Voet D., Voet Ju.G. Biochemistry

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any other movement—also produces heat. Nonshivering

thermogenesis through substrate cycling is discussed in

Section 17-4Fi.)

The mechanism of heat generation in brown fat involves

the regulated uncoupling of oxidative phosphorylation in

their mitochondria. These mitochondria contain the pro-

tein thermogenin [also called uncoupling protein (UCP)],

a transmembrane homodimer of 307-residue subunits that

acts as a channel to control the permeability of the inner

mitochondrial membrane to protons. In cold-adapted ani-

mals, thermogenin constitutes up to 15% of brown fat in-

ner mitochondrial membrane proteins.The flow of protons

through this channel protein is inhibited by physiological

concentrations of purine nucleotides (ADP, ATP, GDP,

GTP), but this inhibition can be overcome by free fatty

acids. The components of this system interact under hor-

monal control.

Thermogenesis in brown fat mitochondria is activated by

free fatty acids. These counteract the inhibitory effects of

purine nucleotides, thereby stimulating the flux through

the proton channel and uncoupling electron transport from

oxidative phosphorylation. The concentration of fatty acids

in brown adipose tissue is controlled by the adrenal hor-

mone norepinephrine (noradrenaline; Section 18-3E) with

cAMP acting as a second messenger (Section 18-3). Norep-

inephrine binds to the ␤

-adrenergic receptor, a G-protein

coupled receptor (GPCR) that, via an associated het-

erotrimeric G protein, stimulates adenylate cyclase to syn-

thesize cAMP (Fig.22-48), as described in Section 19-2.The

cAMP, in turn, activates protein kinase A (PKA), which ac-

tivates hormone-sensitive triacylglycerol lipase by phos-

phorylating it (Section 25-5). Finally, the activated lipase

hydrolyzes triacylglycerols to yield the free fatty acids that

open thermogenin’s proton channel. The transcription of

Section 22-3. Oxidative Phosphorylation 861

Figure 22-48 Mechanism of hormonally induced uncoupling

of oxidative phosphorylation in brown fat mitochondria.

(1) Norepinephrine binds to a ␤

-adrenergic receptor. (2) This

stimulates the associated heterotrimeric G protein to activate

adenylate cyclase (upper green arrow) to synthesize cAMP. (3)

cAMP binding activates protein kinase A (PKA). (4) PKA

ATP

Norepinephrine

cAMP

+ P

2C + R

(cAMP)

Cytosol

PKA

(inactive)

ATP

ADP

triacylglycerol

lipase

(active)

triacylglycerol

lipase

(inactive)

ATP

Thermogenin

proton

channel

opens

channel

Free

fatty

acids

Triacylglycerols

Mitochondrion

ATPase

ATP, ADP, GTP, GDP

block channel

ADP + P

PKA

(active)

Electron

transport

Adenylate

cyclase

+2H

-Adrenergic

receptor

phosphorylates hormone-sensitive triacylglycerol lipase, thereby

activating it. (5) Triacylglycerols are hydrolyzed, yielding free

fatty acids. (6) Free fatty acids overcome the purine nucleotide

block of thermogenin’s proton channel (lower green arrow),

allowing H

⫹

to enter the mitochondrion uncoupled from ATP

synthesis.

JWCL281_c22_823-870.qxd 3/19/10 11:00 AM Page 861

the gene encoding thermogenin is stimulated by the thy-

roid hormone triiodothyronine (T3; Section 19-1D).

b. Other Tissues Contain UCP Homologs

Although it originally seemed that only brown fat mito-

chondria contain an uncoupling protein, it is now apparent

that other tissues contain homologs of UCP1. Thus, UCP2

is expressed in many tissues including white adipose tissue,

whereas UCP3 occurs in both brown and white adipose tis-

sues as well as in muscle. These proteins may help regulate

metabolic rates, and variations in UCP levels or activity

might explain why some people seem to have a “fast” or

“slow” metabolism (Section 27-3E). UCPs are being stud-

ied as targets for treating obesity, since increasing the activ-

ity of UCPs could uncouple respiration from ATP synthe-

sis, thus permitting stored metabolic fuels (especially fat)

to be metabolized.The recent discovery that adult humans

have small depots of brown fat that are activated by cold

has made this an attractive weight loss strategy, although it

is possible that stimulating UCPs will cause a compensa-

tory increase in appetite.

Uncoupling proteins are not limited to animals. Some

plants express uncoupling proteins in response to cold

stress or to increase flower temperature, possibly to en-

hance the vaporization of scent to attract pollinators.

