Reactive Oxygen and Nitrogen Species: Interactions with Mitochondria and Pathophysiology

Reactive Oxygen and Nitrogen Species: Interactions with Mitochondria and Pathophysiology B R Zelickson, S W Ballinger, L J Dell’Italia, J Zhang, and V M Darley-Usmar, University of Alabama at Birmingham, Birmingham, AL, USA ã 2013 Elsevier Inc. All rights reserved.

Glossary Electron transport chain The site of oxidative phosphorylation in the mitochondrial inner membrane which couples the transfer of electrons from electron donors (such as NADH) to O2 with the pumping of protons (Hþ) from the matrix to the intermembrane space, thus generating an electrochemical proton gradient which is used by adenosine triphosphate (ATP) synthase to produce ATP. Flavin The prosthetic group in flavoproteins which is capable of undergoing oxidation–reduction reactions by accepting/donating either one or two electrons at once. Heme The prosthetic group in hemoproteins which contains an iron atom at the center of a porphyrin ring and serves various roles, such as a source/sink of electrons during electron transport and redox reactions and the transportation of diatomic gases. Iron–sulfur center Moiety in iron–sulfur proteins, often consisting of four iron atoms and four sulfur atoms arranged in a cubane-like structure, which participates in redox reactions, particularly in mitochondrial electron transport.

Introduction The importance of reactive oxygen and nitrogen species (ROS/ RNS) in physiology, as well as in pathology, and their interactions with mitochondria are well established. Originally, ROS/RNS were considered to be simply destructive and cytotoxic, but more recently it has become clear that they have several beneficial effects through their ability to activate specific cell-signaling pathways. ROS have been shown, at low levels, to induce cell proliferation and control the synthesis of endogenous antioxidant enzymes. Mitochondria are both a source and target for ROS and play a key role in redox cell signaling. Under pathological conditions, increased levels of mitochondrial ROS formation can lead to damage to DNA, lipids, and proteins. This can impair bioenergetic function, and, in some cases, initiate programmed cell death or apoptosis (Figure 1). Nitric oxide ( NO) is the best-understood redox signaling molecule. It modulates cell function through its interaction with heme proteins such as soluble guanylate cyclase, cytochrome c oxidase, and hemoglobin. In addition,  NO can lead to protein modification through its ability to interact with ROS leading to S-nitros(yl)ation or oxidation. On the other hand, the reactions of ROS and RNS have been implicated in many chronic pathologies, particularly those involving inflammation. For example, high levels of reactive species have been shown to further the progression of various cardiovascular and neurodegenerative diseases.

Krebs cycle Series of enzyme-catalyzed reactions occurring in the mitochondrial matrix which uses acetyl-coenzyme (CoA) and oxaloacetate to reduce three NADþ to NADH and one Q to QH2, while also producing one GTP and two CO2. Oxidative stress State in which there is an increase in oxidizing species, such as reactive oxygen species (ROS), and/or a decrease in antioxidant defenses, and often leads to protein, lipid, and DNA damage. Redox center Moiety which is either oxidized or reduced during redox reactions (e.g., flavins, iron–sulfur centers, and hemes). Reductive stress State of redox imbalance in which there is an excess in reducing species, leading to a reductive cytosolic environment (e.g., high NADH:NADþ ratio). Thiols Organosulfur compound containing a carbon-bound sulfhydryl group (e.g., cysteine and glutathione) which participates in redox biology and signaling through the formation of various oxidation states.

Nitric oxide is produced by a family of enzymes called the nitric oxide synthases (NOSs) and mediates its cell signaling effects through protein modification and binding to heme iron. Despite its predominantly extra-mitochondrial origin,  NO is also known to modulate mitochondrial function at cytochrome c oxidase and to stimulate mitochondrial biogenesis by activation of soluble guanylate cyclase. The aim of this chapter is to discuss what reactive oxygen and nitrogen species are, how they are formed, and how they interact with the mitochondrion and give key examples of their biological effects in cardiovascular and neurodegenerative pathologies.

What Are Reactive Oxygen and Nitrogen Species? The terms ROS/RNS are freely used in the biological literature; however, it is important to note that they do not have a specific chemical definition and may include free radicals and nonradical species. The general property they all share is that ROS/ RNS react with biological molecules leading to changes in function that may contribute to cell signaling in a controlled fashion or to the pathology of a disease. It seems likely that the modification of protein cysteine residues is the basis of redox cell signaling and can be highly specific. In contrast, nonspecific oxidation reactions such as carbonylation are generally thought to contribute to protein damage, aggregation, and activation of the autophagic process.

