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HIV and her pulmonary tuberculosis, either of which could have caused progressive weakness. In

addition, she could have been suffering from a congenital mtDNA myopathy, symptomatic only as she

ages. If she was being treated with zidovudine (AZT), an older drug not used very commonly anymore,

her myopathy could have been caused by a disorder of oxidative phosphorylation induced by AZT. The

lamivudine she was taking has rarely caused this myopathy. A systematic diagnostic process finally led

her physician to conclude that she had HIV-associated myopathy.

As infusion of TPA lysed the clot blocking blood flow to Cora N.’s heart, oxygenated blood

was reintroduced into the ischemic heart. Although oxygen may rapidly restore the capacity

to generate ATP, it often increases cell death, a phenomenon called ischemia–reperfusion injury.

During ischemia, several factors may protect heart cells against irreversible injury and cell

death until oxygen is reintroduced. The stimulation of anaerobic glycolysis in the cytosol generates

ATP without oxygen as glucose is converted to lactic acid. Lactic acid decreases cytosolic pH.

Both cytosolic ATP and a lowering of the pH protect against opening of the MPTP. In addition,

Ca2+ uptake by mitochondria requires a membrane potential, and it is matrix Ca2+ that activates

opening of the MPTP. Thus, depending on the severity of the ischemic insult, the MPTP may not

open, or may open and reseal, until oxygen is reintroduced. Then, depending on the sequence of

events, reestablishment of the proton gradient, mitochondrial uptake of Ca2+, or an increase of pH

>7.0 may activate the MPTP before the cell has recovered. In addition, the reintroduction of O2

generates oxygen free radicals, particularly through free-radical forms of CoQ in the ETC. These

also may open the MPTP. The role of free radicals in ischemia–reperfusion injury is discussed in

more detail in Chapter 25. BIOCHEM ICAL COM M ENTS

Mitochondria and Apoptosis. The loss of mitochondrial integrity is a major route initiatingapoptosis (see Chapter 18, Section V). The intermembrane space contains procaspases 2, 3, and 9, which

are proteolytic enzymes that are in the zymogen form (i.e., they must be proteolytically cleaved to be

active). It also contains apoptosis-initiating factor (AIF) and caspase-activated DNase (CAD). AIF has a

nuclear targeting sequence and is transported into the nucleus under appropriate conditions. Once AIF is

inside the nucleus, it initiates chromatin condensation and degradation. Cytochrome c, which is loosely

bound to the inner mitochondrial membrane, may also enter the intermembrane space when the

electrochemical potential gradient is lost. The release of cytochrome c and the other proteins into the

cytosol initiates apoptosis (see Chapter 18).

What is the trigger for the release of cytochrome c and the other proteins from the mitochondria? The

VDAC pore is not large enough to allow the passage of proteins. Several theories have been proposed,

each supported and contradicted by experimental evidence. One is that Bax (a member of the Bcl-2 family

of proteins that forms an ion channel in the outer mitochondrial membrane) allows the entry of ions into

the intermembrane space, causing swelling of this space and rupture of the outer mitochondrial membrane.

Another theory is that Bax and VDAC (which is known to bind Bax and other Bcl-2 family members)

combine to form an extremely large pore, much larger than is formed by either alone. Finally, it is

possible that the MPTP or ANT participate in rupture of the outer membrane but that they close in a way

that still provides the energy for apoptosis.

In addition to increased transcription of genes that encode TCA cycle enzymes and certain

other enzymes of fuel oxidation, thyroid hormones increase the level of the uncoupling

proteins UCP2 and UCP3. In hyperthyroidism, the efficiency with which energy is derived from

the oxidation of these fuels is significantly less than normal. As a consequence of the increased

rate of the ETC, hyperthyroidism results in increased heat production. Patients with hyperthyroidism, such as Stanley T., complain of constantly feeling hot and sweaty. KEY CONCEP TS

The reduced cofactors generated during fuel oxidation donate their electrons to the mitochondrial

electron-transport chain.

The electron-transport chain transfers the electrons to O2, which is reduced to water.