4 CONTROL OF ATP PRODUCTION

A typical adult woman requires some 1500 to 1800 kcal

(6300–7500 kJ) of metabolic energy per day. This corre-

sponds to the free energy of hydrolysis of over 200 mol of

ATP to ADP and P

. Yet the total amount of ATP present

in the body at any one time is ⬍0.1 mol; obviously, this

sparse supply of ATP must be continually recycled. As we

have seen, when carbohydrates serve as the energy supply

and aerobic conditions prevail, this recycling involves

glycogenolysis, glycolysis, the citric acid cycle, and oxida-

tive phosphorylation.

Of course the need for ATP is not constant. There is a

100-fold change in ATP utilization between sleep and vigor-

ous activity. The activities of the pathways that produce ATP

are under strict coordinated control so that ATP is never pro-

duced more rapidly than necessary. We have already dis-

cussed the control mechanisms of glycolysis, glycogenolysis,

and the citric acid cycle (Sections 17-4, 18-3, and 21-4). In

this section we discuss the mechanisms through which ox-

idative phosphorylation is controlled and observe how all

four systems are synchronized to produce ATP at precisely

the rate required at any particular moment.

A. Control of Oxidative Phosphorylation

In our discussion of the control of glycolysis, we saw that

most of the reactions in a metabolic pathway function close

to equilibrium. The few irreversible reactions constitute the

potential control points of the pathway and usually are cat-

alyzed by regulatory enzymes that are under allosteric con-

trol. In the case of oxidative phosphorylation, the pathway

from NADH to cytochrome c functions near equilibrium

(⌬G ⬇ 0):

for which

[22.2]

This pathway is therefore readily reversed by the addition

of ATP. In the cytochrome c oxidase reaction, however, the

terminal step of the electron-transport chain is irreversible

and is thus one of the important regulatory sites of the path-

way. Cytochrome c oxidase, in contrast to most regulatory

enzyme systems, appears to be controlled exclusively by

the availability of one of its substrates, reduced cytochrome

c (c

2⫹

). Since this substrate is in equilibrium with the rest of

the coupled oxidative phosphorylation system (Eq. [22.2]),

its concentration ultimately depends on the intramitochon-

drial [NADH]/[NAD

⫹

] ratio and the ATP mass action

ratio ([ATP]/[ADP][P

]). By rearranging Eq. [22.2], the ratio

of reduced to oxidized cytochrome c is expressed

[22.3]

Consequently, the higher the [NADH]/[NAD

⫹

] ratio and

the lower the ATP mass action ratio, the higher is the [c

2⫹

]

(reduced cytochrome c) and thus the higher is the cy-

tochrome c oxidase activity.

How is this system affected by changes in physical activ-

ity? In an individual at rest,ATP hydrolysis to ADP and P

is minimal and the ATP mass action ratio is high; the con-

centration of reduced cytochrome c is therefore low and

oxidative phosphorylation is minimal. Increased activity

results in hydrolysis of ATP to ADP and P

, thereby de-

creasing the ATP mass action ratio and increasing the con-

centration of reduced cytochrome c. This results in an in-

crease in the electron-transport rate and its coupled

phosphorylation. Such control of oxidative phosphoryla-

tion by the ATP mass action ratio is called acceptor control

because the rate of oxidative phosphorylation increases

with the concentration of ADP, the phosphoryl group

acceptor. In terms of a supply–demand system (Section

17-4D), acceptor control is understood as control by the

demand block.

The compartmentalization of the cell into mitochon-

dria, where ATP is synthesized, and cytoplasm, where ATP

is utilized, presents an interesting control problem: Is it the

ATP mass action ratio in the cytosol or in the mitochondr-

ial matrix that ultimately controls oxidative phosphoryla-

tion? Clearly the ATP mass action ratio that exerts direct

control must be that of the mitochondrial matrix where

ATP is synthesized. However, the inner mitochondrial

membrane, which is impermeable to adenine nucleotides

and P

, depends on specific transport systems to maintain

communication between the two compartments (Section

20-4C).This organization makes it possible for the transport

2⫹

]

3⫹

]

⫽ a

[NADH]

[NAD

⫹

]

[ADP][P

]

[ATP]

⫽ a

[NAD

⫹

]

[NADH]

Ⲑ

2⫹

]

3⫹

]

[ATP]

[ADP] [P

]

NAD

⫹

⫹ cytochrome c

2⫹

⫹ ATP

NADH ⫹ cytochrome c

3⫹

⫹ ADP ⫹ P

862 Chapter 22. Electron Transport and Oxidative Phosphorylation

JWCL281_c22_823-870.qxd 3/19/10 11:00 AM Page 862

of adenine nucleotides or P

to participate in the control of

oxidative phosphorylation.