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Bioenergetics | Reactive Oxygen and Nitrogen Species: Interactions with Mitochondria and Pathophysiology

e ut O

e ran mb e rm

Electron transport chain

C

Intermembrane space Inn er me m

e an br

mPTP

O2•–, H2O2

Lipid peroxidation

mtDNA

C

Cell death

Mitochondrial dysfunction

Redox signaling

Pathology Figure 1 Effects of mitochondrial ROS. Mitochondrial ROS production can alter the function through a variety of pathways. Lower levels of ROS can act through several redox signaling mechanisms, while higher levels can directly damage mitochondrial proteins, leading to the further production of ROS. ROS can also damage mtDNA and induce lipid peroxidation in the membrane, and can also cause mitochondrial permeability transition pore (mPTP) opening, which leads to cytochrome c release and apoptosis induction.

Reactive Oxygen Species The ROS are the partially reduced intermediates of oxygen and can be radical or nonradical molecules. Free radicals are denoted in a chemical formula using a superscript dot which is usually written next to the symbol of the atom which carries the unpaired electron. ROS include the superoxide radical anion (O 2 ) and its protonated form, the hydroperoxyl radical (HO2 ), hydrogen peroxide (H2O2), and the hydroxyl radical ( OH). As discussed below, O 2 is formed by the mitochondrial respiratory chain at a number of sites within electron transport and the Krebs cycle. As the mitochondrion contains high levels of the enzyme superoxide dismutase (SOD), most of the O 2 that is formed is converted into H2O2.

Reactive Nitrogen Species As with reactive oxygen species, this is a term commonly used by biologists to describe  NO-derived molecules which show biological activity. Nitric oxide is an uncharged free radical which can diffuse freely across membranes. It reacts rapidly with other free radicals and has a high affinity for ferrous heme groups. The RNS peroxynitrite (ONOOH) is formed from the reaction of superoxide with  NO and has been studied in depth. The reactivity of peroxynitrite is enhanced in biological systems through its reaction with carbon dioxide to form nitrosoperoxycarbonate, which can then undergo homolytic fission to generate the nitrogen dioxide (NO2 ) and carbonate

(CO 3 ) radicals, both of which are reactive and can cause damage to biomolecules. For example, NO2 can react with tyrosine residues or tyrosyl radicals on proteins, resulting in 3-nitrotyrosine formation. This adduct can be detected in a broad range of pathologies, and is often used as a marker of nitrative stress. Nitrated proteins have been detected in mitochondria isolated from a broad range of pathological conditions. Mitochondrial DNA damage is associated with several diseases associated with oxidative and nitrative stress, leading to increased mitochondrial ROS production and dysfunction.

Generation of ROS from Mitochondria Mitochondria can generate ROS from a number of different redox centers in several metabolic pathways. The basic principle for ROS generation from these sites is shown in Figure 2(a). Electrons are transferred to a redox center which then acts as an intermediate carrier to another redox active member of the metabolic pathway. The more reduced the redox center is, the greater the potential production of ROS. The detailed mechanisms for ROS generation from each redox center are described below.

Complex I NADH-ubiquinone oxidoreductase (complex I) consists of 45 subunits with a combined mass of close to 1 MDa. Complex I accepts two electrons from NADH, which is produced by the

Bioenergetics | Reactive Oxygen and Nitrogen Species: Interactions with Mitochondria and Pathophysiology

Redox center

Electron donor

Electron acceptor

FMN FAD CoQ/Q

e−

19

e− O2• –

H2O2

(a) Complex I

Complex I:

CoQ

2e− 2NADH

QH2

O2• –

(b)

RET:

H2O2

Complex I

2NAD+

2e− Complex II

QH2

Complex III

2e−

FMN

CoQ

2NADH

2e−

FMN

O2• –

H2O2

(c)

Complex III:

2e−

Complex III

Rieske Fe-S

Cytochrome c

2e− QH2

bL heme

Qo

O2• –

QH2 H2O2

(d)

Complex II:

Complex II 2e−

2Succinate

CoQ

QH2

Complex III

2e−

FAD

O2• –

H2O2

(e)

αKGDH/PDH: αKG pyruvate

Complex I E3 subunit (DLD)

e−

(f)

EFT-QOR:

EFT-QOR 2e− EFT

NAD+

NADH

O2• –

H2O2

Complex I

e−

Q

QH2

Complex III

2e−

FAD

O2• –

H2O2

(g) GDPH

GPDH: 2e− 2G3P (h)