As electrons travel through the electron-transport chain, protons are transferred from the

mitochondrial matrix to the cytosolic side of the inner mitochondrial membrane. The asymmetric distribution of protons across the inner mitochondrial membrane generates an

electrochemical gradient across the membrane.

The electrochemical gradient consists of a change in pH (ΔpH) across the membrane and a

difference in charge (ΔΨ) across the membrane.

Proton entry into the mitochondrial matrix is energetically favorable and drives the synthesis of ATP

via the ATP synthase.

Respiration (oxygen consumption) is normally coupled to ATP synthesis; if one process is inhibited,

the other is also inhibited.Uncouplers allow respiration to continue in the absence of ATP synthesis, as the energy inherent in

the proton gradient is released as heat.

OXPHOS diseases are caused by mutations in either nuclear or mitochondrial DNA that lead to a

decrease in mitochondrial capacity for synthesizing ATP via oxidative phosphorylation.

As the inner mitochondrial membrane is impermeable to virtually all biochemical compounds,

transport systems exist to allow entry and exit of appropriate metabolites.

The transfer of cytoplasmic reducing equivalents into the mitochondria occurs via shuttle systems;

either the glycerol 3-phosphate shuttle or the malate-aspartate shuttle.

Under appropriate stress, mitochondria will generate a nonspecific channel across both the inner

and outer membranes that is known as the mitochondrial permeability transition pore. The opening

of the pore is associated with events that lead to necrotic cell death. Diseases discussed in this chapter are summarized in Table 24.3.

REVIEW QUESTIONS—CHAPTER 241. Consider the following experiment. Carefully isolated liver mitochondria are incubated in the

presence of a limiting amount of malate. Three minutes after adding the substrate, cyanide is added,

and the reaction is allowed to proceed for another 7 minutes. At this point, which of the following

components of the electron-transfer chain will be in an oxidized state?

A.Complex I

B.Complex II

C.Complex III

D.CoQ

E.Cytochrome c

2.Consider the following experiment. Carefully isolated liver mitochondria are placed in a weakly

buffered solution. Malate is added as an energy source, and an increase in oxygen consumption

confirms that the ETC is functioning properly within these organelles. Valinomycin and potassium

are then added to the mitochondrial suspension. Valinomycin is a drug that allows potassium ions to

freely cross the inner mitochondrial membrane. What is the effect of valinomycin on the proton

motive force that had been generated by the oxidation of malate? A. The proton motive force will be reduced to a value of zero. B. There will be no change in the proton motive force.

C. The proton motive force will be increased.

D. The proton motive force will be decreased but to a value greater than zero. E. The proton motive force will be decreased to a value less than zero.

3.Dinitrophenol, which was once tested as a weight-loss agent, acts as an uncoupler of oxidative

phosphorylation by which one of the following mechanisms? A. Activating the H+-ATPase

B. Activating CoQ

C. Blocking proton transport across the inner mitochondrial membrane D. Allowing for proton exchange across the inner mitochondrial membrane E. Enhancing oxygen transport across the inner mitochondrial membrane

4.A 25-year-old woman presents with chronic fatigue. A series of blood tests is ordered, and the

results suggest that her red blood cell count is low because of iron-deficiency anemia. Such a

deficiency would lead to fatigue because of which one of the following?

A. Her decrease in Fe–S centers is impairing the transfer of electrons in the ETC. B. She is not producing enough H2O in the electron-transport chain, leading to dehydration, which

has resulted in fatigue.

C. Iron forms a chelate with NADH and FAD(2H) that is necessary for them to donate their

electrons to the ETC.

D. Iron acts as a cofactor for α-ketoglutarate dehydrogenase in the TCA cycle, a reaction required

for the flow of electrons through the ETC.

E. Iron accompanies the protons that are pumped from the mitochondrial matrix to the cytosolic side

of the inner mitochondrial membrane. Without iron, the proton gradient cannot be maintained to

produce adequate ATP.