Considerable research effort has been aimed at deter-

mining how oxidative phosphorylation is controlled in

terms of metabolic control analysis. For example, Hans

Westerhoff and Martin Kushmerick employed

P NMR to

measure the ATP/ADP ratios in human forearm muscle at

rest and during twitch contractions caused by external

electrical stimulation (the

P NMR spectrum of ATP is

shown in Fig. 16-15). Under conditions of low to moderate

ATP demand, the cytosolic mass action ratio as controlled

by the demand block of the system appears to be the major

control factor for mitochondrial oxidation. However, as

other laboratories have shown, as the demand for ATP in-

creases, the ADP–ATP translocator exerts greater control

until finally, when the demand for ATP is high, control

shifts to the supply block of the system, oxidative phospho-

rylation itself.

B. Coordinated Control of ATP Production

Glycolysis, the citric acid cycle, and oxidative phosphoryla-

tion constitute the major pathways for cellular ATP pro-

duction. Control of oxidative phosphorylation by the ATP

mass action ratio depends, of course, on an adequate sup-

ply of electrons to fuel the electron-transport chain. This

aspect of the system’s control is, in turn, dependent on the

[NADH]/[NAD

⫹

] ratio (Eq. [22.3]), which is maintained

high by the combined action of glycolysis and the citric acid

cycle in converting 10 molecules of NAD

⫹

to NADH per

molecule of glucose oxidized (Fig. 22-1). It is clear, there-

fore, that coordinated control is necessary for the three

processes. This is provided by the regulation of each of the

control points of glycolysis [hexokinase, phosphofructoki-

nase (PFK), and pyruvate kinase] and the citric acid cycle

(pyruvate dehydrogenase, citrate synthase, isocitrate dehy-

drogenase, and ␣-ketoglutarate dehydrogenase) by ade-

nine nucleotides or NADH or both as well as by certain

metabolites (Fig. 22-49).

a. Citrate Inhibits Glycolysis

The main control points of glycolysis and the citric acid cy-

cle are regulated by several effectors besides adenine nu-

cleotides or NADH (Fig. 22-49). This is an extremely com-

plex system with complex demands. Its many effectors, which

are involved in various aspects of metabolism, increase its

regulatory sensitivity. One particularly interesting regulatory

effect is the inhibition of PFK by citrate. When demand for

ATP decreases, [ATP] increases and [ADP] decreases. The

Section 22-4. Control of ATP Production 863

Figure 22-49 Schematic diagram depicting the coordinated

control of glycolysis and the citric acid cycle by ATP, ADP, AMP,

,Ca

2ⴙ

, and the [NADH]/[NAD

ⴙ

] ratio (the vertical arrows

indicate increases in this ratio). Here a green dot signifies

activation and a red octagon represents inhibition. [After

Newsholme, E.A. and Leech, A.R., Biochemistry for the Medical

Sciences, pp. 316 and 320, Wiley (1983).]

See the Animated

Figures

Glycolysis

Glucose

Glucose-6-phosphate

Fructose-6-phosphate

Fructose-1,6-bisphosphate

PEP

Pyruvate

hexokinase

phosphoglucose isomerase

phosphofructokinase

pyruvate kinase

AMP

ATP

ADP

ATP

ADP

NADH

NAD

]

Acetyl-CoA

citrate synthase

Citrate

Isocitrate

α-KetoglutarateSuccinyl-CoA

Fumarate

Malate

Oxaloacetate

Succinate

ADP

ATP

CoASH

succinyl-CoA

]

isocitrate

dehydrogenase

α-ketoglutarate

dehydrogenase

Citric acid

cycle

NADH

NAD

]

NADH

NAD

]

NADH

NAD

]

pyruvate dehydrogenase

JWCL281_c22_823-870.qxd 6/8/10 9:19 AM Page 863

citric acid cycle slows down at its isocitrate dehydrogenase

(activated by ADP) and ␣-ketoglutarate dehydrogenase (in-

hibited by ATP) steps, thereby causing the citrate concentra-

tion to build up. Citrate can leave the mitochondrion via a

specific transport system and, once in the cytosol, acts to re-

strain further carbohydrate breakdown by inhibiting PFK.

b. Fatty Acid Oxidation Inhibits Glycolysis

As we shall see in Section 25-2, the oxidation of fatty

acids is an aerobic process that produces acetyl-CoA, which

enters the citric acid cycle, thereby increasing both the mi-

tochondrial and cytoplasmic concentrations of citrate. The

increased [acetyl-CoA] inhibits the pyruvate dehydroge-

nase complex, whereas the increased [citrate] inhibits

phosphofructokinase, leading to a buildup of glucose-6-

phosphate, which inhibits hexokinase (Fig. 22-49). This in-

hibition of glycolysis by fatty acid oxidation is called the

glucose–fatty acid cycle or Randle cycle (after its discov-

erer, Philip Randle), although it is not, in fact, a cycle. The

Randle cycle allows fatty acids to be utilized as the major

fuel for oxidative metabolism in heart muscle, while con-

serving glucose for organs such as the brain,which require it.