FAD

Q

QH2

Complex III

2e− O2• –

H2O2

Figure 2 Mechanisms of ROS formation in the mitochondria. The sites of ROS production within the mitochondria all follow a similar mechanism. An electron donor donates an electron to a redox center, which then reduces the electron acceptor. However, electrons can also be transferred from the redox center to reduce oxygen into superoxide (a). Complex I oxidizes NADH through the FMN site, and can transfer the electrons to either the CoQ site to make ubiquinol (QH2) or, if the CoQ site is inhibited, to O2 to make superoxide (b). In reverse electron transport (RET), QH2 produced by complex II can be transferred back into complex I through the CoQ site to the FMN, which can then either reduce NADþ back to NADH or reduce O2 to form superoxide (c). Complex III uses QH2 produced predominantly by complex I and II as the electron donor, transferring the electrons to the Rieske Fe–S and then on to cytochrome c1 and finally cytochrome c. Complex III also transfers electrons to the bL heme, which participates in the Q cycle. However, when the Q cycle is inhibited, by antimycin A, for example, the electrons can leak out of the Qo site to reduce O2 to make superoxide (d). Complex II has also been shown to produce superoxide when electron transfer to the CoQ site is inhibited, or when the enzyme is solubilized, causing electrons to leak from the reduced FAD site to make superoxide (e). PDH and aKGDH use their substrates to transfer electrons through their E3 subunit to reduce NADþ; however, electrons have also been shown to reduce O2 to make ROS (f). EFT–QOR oxidizes EFT and transfers electrons through its FAD domain to reduce Q to QH2, but it has also been shown that it can also reduce O2 to superoxide as well (g). GDPH reduces G3P via its FAD site to make QH2, although it has also been shown to leak electrons to make ROS as well (h).

Krebs cycle, via a flavin mononucleotide (FMN) and transfers them through a chain of seven iron–sulfur centers to the CoQ reduction site. There the electrons are transferred to the lipidsoluble electron carrier ubiquinone (Q), reducing it to ubiquinol (QH2). One mechanism by which complex I produces ROS is by the reaction of O2 with the fully reduced FMN

forming O 2 on the matrix side of the complex. The rate of O 2 production at the FMN can be increased by inhibiting the CoQ site using the small molecule inhibitor rotenone. Similarly, this reaction is dependent on the NADH/NADþ ratio, which determines the proportion of FMN that is fully reduced. Under physiological conditions, O generation 2

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Bioenergetics | Reactive Oxygen and Nitrogen Species: Interactions with Mitochondria and Pathophysiology

from complex I is sensitive to the redox state of the respiratory chain (Figure 2(b)). Pathologically, there are several conditions which result in a reductive stress, causing the NADH/NADþ ratio to increase, and this in turn results in increased O 2 production. Some of the best-understood examples of this condition are the reductive stresses associated with alcohol metabolism and ischemia. An additional mechanism through which complex I can produce ROS is called reverse electron transport (RET) (Figure 2(c)). This occurs when the mitochondria are utilizing succinate as a substrate through complex II. Under conditions of low electron flux (when adenosine diphosphate (ADP) levels are low), the larger pool of reduced QH2 is sufficient to reduce complex I at the CoQ site. This reverses the normal flow of electrons back through the complex to reduce the FMN site, thereby reducing NADþ to form NADH, while also reducing O2 to form O 2 . RET results in the generation of O 2 in the matrix and can be inhibited with rotenone treatment. Interestingly, RET generates the highest rate of O 2 formation detected thus far in the mitochondrion. Whether RET plays a physiological role in cell signaling through ROS is not clear. Since complex I appears to play a central role in mitochondrial ROS formation, it is not surprising that it has been implicated in a number of pathological processes. For example, the complex I (and RET) inhibitor rotenone has been shown to induce Parkinson’s disease-related pathologies in rodents. It may also be an important environmental toxicant which can contribute to Parkinson’s disease pathogenesis in humans. Furthermore, many studies have consistently found decreases in complex I activity in Parkinson’s disease brains. Other compounds have been shown to interact with complex I, modifying both enzyme activity and ROS formation. An important example is 1-methyl-4-phenyl1,2,3,6-tetrahydropyridine (MPTP), originally discovered as a by-product of illegal heroin production, which was shown to cause Parkinsonism in drug users. MPTP toxicity is mediated by its metabolite MPPþ, which is selectively taken up by dopaminergic neurons and inhibits complex I activity, leading to their eventual death.