5.Which one of the following would be expected for a patient with an OXPHOS disease?A. A high ATP:ADP ratio in the mitochondria

B. A high NADH:NAD+ ratio in the mitochondria C. A deletion on the X chromosome

D. A high activity of complex II of the ETC

E. A defect in the integrity of the inner mitochondrial membrane

6.A 5-year-old boy was eating paint chips from the windowsill in his 125-year-old home, and he

developed an anemia. Bloodwork indicated high levels of lead, which interfere with heme synthesis.

Reduced heme synthesis would have little effect on the function of which one of the following

proteins or complexes? A. Myoglobin

B. Hemoglobin C. Complex I

D.Complex III

E.Complex IV

7.Rotenone, an inhibitor of NADH dehydrogenase, was originally used for fishing. When it was

sprinkled on a lake, fish would absorb it through their gills and die. Until recently, it was used in the

United States as an organic pesticide and was recommended for tomato plants. It was considered

nontoxic to mammals and birds, neither of which can readily absorb it. What effect would rotenone

have on ATP production by heart mitochondria, if it could be absorbed? A. There would be no reduction in ATP production.

B. There would be a 95% reduction in ATP production. C. There would be a 10% reduction in ATP production. D. There would be a 50% reduction in ATP production. E. There would be a 50% increase in ATP production.

8.In order for cells to function properly, energy is required; for most cells, the energy is primarily

derived from the high-energy phosphate bonds of ATP, which is produced through oxidative

phosphorylation. Which one of the following is a key component of oxidative phosphorylation?

A. Using NADH and FAD(H2) to accept electrons as substrates are oxidized.

B. Creating a permeable inner mitochondrial membrane to allow mitochondrial ATP to enter the

cytoplasm as it is made.

C. An ATP synthase to synthesize ATP D. An ATP synthetase to synthesize ATP

E. A source of electrons, which is usually oxygen in most tissues

9.Carefully isolated intact mitochondria were incubated with a high-salt solution, which is capable of

disrupting noncovalent interactions between molecules at the membrane surface. After washing the

mitochondria, pyruvate and oxygen were added to initiate electron flow. Oxygen consumption was

minimal under these conditions because of the loss of which one of the following components from

the electron-transfer chain? A. Complex I

B. CoQ

C. Complex IIID. Cytochrome C E. Complex IV

10.UCPs allow oxidation to be uncoupled from phosphorylation. Assume that a drug company has

developed a reagent that can activate several UCPs with the goal being the development of a weightloss drug. A potential side effect of this drug could be which one of the following?

A. Decreased oxidation of acetyl coenzyme A B. Decreased glycolytic rate

C. Increase in body temperature

D. Increased ATP production by the ATP synthase E. Inhibition of the ETC

ANSWERS TO REVIEW QUESTIONS

1.The answer is B. For a component to be in the oxidized state, it must have donated, or never

received, electrons. Complex II will metabolize succinate to produce fumarate (generating

FAD[2H]), but no succinate is available in this experiment. Thus, complex II never sees any

electrons and is always in an oxidized state. The substrate malate is oxidized to oxaloacetate,

generating NADH, which donates electrons to complex I of the ETC. These electrons are

transferred to CoQ, which donates electrons to complex III, to cytochrome c, and then to complex

IV. Cyanide will block the transfer of electrons from complex IV to oxygen, so all previous

complexes containing electrons will be backed up and the electrons will be “stuck” in the

complexes, making these components reduced. Thus, answers A and C through E must be incorrect.

2.The answer is D. The proton motive force consists of two components, a ΔpH, and a

Δψ

(electrical component). The addition of valinomycin and potassium will destroy the electrical

component but not the pH component. Thus, the proton motive force will decrease but will still be

greater than zero. Thus, the other answers are all incorrect.

3.The answer is D. Dinitrophenol equilibrates the proton concentration across the inner

mitochondrial membrane, thereby destroying the proton motive force. Thus, none of the other

answers is correct.

4.The answer is A. A deficiency of Fe–S centers in the ETC would impair the transfer of electrons

down the chain and reduce ATP production by oxidative phosphorylation. Answer B is incorrect

because the decreased production of water from the ETC is not of sufficient magnitude to cause

her to become dehydrated. Answer C is incorrect because iron does not form a chelate with

NADH and FAD(2H). Answer D is incorrect because iron is not a cofactor for α-ketoglutarate

dehydrogenase. Answer E is incorrect because iron does not accompany the protons that make up

the proton gradient.