C. Physiological Implications of Aerobic versus

Anaerobic Metabolism

In 1861, Louis Pasteur observed that when yeast are exposed

to aerobic conditions, their glucose consumption and ethanol

production drop precipitously (the Pasteur effect; alcoholic

fermentation in yeast to produce ATP, CO

, and ethanol are

discussed in Section 17-3B). An analogous effect is ob-

served in mammalian muscle; the concentration of lactic

acid, the anaerobic product of muscle glycolysis, drops dra-

matically when cells switch to aerobic metabolism.

a. Hypoxia Causes an Increase in Glycolysis

In the presence of sufficient oxygen, oxidative phospho-

rylation supplies most of the body’s ATP needs. However,

during hypoxia (when oxygen is limiting), glycolysis must be

stimulated (with its inherent increased rate of glucose con-

sumption; the reverse of the Pasteur effect) to supply the

necessary ATP. F2,6P, the most potent activator of PFK-1,

also participates in this process. The concentration of F2,6P,

as we have seen (Section 18-3Fc), is regulated by the bifunc-

tional enzyme PFK-2/FBPase-2. In its heart isozyme, the

PFK-2 activity is stimulated by phosphorylation at its Ser

466. Among the enzymes that do so is AMP-activated pro-

tein kinase (AMPK; Sections 25-4Ba, 25-5, and 27-1). When

oxygen deficiency prevents oxidative phosphorylation from

providing sufficient ATP for heart function, as occurs in is-

chemia (insufficient blood flow), the resulting increased

[AMP] activates AMPK. The consequent phosphorylation

and hence activation of PFK-2 results in an increase of

[F2,6P], thereby activating PFK-1 and thus glycolysis.

b. Aerobic ATP Production Is Far More Efficient

than Anaerobic ATP Production

One reason for the decrease in glucose consumption on

switching from anaerobic to aerobic metabolism is clear

from an examination of the stoichiometries of anaerobic

and aerobic breakdown of glucose (C

Anaerobic glycolysis:

Aerobic metabolism of glucose:

(2.5 ATP for each of the 10 NADH generated per glucose

oxidized, 1.5 ATP for each of the 2 FADH

generated,

2 ATP produced in glycolysis, and 2GTP 34 2ATP pro-

duced in the citric acid cycle.) Thus aerobic metabolism is

16 times more efficient than anaerobic glycolysis in produc-

ing ATP. The switch to aerobic metabolism therefore rap-

idly increases the ATP mass action ratio. As the ATP mass

action ratio increases, the rate of electron transport

decreases, which has the effect of increasing the [NADH]/

[NAD

⫹

] ratio. The increases in [ATP] and [NADH] inhibit

their target enzymes in the citric acid cycle and in the gly-

colytic pathway. The activity of PFK, which is citrate- and

adenine nucleotide-regulated and one of the rate-controlling

enzymes of glycolysis, decreases manyfold on switching

from anaerobic to aerobic metabolism.This accounts for the

dramatic decrease in glycolysis.

c. Anaerobic Glycolysis Has Advantages as

Well as Limitations

Animals can sustain anaerobic glycolysis for only short

periods of time.This is because PFK, which cannot function

effectively much below pH 7, is inhibited by the acidifica-

tion arising from lactic acid production. Despite this limita-

tion and the low efficiency of glycolytic ATP production, the

enzymes of glycolysis are present in such great concentra-

tions that when they are not inhibited, ATP can be produced

much more rapidly than through oxidative phosphorylation.

The different characteristics of aerobic and anaerobic

metabolism permit us to understand certain aspects of can-

cer cell metabolism and cardiovascular disease.

d. Cancer Cell Metabolism

As Warburg first noted in 1926, certain cancer cells pro-

duce more lactic acid under aerobic conditions than do

normal cells.This is because the glycolytic pathway in these

cells produces pyruvate more rapidly than the citric acid

cycle can accommodate. How can this happen given the in-

terlocking controls on the system? One explanation is that

these controls have broken down in cancer cells.Another is

that their ATP utilization occurs at rates too rapid to be re-

plenished by oxidative phosphorylation. This would alter

the ratios of adenine nucleotides so as to relieve the inhibi-

tion of PFK-1. In addition, many cancer cell lines have a

much larger [F2,6P] than do normal cells. These cells con-

tain an inducible isozyme of PFK-2/FBPase-2 that has an

AMPK-phosphorylatable site for activating PFK-2. Conse-

quently, an [AMP] increase in these cells results in an in-

crease in their [F2,6P], which further activates PFK-1 and

6CO

⫹ 38H

O ⫹ 32ATP

⫹ 32ADP ⫹ 32P

⫹ 6O

2 lactate ⫹ 2H

⫹

⫹ 2H

O ⫹ 2ATP

⫹ 2ADP ⫹ 2P

864 Chapter 22. Electron Transport and Oxidative Phosphorylation

JWCL281_c22_823-870.qxd 3/19/10 11:00 AM Page 864

Section 22-4. Control of ATP Production 865

glycolysis. Efforts to understand the metabolic differences

between cancer cells and normal cells may eventually lead

to a treatment of certain forms of this devastating disease.

e. Cardiovascular Disease

Oxygen deprivation of certain tissues resulting from

cardiovascular disease is of major medical concern. For ex-

ample, two of the most common causes of human death,

myocardial infarction (heart attack) and stroke, are caused

by interruption of the blood (O

) supply to a portion of the

heart or the brain, respectively. It seems obvious why this

should result in a cessation of cellular activity, but why does

it cause cell death?