Complex III Complex III is approximately 240 kDa and is made up of 11 subunits, three heme groups, and the Rieske iron–sulfur (Fe–S) center. It functions by oxidizing QH2 in the inner membrane and transferring the electrons to reduce cytochrome c in the intermembrane space. The rates of O 2 production from complex III are relatively low during RET compared to that of complex I. However, the further inhibition of complex III with the Qo site inhibitor myxothiazol attenuates most of this O 2 production, which suggests that complex III does produce some O 2 during RET, albeit a small amount. Alternatively, when the Qi site is inhibited using antimycin A, complex III produces greater amounts of O 2 , which are released to both sides of the inner membrane. This formation of O 2 is due to the reaction of O2 with the QH bound to the Qo site. This O 2 formation due to antimycin A can be abrogated by treatment with myxothiazol, which inhibits the Qo site, suggesting that QH bound to the Qo site is the main site of O 2 production (Figure 2(d)).

Complex II Succinate dehydrogenase, also known as complex II, is embedded in the matrix side of the inner mitochondrial membrane and consists of four subunits. It contains a flavin adenine dinucleotide (FAD) site, three Fe–S clusters, a heme group, and a Q binding site. Complex II oxidizes succinate to fumarate as part of the Krebs cycle, which is coupled to the reduction of Q to QH2. Complex II readily produces O 2 when solubilized, probably by the reaction of O2 with the reduced FAD site, due to the lack of Q as an electron acceptor (Figure 2(e)). This can occur in vivo, and only when complex II has been damaged by oxidative stress or aging. There are also several genetic mutations of the various subunits of complex II which can cause increased O 2 production. Several of these mutations have been shown to lead to cancer, particularly cancers of neuroendocrine lineage. However, complex II produces very little O 2 during normal physiological conditions.

Other Sources of ROS Formation within the Mitochondrion Other mitochondrial metabolic pathways, including the Krebs cycle, contain proteins with redox centers which are also capable of generating ROS. For example, the enzyme glycerol3-phosphate dehydrogenase (GPDH) has been shown to produce O 2 . This enzyme participates in the glycerol-3-phosphate (G3P) shuttle, which provides a mechanism for the transport of electrons from cytosolic NADH to the electron transport chain, by oxidizing NADH on the cytosolic face of the outer mitochondrial membrane and reducing Q to QH2 in the inner mitochondrial membrane. GPDH has been reported to have some G3P-dependent production of O 2 , most likely at the FAD site within the enzyme (Figure 2(h)). Approximately 70% of the O 2 produced is directed to the intermembrane space, while 30% is directed toward the matrix. Similarly, a-ketoglutarate dehydrogenase (aKGDH), which is responsible for the conversion of a-ketoglutarate to succinyl CoA in a reaction which is coupled to the reduction of NADþ þ to NADH, can generate O 2 when the NADH/NAD ratio is elevated. This occurs in the E3 subunit of the enzyme complex, which is a flavoprotein known as dihydrolipoamide dehydrogenase (DLD). Pyruvate dehydrogenase (PDH) has been shown to produce ROS via a similar mechanism (Figure 2(f)). In addition, many of the key enzymes controlling fatty acid oxidation are located in the mitochondrial matrix and inner membrane. In this metabolic pathway, reduction of the electron transfer flavoprotein (ETF) results in the conversion of Q to QH2 via the enzyme ETF:ubiquinone oxidoreductase (ETF– QOR). ETF–QOR is located on the mitochondrial inner membrane and contains an Fe–S cluster and an FAD site, and has been shown to produce O 2 directed toward the matrix when in the presence of palmitoyl carnitine as a substrate. ETF–QOR has also been shown to increase RET and so can enhance O 2 production from complex I (Figure 2(g)).

Can the Mitochondria Sense the Site of ROS Formation? If mitochondrial ROS play a role in cell signaling, it implies that the site of ROS formation within the respiratory chain will lead to differential modification of mitochondrial proteins.