5.The answer is B. NADH would not be reoxidized as efficiently by the ETC, and the NADH/NAD+ ratio would increase. Answer A is incorrect because ATP would not be produced

at a high rate. Therefore, ADP would build up and the ATP:ADP ratio would be low. Answer C is

incorrect because OXPHOS diseases can be caused by mutations in nuclear or mtDNA, and not

all OXPHOS proteins are encoded by the X chromosome. Answer D is incorrect because,depending on the nature of the mutation, the activity of complex II of the ETC might be normal or

decreased, but there is no reason to expect increased activity. Answer E is incorrect because the

integrity of the inner mitochondrial membrane would not necessarily be affected. It could be, but it

would not be expected for all patients with OXPHOS disorders.

6.The answer is C. Heme is required for the synthesis of cytochromes. Complex I, although

containing iron, does so in iron–sulfur centers and contains no cytochromes. Complexes III and IV

contain cytochromes, whereas myoglobin and hemoglobin contain heme as the oxygen binding

component of those proteins. Defects in heme synthesis, then, would have a negative impact on the

function of complexes III and IV, as well as hemoglobin and myoglobin, without greatly affecting

the functioning of complex I.

7.The answer is B. Because rotenone inhibits the oxidation of NADH, it would completely block

the generation of the electrochemical potential gradient in vivo and, therefore, it would block ATP

generation. In the presence of rotenone, NADH would accumulate and NAD+ concentrations

would decrease. Although the mitochondria might still be able to oxidize compounds like

succinate, which transfer electrons to FAD, no succinate would be produced in vivo if the NAD+-

dependent dehydrogenases of the TCA cycle were inhibited. Thus, very shortly after rotenone

administration, there would not be any substrates available for the electron transfer chain, and

NADH dehydrogenase would be blocked, so oxidative phosphorylation would be completely

inhibited. If glucose supplies were high, anaerobic glycolysis could provide some ATP, but not

nearly enough to keep the heart pumping. Anaerobic glycolysis produces 2 ATP per glucose

molecule, compared with 32 ATP molecules generated by oxidative phosphorylation, which is a

reduction of approximately 95%.

8.The answer is C. NAD+ and FAD are electron acceptors and NADH and FAD(2H) are electron

donors. Oxygen is the terminal electron acceptor and is not an electron donor. The inner

mitochondrial membrane must be impermeable to most compounds, including protons; otherwise,

the proton gradient that drives ATP synthesis could not be created or maintained. The enzyme ATP

synthase contains a proton pore that spans the inner mitochondrial membrane and a catalytic

headpiece that protrudes into the matrix. Protons are driven through the pore and change the

conformation of the subunits in the headpiece, thereby producing ATP. If protons enter the

mitochondria other ways then through the pore, no ATP is generated by the ATP synthase (partial

uncoupling). A synthetase is an enzyme that uses high-energy phosphate bonds (usually from ATP)

to catalyze its reaction, and the ATP synthase creates high-energy phosphate bonds and does not

use them.

9.The answer is D. Complexes I, III, and IV are protein complexes that span the inner mitochondrial

membrane, and their location within the membrane would not be disrupted by a high-salt solution.

Cytochrome c is a small protein in the intermembrane space that binds to the inner mitochondrial

membrane through noncovalent interactions. High salt can dislodge cytochrome c from the inner

membrane, and the mitochondria would become cytochrome c–deficient. Electron flow would

stop in the absence of cytochrome c owing to an inability to transfer electrons from complexes III

to complex IV. CoQ is a lipid-soluble quinone that diffuses in the lipid membrane, and it would

not be removed from the membrane by a high-salt solution.10. The answer is C. Uncouplers uncouple oxidation from phosphorylation such that oxygen consumption is increased, there is an increased flow of electrons through the electron-transfer

chain, but ATP synthesis via oxidative phosphorylation is diminished. ATP production is reduced

owing to the reduction in the size of the proton gradient across the inner mitochondrial membrane

because the UCPs allow protons to enter the matrix of the mitochondria without going through the

