisemiquinone that operates as a free radical reaction center able to generate O₂^- by autooxidation in a propagation reaction (Boveris and Cadenas, 1997) (Fig. 2). The addition reaction of NO with cytochrome oxidase, similar to the reactions of cytochrome oxidase with O₂ and CO, inhibits the main pathway of oxygen uptake and energy production. The a₃-heme is able to transfer one electron to NO yielding nytroxyl anion (NO-) in a slow first order reaction.

FIGURE 2. The crossed pathways of the biochemical free radical reaction. The primary production of superoxide anion and nitric oxide leads to the generation of hydroxyl radical, which initiates the lipoperoxidation process.

5. The steady-state concentrations of oxygen and nitrogen reactive species

Oxygen and nitrogen reactive species are kept in biological systems at steady-state concentrations that can be estimated by using the steady-state approach with the assumption that the rate of production is equal to the rate of utilization. The primary production of O₂^-, cytosolic Cu-Zn-SOD and mitochondrial Mn-SOD keep steady-state concentrations of 10^-10 M in the mitochondrial matrix and 10^-11 M in the cytosol (Boveris and Cadenas, 1997). The cytosolic steady-state concentration of H2O2 estimated from the rate of H₂O₂ generation by subcellular sources and its removal by catalase and glutathione peroxidase is about 10^-7 - 10^-8 M. Taking into account the rate of H₂O₂ production, its removal by intramitochondrial glutathione peroxidase and its diffusion to the cytosolic space, H₂O₂ steady-state concentration in the mitochondrial matrix results in approximately 10^-8 M (Boveris and Cadenas, 1997). The balance between NO production by mtNOS and its utilization by the reactions with the components of the respiratory chain and with O₂^- regulates the intra-mitochondrial steady-state concentration of NO at about 50 nM (Poderoso et al., 1998; Lores Arnaiz et al., 1999; Boczkowski et al., 1999), which in turn regulates mitochondrial oxygen uptake and energy supply (Fig. 3).

FIGURE 3. Intramitochondrial metabolism of nitric oxide and superoxide radicals. The steady-state concentrations are indicated.

6. Mitochondria and aging

The concept that mitochondria are involved in aging derives from the views of Gerschman et al. (1954) and Harman (1956) linking senescence to the injurious effects of free radicals produced in the univalent reduction of oxygen in cell and tissues. This hypothesis is more attractive when restrictively applied to mitochondria than to cells of tissues because 1) mitochondria are the relatively more important subcellular site of oxyradical production, 2) O₂^- steady-state concentration in the mitochondrial matrix is about 5 to 10 times higher than in the cytosolic and nuclear spaces, 3) mtDNA is in close proximity to the sites of oxyradical generation and is not protected by histones, and 4) the accumulation of faulty synthetized proteins might compromise energy transduction (Boveris et al., 1999a). Additional support for the free radical theory of aging came from the linear relationship observed by Cutler after plotting the ratio of SOD activity (protection against oxyradicals) over basal metabolic rate (aggression due to the metabolic generation of oxyradicals) against maximal life span in a series of mammals and primates (Cutler, 1994).

Experimental evidence has appeared in recent years to indicate that mitochondria evolve during aging to a state of oxidative stress and dysfunctionality. Mitochondrial aging by oxyradical-induced damage would occur through mtDNA damage and relatively specific protein inactivation, such as adenine nucleotide translocase. This process would lead to a state of dysfunctional mitochondria that are not able to maintain membrane potential and ATP synthesis. Dysfunctional mitochondria release Ca²⁺ and cytochrome c to the cytosol and signal for organelle digestion by primary lisosomes and apoptosis.

7. Mitochondrial nitric oxide and apoptosis

Nitric oxide plays different roles in physiological functions acting as an intracellular signal in several cellular pathways (Moncada et al., 1991). However, increases in NO production and in NO intracellular steady-state concentrations can turn its physiological effects into cytotoxicity. Bustamante et al. (1997) have reported the early appearance of oxidant species during apoptosis, which could alter the redox balance and cellular NO effects. The regulation of cellular respiration and energy production by NO and its ability to switch the pathway of cell death from apoptosis to necrosis in physiological and pathological conditions could take place primarily through the inhibition of mitochondrial ATP production. Modulation of programmed cell death by exogenous addition of NO has been observed in different cell types (Bustamante et al., 1998). It was recently reported that exogenous NO can inhibit apoptosis by S-nitrosation of caspase-3 (Rossig et al., 1999). Evidence of pro-apoptotic and anti-apoptotic effects of NO are conflicting for the moment and require better knowledge of intracellular NO concentrations. Recent results from our laboratory show that an increased mitochondrial NO production is associated with a decrease of mitochondrial respiratory rate in state 3, with a decrease in mitochondrial GSH and with cytochrome c release during thymocyte apoptosis. It seems that NO is involved in the initial steps of the mitochondrial signaling that leads to apoptosis.

ACKNOWLEDMENTS

This research was supported by grants from CONICET (PIP 4110/97), ANPCYT (PICT 01608) and the TB11 from the University of Buenos Aires (Argentina).

Received: November 4, 1999. Accepted: June 7, 2000

Corresponding Author: Dr. Laura Valdez/Alberto Boveris Cátedra de Fisicoquímica Facultad de Farmacia y Bioquímica Universidad de Buenos Aires Junín 956, 1113, Buenos Aires, Argentina Tel/fax: (54-11) 4508-3646 e-mail: lbvaldez@ffyb.uba.ar

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