In the absence of O

, a cell, which must then rely only on

glycolysis for ATP production, rapidly depletes its stores of

phosphocreatine (a source of rapid ATP production; Sec-

tion 16-4Cd) and glycogen. As the rate of ATP production

falls below the level required by membrane ion pumps for

the maintenance of proper intracellular ionic concentra-

tions, the osmotic balance of the system is disrupted, so that

the cell and its membrane-enveloped organelles begin to

swell.The resulting overstretched membranes become per-

meable, thereby leaking their enclosed contents. [In fact, a

useful diagnostic criterion for myocardial infarction is the

presence in the blood of heart-specific enzymes, such as the

H-type isozyme of lactate dehydrogenase (vs the M-type

isozyme, which predominates in skeletal muscle; Section

17-3A), which leak out of necrotic (dead) heart tissue.]

Moreover, the decreased intracellular pH that accompa-

nies anaerobic glycolysis (because of lactic acid produc-

tion; Section 17-3A) permits the released lysosomal en-

zymes (which are active only at acidic pH’s) to degrade the

cell contents. Thus, the cessation of metabolic activity re-

sults in irreversible cell damage. Rapidly respiring tissues,

such as those of heart and brain, are particularly suscepti-

ble to such damage.

f. IF

Inhibits F

–ATPase during Hypoxia

Under hypoxic conditions, the proton-motive force

across the inner mitochondrial membrane is reduced to the

point that F

–ATPase would switch from the synthesis of

ATP to its hydrolysis, resulting in a catastrophic loss of

ATP. This is prevented through the interaction of the

–ATPase with an 84-residue regulatory protein named

. Under normal physiological conditions, IF

forms inac-

tive tetramers and higher order oligomers. However, when

the pH drops below 6.5, which occurs under anaerobic con-

ditions due to lactic acid production, IF

forms dimers in

which its almost entirely ␣ helical subunits associate via an

antiparallel coiled coil involving its residues 48 to 84. The

X-ray structure of F

in complex with AMPPNP and IF

determined by Leslie and Walker, reveals that each N-

terminal segment of the IF

dimer has bound to the

␣

–␤

interface (Fig. 22-38b) of a separate F

. This traps

AMPPNP and presumably ATP in the ␤

binding site,

which would prevent it from hydrolyzing ATP (which,

since AMPPNP rather than ADP is bound to ␤

, suggests

that this structure is that of a prehydrolysis step in the cat-

alytic reaction). When oxygen becomes available, the cell

re-energizes and its pH increases, thereby causing IF

dissociate from F

, which then commences synthesizing

ATP.

g. Partial Oxygen Reduction Produces Reactive

Oxygen Species (ROS)

Although the four-electron reduction of O

by cy-

tochrome c oxidase normally goes to completion, the en-

zyme infrequently releases partially reduced reactive oxygen

species (ROS) that readily react with a variety of cellular

components. The best known ROS is the superoxide

radical, . It is also produced by the occasional leakage

of electrons from Complexes I and III:

Its production is enhanced under hypoxic conditions.

Superoxide radical is a precursor of other reactive

species. Protonation of yields HO

ⴢ, a much stronger

oxidant than . The most potent oxygen species in bio-

logical systems is probably the hydroxyl radical, which

forms from the relatively harmless hydrogen peroxide

The hydroxyl radical also forms through the reaction of su-

peroxide with H

ROS readily extract electrons from other molecules, con-

verting them to free radicals and thereby initiating a chain

reaction.

The random nature of ROS attacks makes it difficult to

characterize their reaction products, but all classes of bio-

logical molecules are susceptible to oxidative damage

caused by free radicals. The oxidation of polyunsaturated

lipids in cells may disrupt the structures of membranes, and

oxidative damage to DNA may result in point mutations.

Enzyme function may also be compromised through radi-

cal reactions with amino acid side chains. Because the mi-

tochondrion is the site of the bulk of the cell’s oxidative

metabolism, its lipids, DNA, and proteins bear the brunt of

free radical–related damage.