ATP synthase. Because the energy of electron transfer is no longer being used to generate a proton

gradient, it is released as heat, and an individual taking such a drug would be expected to exhibit

an increased body temperature. Owing to the uncoupling, the oxidation of acetyl

coenzyme A

(acetyl-CoA) would increase, as would the glycolytic rate, in attempts to generate ATP for the

cell. The ATP synthase would be producing less ATP, and glycolysis would be producing more

ATP. The weight loss would come about because of the inefficiency in ATP generation via acetylCoA oxidation, as more fatty acids would have to be metabolized in order to generate a certain

amount of ATP from the acetyl-CoA derived from the fatty acids.25 Oxygen Toxicity and Free-Radical

Injury

For additional ancillary materials related to this chapter, please visit thePoint. O2 is both essential to human life and toxic. We are dependent on O2 for oxidation reactions in the

pathways of adenosine triphosphate (ATP) generation, detoxification, and biosynthesis. However, when

O2 accepts single electrons, it is transformed into highly reactive oxygen radicals that damage cellular

lipids, proteins, and DNA. Damage by reactive oxygen radicals contributes to cellular death and

degeneration in a wide range of diseases (Table 25.1).

Radicals are compounds that contain a single electron, usually in an outside orbital. Oxygen is a

biradical, a molecule that has two unpaired electrons in separate orbitals (Fig. 25.1). Through several

enzymatic and nonenzymatic processes that routinely occur in cells, O2 accepts single electrons to form

reactive oxygen species (ROS). ROS are highly reactive oxygen radicals or compounds that are readily

converted in cells to these reactive radicals. The ROS formed by reduction of O2 are the radicalsuperoxide (O2−), the nonradical hydrogen peroxide (H2O2), and the hydroxyl radical (OH•).

ROS may be generated nonenzymatically or enzymatically as accidental by-products or major

products of reactions. Superoxide may be generated nonenzymatically from coenzyme Q (CoQ) or from

metal-containing enzymes (e.g., cytochrome P450, xanthine oxidase, and reduced nicotinamide adenine

dinucleotide phosphate [NADPH] oxidase). The highly toxic hydroxyl radical is formed nonenzymatically from superoxide in the presence of Fe2+ or Cu+ by the Fenton reaction and from

hydrogen peroxide in the Haber–Weiss reaction.

Oxygen radicals and their derivatives can be deadly to cells. The hydroxyl radical causes oxidative

damage to proteins and DNA. It also forms lipid peroxides and malondialdehyde from membrane lipids

containing polyunsaturated fatty acids. In some cases, free-radical damage is the direct cause of a

disease state (e.g., tissue damage initiated by exposure to ionizing radiation). In neurodegenerative

diseases, such as Parkinson disease, or in ischemia–reperfusion injury, ROS may perpetuate the cellular

damage caused by another process.

Oxygen radicals are joined in their destructive damage by the free-radical nitric oxide (NO) and the

ROS hypochlorous acid (HOCl). NO combines with O2 or superoxide to form reactive nitrogen–

oxygen species (RNOS), such as the nonradical peroxynitrite or the radical nitrogen dioxide. RNOS are

present in the environment (e.g., cigarette smoke) and are generated in cells. During phagocytosis of

invading microorganisms, cells of the immune system produce O2−, HOCl, and NO through the actions of

NADPH oxidase, myeloperoxidase, and inducible nitric oxide synthase, respectively. In addition to

killing phagocytosed invading microorganisms, these toxic metabolites may damage surrounding tissue

components.