Bioenergetics | Reactive Oxygen and Nitrogen Species: Interactions with Mitochondria and Pathophysiology

This is a relatively new concept, but in support of this idea it has been shown that ROS produced by complex III upon treatment with antimycin A induces the modification of protein thiols on complex III core protein I, which does not occur with exogenous hydrogen peroxide treatment or with the induction of RET. These modifications are reversible by the peroxiredoxin/thioredoxin system, as well as by glutathione, which further support their roles as mediators of redox signaling. Reversible thiol modification due to ROS production by specific respiratory chain complexes may be responsible for regulation of carbohydrate and fatty acid metabolism. Mitochondrial protein thiols may be more susceptible to modification than cytosolic protein thiols because of both the proximity to the source of ROS and the fact that the mitochondrial matrix has a high pH (8), which will result in more thiols being in their reactive form, the thiolate anion. Mitochondria have also been shown to sense ROS levels through cardiolipin oxidation. Cardiolipin is an abundant lipid which is found in the mitochondrial inner membrane. Cardiolipin participates in redox signaling due to its propensity for peroxidation in the presence of high levels of ROS. The peroxidation of cardiolipin then enables it to induce apoptosis by aiding in mitochondrial pore opening and cytochrome c release from the intermembrane space.

Can Mitochondrial ROS be Controlled? The mitochondrion has several ways through which it can control the levels of ROS it produces. Superoxide can be converted to hydrogen peroxide by the enzyme SOD. The SOD located in the mitochondrial matrix contains a Mn prosthetic group and is known as SOD2 or MnSOD. The SOD located in the cytosol contains Cu/Zn prosthetic groups and is known as Cu/Zn SOD or SOD1. SOD1 may also be present in the mitochondrial inter-membrane space. SOD2 provides a major mechanism by which the mitochondrion can control the levels of ROS, through its ability to catalyze the dismutation of O 2 to hydrogen peroxide, which can be further metabolized to H2O by peroxiredoxin, thioredoxin, or glutathione peroxidase. In addition, these systems can also help to remove other peroxides, such as those that have formed on lipids and proteins. Mitochondria also control reactive oxygen species production through the ability of uncoupling proteins (UCPs) to increase proton leak and decrease the mitochondrial membrane potential and the redox state of sites of O 2 formation. UCP3 has also been suggested to lower mitochondrial levels of lipid peroxidation by causing fatty acid efflux out of the matrix when fatty acid levels are in excess.

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ATP formation and can be pathological. In addition to modulating mitochondrial function, low levels of  NO are now known to be able to induce mitochondrial biogenesis. This occurs through  NO activating soluble guanylate cyclase (sGC), which then produces cyclic GMP (cGMP). The cGMP then leads to the activation of PGC-1a, which is the principal transcription regulator of mitochondrial biogenesis. However, when  NO is produced in excess, it impacts on pathologies with an underlying bioenergetic defect, including ischemia reperfusion. One prominent example of  NO production in the central nervous system is associated with ischemic stroke. Initial brain damage is caused by lack of blood flow, oxygen, and nutrients. Further exacerbating the initial insult, injured ischemic brain cells release excitatory amino acids, such as glutamate, into the synapses between neurons. Excitatory amino acids bind to and induce over-excitation of neighboring neurons, leading to calcium influx. Both the primary ischemia itself and the secondary over-excitation of neighboring neurons followed by calcium influx induce NOS, resulting in overproduction of  NO. The excessive  NO in neurons, in turn, causes DNA, protein, and organelle damage, and promotes additional cell death with a major contribution from mitochondrial calcium overload and bioenergetic failure.

Summary In this short overview, we have outlined how ROS/RNS are formed as well as how they function in (patho)physiology. Although much is already known about the production and function of these reactive species, many aspects of how ROS and RNS are formed by and interact with the mitochondrion are still unclear, particularly in vivo. A greater understanding of how these species are formed, and how they affect mitochondrial and cellular function, is important for the development of mitochondrial therapeutics.

Acknowledgments The authors are funded by T32 HL007918 (BRZ), R01 HL77419 (SWB), R01 HL94518 (SWB), R01 NS064090 (JZ), R01 ES010167 (VDU), R01 DK075865 (VDU), R01 AA013395 (VDU), and T32 HL007918 (VDU).

See also: Protein/Enzyme Structure Function and Degradation: Flavins; Heme Proteins.

Further Reading Nitric Oxide and Mitochondria Nitric oxide partitions into the mitochondrial inner membrane and inhibits cytochrome c oxidase by competing with O2 for binding at the binuclear CuB/cytochrome a3 site.  NO binding to complex IV is O2 dependent, which may be beneficial because it can then allow O2 to diffuse further into tissues, thus extending the O2 gradient. However, excessive inhibition of mitochondrial respiration by  NO may lead to inhibition of

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Bioenergetics | Reactive Oxygen and Nitrogen Species: Interactions with Mitochondria and Pathophysiology

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