Several degenerative diseases, including Parkinson’s,

Alzheimer’s, and Huntington’s diseases, are associated

with oxidative damage to mitochondria. Such observations

have led to the free-radical theory of aging, which holds

that free-radical reactions arising during the course of nor-

mal oxidative metabolism are at least partially responsible

for the aging process. In fact, individuals with congenital

defects in their mitochondrial DNA suffer from a variety

of symptoms typical of old age, including neuromotor

difficulties, deafness, and dementia. These genetic defects

may increase the susceptibility of mitochondria to ROS-

generated damage.

h. Cells Are Equipped with Antioxidant Mechanisms

Antioxidants eliminate oxidative free radicals such as

and ⴢOH. In 1969, Irwin Fridovich discovered that the

⫺

ⴢ

⫺

ⴢ ⫹ H

⫹ OH

⫺

⫹ ⴢOH

⫹ Fe

2⫹

ⴢOH ⫹ OH

⫺

⫹ Fe

3⫹

⫺

ⴢ

⫺

ⴢ

⫹ e

⫺

ⴢ

⫺

ⴢ

JWCL281_c22_823-870.qxd 7/5/10 11:43 AM Page 865

enzyme superoxide dismutase (SOD), which is present in

nearly all cells, catalyzes the conversion of to H

Mitochondrial and bacterial SOD are both Mn

2⫹

-containing

tetramers; eukaryotic cytosolic SOD is a dimer that contains

both Cu

2⫹

and Zn

2⫹

ions.Although the rate of nonenzymatic

superoxide breakdown is ⬃2 ⫻ 10

⫺1

ⴢ s

⫺1

, that of the

Cu,Zn-SOD–catalyzed reaction is ⬃2 ⫻ 10

⫺1

ⴢ s

⫺1

, close

to the diffusion-controlled limit (Section 14-2Bb). This is

apparently accomplished by electrostatic guidance of the

negatively charged superoxide substrate into the enzyme’s

active site (Fig. 14-10).

is degraded to water and oxygen by enzymes such

as catalase, which catalyzes the reaction

and glutathione peroxidase, which uses glutathione (GSH;

Section 21-2Ba) as a reducing agent:

2GSH ⫹ H

GSSG ⫹ 2H

O ⫹ O

⫺

ⴢ ⫹ 2H

⫹

⫹ O

⫺

ⴢ

The latter enzyme also catalyzes the breakdown of organic

hydroperoxides. Some types of glutathione peroxidase re-

quire Se for activity, which is one reason why Se appears to

have antioxidant activity.

Other potential antioxidants are plant-derived com-

pounds such as ascorbic acid (vitamin C; Section 11-1Cb)

and vitamin E, a group of compounds whose most promi-

nent member is ␣-tocopherol.

These compounds may help protect plants from oxidative

damage during photosynthesis, a process in which H

O is ox-

idized to O

(Section 24-2). However, clinical trials indicate

that their use does not contribute to longevity in humans.

␣-Tocopherol (vitamin E)

()

866 Chapter 22. Electron Transport and Oxidative Phosphorylation

1 The Mitochondrion Oxidative phosphorylation is the

process through which the NADH and FADH

produced by

nutrient oxidation are oxidized with the concomitant forma-

tion of ATP. The process takes place in the mitochondrion, an

ellipsoidal organelle that is bounded by a permeable outer

membrane and contains an impermeable and highly invagi-

nated inner membrane that encloses the matrix. Enzymes of

oxidative phosphorylation are embedded in the inner mito-

chondrial membrane. P

is imported into the mitochondrion by

a specific transport protein. Ca

2⫹

import and Ca

2⫹

export pro-

teins operate to maintain a constant cytosolic [Ca

2⫹

]. NADH’s

electrons are imported into the mitochondrion by shuttle sys-

tems such as the glycerophosphate shuttle and the malate–

aspartate shuttle.

2 Electron Transport The standard free energy change

for the oxidation of NADH by O

is ⌬G°¿ ⫽ –218 kJ ⴢ mol

⫺1

whereas that for the synthesis of ATP from ADP and P

⌬G°¿ ⫽ 30.5 kJ ⴢ mol

⫺1

. Consequently, the molar free energy of

oxidation of NADH by O

is sufficient to power the synthesis

of several moles of ATP under standard conditions. The elec-

trons generated by oxidation of NADH and FADH

pass

through four protein complexes, the electron-transport chain,

with the coupled synthesis of ATP. Complexes I, III, and IV

participate in the oxidation of NADH, producing ⬃2.5 ATPs

per NADH, whereas FADH

oxidation, which involves Com-

plexes II, III, and IV, produces only ⬃1.5 ATPs per FADH

Thus, the ratio of moles of ATP produced per mole of coenzyme

oxidized by O

, the P/O ratio, is ⬃2.5 for NADH oxidation and

⬃1.5 for FADH

oxidation. The route taken by electrons

through the electron-transport chain was elucidated, in part,

through the use of electron-transport inhibitors. Rotenone and

amytal inhibit Complex I, antimycin inhibits Complex III, and

⫺

inhibits Complex IV.Also involved were measurements of

the reduction potentials of the electron-carrying prosthetic

groups contained in the electron-transport complexes.