Cells protect themselves against damage by ROS and other radicals through repair processes,

compartmentalization of free-radical production, defense enzymes, and endogenous and exogenous

antioxidants (free-radical scavengers). The defense enzyme superoxide dismutase (SOD) removes the

superoxide free radical. Catalase and glutathione peroxidase remove hydrogen peroxide and lipid

peroxides. Vitamin E, vitamin C, and plant flavonoids act as antioxidants. Oxidative stress occurs

when the rate of ROS generation exceeds the capacity of the cell for their removal (Fig. 25.2).THE WAITING ROOM

Two years ago, Les G., a 62-year-old man, noted an increasing tremor of his right hand when

sitting quietly (resting tremor). The tremor disappeared if he actively used this hand to do

purposeful movement. As this symptom progressed, he also complained of stiffness in his muscles that

slowed his movements (bradykinesia). His wife noticed a change in his gait; he had begun taking short,

shuffling steps and leaned forward as he walked (postural imbalance). He often appeared to be staring

ahead with a rather immobile facial expression. She noted a tremor of his eyelids when he was asleep

and, recently, a tremor of his legs when he was at rest. Because of these progressive symptoms and some

subtle personality changes (anxiety and emotional lability), she convinced Les to see their family doctor.

The doctor suspected that her patient probably had primary or idiopathic parkinsonism (Parkinson

disease) and referred Mr. G. to a neurologist. In Parkinson disease, neurons of the substantia nigra pars

compacta, containing the pigment melanin and the neurotransmitter dopamine, degenerate.

Cora N. had done well since the successful lysis of blood clots in her coronary arteries with the

use of intravenous recombinant tissue plasminogen activator (TPA) (see Chapters 20 and 24). This

therapy quickly relieved the crushing chest pain (angina) she had experienced when she won the lottery.

At her first office visit after her discharge from the hospital, Cora’s cardiologist told her she had

developed multiple premature contractions of the ventricular muscle of her heart as the clots were being

lysed. This process could have led to a life-threatening arrhythmia known as ventricular tachycardia or

ventricular fibrillation. However, Cora’s arrhythmia responded quickly to pharmacologic suppression

and did not recur during the remainder of her hospitalization.

The basal ganglia are part of a neuronal feedback loop that modulates and integrates the

flow of information from the cerebral cortex to the motor neurons of the spinal cord. The

neostriatum is the major input structure from the cerebral cortex. The substantia nigra parscompacta consists of neurons that provide integrative input to the neostriatum through pigmented

neurons that use dopamine as a neurotransmitter (the nigrostriatal pathway). Integrated information

feeds back to the basal ganglia and to the cerebral cortex to control voluntary movement. In

Parkinson disease, a decrease in the amount of dopamine reaching the basal ganglia results in the

movement disorder.

In ventricular tachycardia, rapid premature beats from an irritative focus in ventricular

muscle occur in runs of varying duration. Persistent ventricular tachycardia compromises

cardiac output, leading to death. This arrhythmia can result from severe ischemia (lack of blood

flow) in the ventricular muscle of the heart caused by clots forming at the site of a ruptured

atherosclerotic plaque. However, Cora N.’s rapid beats began during the infusion of tissue TPA as

the clot was lysed. Thus, they probably resulted from reperfusing a previously ischemic area of

her heart with oxygenated blood. This phenomenon is known as ischemia–reperfusion injury, and

it is caused by cytotoxic ROS derived from oxygen in the blood that reperfuses previously hypoxic

cells. Ischemia–reperfusion injury also may occur when tissue oxygenation is interrupted during

surgery or transplantation.

I. O2 and the Generation of Reactive Oxygen Species

The generation of ROS from O2 in our cells is a natural, everyday occurrence. The electrons that

contribute to their formation are usually derived from reduced electron carriers of the electron-transport

chain (ETC). ROS are formed as accidental products of nonenzymatic and enzymatic reactions.

Occasionally, they are deliberately synthesized in enzyme-catalyzed reactions. Ultraviolet radiation and

pollutants in the air can increase formation of toxic oxygen-containing compounds. Catecholamine (epinephrine, norepinephrine, dopamine) measurements, which were ordered for Mr. G., use either serum or a 25-hour urine collection as samples for assay.

After appropriate removal of cells and/or particulate matter, the sample is placed over an ionexchange high-pressure liquid chromatography (HPLC) column and the eluate from the column is

analyzed by sensitive electrochemical detection. Through comparison with retention times of

standard catecholamines on the column, the various catecholamine species can be clearly resolved

from each other. The electrochemical detection uses electrodes that are oxidized by the samples,

and the amplitude current that is generated via the redox reaction allows one to determine the

concentration of catecholamines in the specimen. A. The Radical Nature of O2

A radical, by definition, is a molecule that has a single unpaired electron in an orbital. A free radical is a

radical that is capable of independent existence. (Radicals formed in an enzyme active site during a

reaction, for example, are not considered free radicals unless they can dissociate from the protein tointeract with other molecules.) Radicals are highly reactive and initiate chain reactions by extracting an

electron from a neighboring molecule to complete their own orbitals. Although the transition metals (e.g.,

Fe, Cu, Mo) have single electrons in orbitals, they are not usually considered free radicals because they

are relatively stable, do not initiate chain reactions, and are bound to proteins in the cell.

The oxygen molecule is a biradical, which means it has two single electrons in different orbitals.

These electrons cannot both travel in the same orbital because they have parallel spins (they spin in the

same direction). Although oxygen is very reactive from a thermodynamic standpoint, its single electrons

cannot react rapidly with the paired electrons found in the covalent bonds of organic molecules. As a

consequence, O2 reacts slowly through the acceptance of single electrons in reactions that require a

catalyst (such as a metal-containing enzyme).

Because the two unpaired electrons in oxygen have the same (parallel) spin, they are called

antibonding electrons. In contrast, carbon–carbon and carbon–hydrogen bonds each contain two

electrons, which have antiparallel spins and form a thermodynamically stable pair. As a consequence, O2

cannot readily oxidize a covalent bond because one of its electrons would have to flip its spin around to

make new pairs. The difficulty in changing spins is called spin restriction. Without spin restriction,

organic life forms could not have developed in the oxygen atmosphere on earth because they would be

spontaneously oxidized by O2.

O2 is capable of accepting a total of four electrons, which reduces it to water (Fig. 25.3). When O2

accepts one electron, superoxide is formed. Superoxide is still a radical because it has one unpaired

electron remaining. This reaction is not thermodynamically favorable and requires a moderately strong

reducing agent that can donate single electrons (e.g., the radical form of coenzyme Q [CoQH•] in the

ETC). When superoxide accepts an electron, it is reduced to hydrogen peroxide (H2O2), which is not a

radical. The hydroxyl radical is formed in the next one-electron reduction step in the reduction sequence.

Finally, acceptance of the last electron reduces the hydroxyl radical to H2O.B. Characteristics of Reactive Oxygen Species

ROS are oxygen-containing compounds that are highly reactive free radicals or compounds that are

readily converted to these oxygen free radicals in the cell. The major oxygen metabolites produced by

one-electron reduction of oxygen (superoxide, hydrogen peroxide, and the hydroxyl radical) are classified

as ROS (Table 25.2).Reactive free radicals extract electrons (usually as hydrogen atoms) from other compounds to

complete their own orbitals, thereby initiating free-radical chain reactions. The hydroxyl radical is

probably the most potent of the ROS. It initiates chain reactions that form lipid peroxides and organic

radicals and adds directly to compounds. The superoxide anion is also highly reactive, but it has limited

lipid solubility and cannot diffuse far. However, it can generate the more reactive hydroxyl and

hydroperoxy radicals by reacting nonenzymatically with hydrogen peroxide in the Haber–Weiss reaction

(Fig. 25.4).Hydrogen peroxide, although not actually a radical, is a weak oxidizing agent that is classified as an

ROS because it can generate the hydroxyl radical (OH•). Transition metals, such as Fe2+ or Cu+, catalyze

formation of the hydroxyl radical from hydrogen peroxide in the nonenzymatic Fenton reaction (see Fig.

25.4). Because hydrogen peroxide is lipid-soluble, it can diffuse through membranes and generate OH• at

localized Fe2+- or Cu+-containing sites, such as the ETC within the mitochondria. Hydrogen peroxide is

also the precursor of hypochlorous acid (HOCl), a powerful oxidizing agent that is produced

endogenously and enzymatically by phagocytic cells. To decrease occurrence of the Fenton reaction,

accessibility to transition metals, such as Fe2+ and Cu+, are highly restricted in