Complex I contains FMN and nine iron–sulfur clusters in a

45-subunit (in mammals) transmembrane protein complex.

This L-shaped complex passes electrons from NADH to CoQ,

a nonpolar small molecule that diffuses freely within the mem-

brane. Complex II, which is also the citric acid cycle enzyme

succinate dehydrogenase, also passes electrons to CoQ, in this

case from succinate through FAD and three iron–sulfur clus-

ters.The X-ray structure of Complex II indicates that its redox

cofactors are arranged in a linear chain. CoQH

passes elec-

trons to Complex III (cytochrome bc

), a homodimeric com-

plex whose protomers each contain two b-type hemes bound

to a cytochrome b subunit, a Rieske iron–sulfur protein (ISP),

and a cytochrome c

.An electron from cytochrome c

of Com-

plex III is passed to the Cu

center of Complex IV (cy-

tochrome c oxidase) via the peripheral membrane protein cy-

tochrome c. This electron is then passed to cytochrome a,

which, in turn, passes it to a binuclear center composed of

heme a

and Cu

, which reduces O

to H

O. This process oc-

curs in four 1-electron steps that pump four protons from the

mitochondrial matrix/bacterial cytoplasm to the intermem-

brane space/periplasm. Complexes I, III, and IV form super-

complexes that increase the efficiency of electron transport.

3 Oxidative Phosphorylation The mechanism by which

the free energy released by the electron-transport chain is

stored and utilized in ATP synthesis is described by the

chemiosmotic hypothesis. This hypothesis states that the free

energy released by electron transport is conserved by the gen-

eration of an electrochemical proton gradient across the inner

mitochondrial membrane (bacterial cell membrane; outside

positive and acidic), which is harnessed to synthesize ATP. The

proton gradient is created and maintained by the obligatory

outward translocation of H

⫹

across the inner mitochondrial

membrane as electrons travel through Complexes I,III, and IV.

Complex III pumps protons via a redox loop mechanism

called the Q cycle, a bifurcated double cycle in which one

CHAPTER SUMMARY

JWCL281_c22_823-870.qxd 7/2/10 11:14 AM Page 866

References 867

molecule of CoQH

is oxidized to CoQ and then is rereduced to

CoQH

by a second molecule of CoQH

in a process that col-

lectively transfers four protons from the inside to the outside

while oxidizing one molecule of CoQH

to CoQ. Electrons are

transferred between the two CoQ’s, which are bound at differ-

ent sites, Q

and Q

, as well as between the CoQH

bound at Q

and cytochrome c

via the ISP, which undergoes a conforma-

tional change in doing so. Complex IV contains no (H

⫹

⫹ e

⫺

)

carriers such as CoQH

and hence translocates proteins via a

proton pump mechanism. Bacteriorhodopsin, the best charac-

terized proton pump, translocates protons in a light driven

process. This involves a trans to cis isomerization of bacterio-

rhodopsin’s retinal prosthetic group on absorbing a photon,

followed by the translocation of a proton through the hy-

drophilic central channel of this transmembrane protein via a

process that involves conformational and pK changes of the

polar groups lining the channel as the retinal relaxes to its

ground state. Complex IV is thought to pump protons via a sim-

ilar mechanism that is driven by the changes in the redox state

of its heme a

–Cu

binuclear center as it reduces O

to H

The energy stored in the electrochemical proton gradient is

utilized by proton-translocating ATP synthase (Complex V,

–ATPase) in the synthesis of ATP via the binding change

mechanism, by coupling this process to the exergonic transport

of H

⫹

back to the inside. Mitochondrial proton-translocating

ATP synthase consists of two oligomeric components: F

(␣

␤

␥␦ε), a peripheral membrane protein that appears as “lol-

lipops” in electron micrographs of the inner mitochondrial

membrane, and F

(ab

in E. coli), an integral membrane

protein that contains the proton channel. The conformational

changes that promote the synthesis of ATP from ADP ⫹ P

arise through the demonstrated rotation of the ␥ subunit rela-

tive to the catalytic ␣

␤

assembly that contains the enzyme’s

three active sites. The ␥ subunit is attached to a ring of c sub-

units in F

, whose rotation is driven by the passage of protons

between it and the a subunit.

Compounds such as 2,4-dinitrophenol are uncouplers of

oxidative phosphorylation because they carry H

⫹

across the

mitochondrial membrane, thereby dissipating the proton gra-

dient and allowing electron transport to continue without con-

comitant ATP synthesis. Brown fat mitochondria contain a

regulated uncoupling system that, under hormonal control,

generates heat instead of ATP.

4 Control of ATP Production Under aerobic conditions,

the rate of ATP synthesis by oxidative phosphorylation is reg-

ulated, in a phenomenon known as acceptor control, by the

ATP mass action ratio. ATP synthesis is tightly coupled to the

oxidation of NADH and FADH

by the electron-transport

chain. Glycolysis and the citric acid cycle are coordinately con-

trolled so as to produce NADH and FADH

only at a rate re-

quired to meet the system’s demand for ATP. IF

inhibits the

ATP hydrolysis by mitochondrial F

–ATPase that would

otherwise occur under hypoxic conditions. Incomplete and

side reactions of Complexes I, III, and IV produce damaging

reactive oxygen species (ROS) that are largely eliminated

through the actions of several cellular enzymes, most notably

superoxide dismutase.

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1. Rank the following redox-active coenzymes and prosthetic

groups of the electron-transport chain in order of increasing affin-

ity for electrons: cytochrome a, CoQ, FAD, cytochrome c, NAD

⫹

2. Why is the oxidation of succinate to fumarate only associ-

ated with the production of two ATPs during oxidative phospho-

rylation, whereas the oxidation of malate to oxaloacetate is asso-

ciated with the production of three ATPs?

3. What is the thermodynamic efficiency of oxidizing FADH

as to synthesize two ATPs under standard biochemical conditions?

4. Sublethal cyanide poisoning may be reversed by the admin-

istration of nitrites. These substances oxidize hemoglobin, which

has a relatively low affinity for CN

⫺

, to methemoglobin, which has

a relatively high affinity for CN

⫺

.Why is this treatment effective?

5. Match the compound with its behavior: (1) rotenone,

(2) dinitrophenol, and (3) antimycin. (a) Inhibits oxidative phos-

phorylation when the substrate is pyruvate but not when the sub-

strate is succinate. (b) Inhibits oxidative phosphorylation when

the substrate is either pyruvate or succinate. (c) Allows pyruvate

to be oxidized by mitochondria even in the absence of ADP.

6. Nigericin is an ionophore (Section 20-2C) that exchanges

⫹

for H

⫹

across membranes. Explain how the treatment of func-

tioning mitochondria with nigericin uncouples electron transport

from oxidative phosphorylation. Does valinomycin, an ionophore

that transports K

⫹

but not H

⫹

, do the same? Explain.

7. Why is it possible for electrons in an electron-transfer com-

plex to flow from a redox center to one with a lesser value of e°¿?

8. How do the P/O ratios for NADH differ in ATP synthases

that contain 10 and 15 c subunits?

9. The difference in pH between the internal and external

surfaces of the inner mitochondrial membrane is 1.4 pH units

(external side acidic). If the membrane potential is 0.06 V (inside

negative), what is the free energy released on transporting 1 mol

of protons back across the membrane? How many protons must

be transported to provide enough free energy for the synthesis of

1 mol of ATP (assume standard biochemical conditions)?

*10. (a) A simplistic interpretation of the Q cycle would pre-

dict that the proton pumping efficiency of cytochrome bc

would

be reduced by no more than 50% in the presence of saturating

amounts of antimycin. Explain. (b) Indicate why cytochrome bc

nearly 100% inhibited by antimycin.

PROBLEMS

JWCL281_c22_823-870.qxd 3/19/10 11:00 AM Page 869

870 Chapter 22. Electron Transport and Oxidative Phosphorylation

11. The antibiotic oligomycin B

binds to the F

subunit of the mitochondrial F

–ATPase and

thereby prevents it from synthesizing ATP [note that oligomycin-

sensitivity conferral protein (OSCP), the mitochondrial counter-

part of the E. coli ␦ subunit (Fig. 22-41), does not bind oligomycin

Oligomycin B

B.] Explain why: (a) Submitochondrial particles from which F

has

been removed are permeable to protons. (b) Addition of

oligomycin B to F

-depleted submitochondrial particles decreases

this permeability severalfold.

12. Oligomycin B (see Problem 11) and cyanide both inhibit

oxidative phosphorylation when the substrate is either pyruvate

or succinate. Dinitrophenol can be used to distinguish between

these inhibitors. Explain.

13. The E. coli F

–ATPase cannot synthesize ATP when

Met 23 of its ␥ subunit is mutated to Lys.Yet the F

component of

this complex still exhibits rotation of its ␥ subunit relative to its

␣

␤

spheroid when it is supplied with ATP. Suggest a reason for

these effects.

14. For the oxidation of a given amount of glucose, does non-

shivering thermogenesis by brown fat or shivering thermogenesis

by muscle produce more heat?

15. What is the advantage of hormones activating a lipase to

stimulate nonshivering thermogenesis in brown fat rather than ac-

tivating UCP1 directly?

16. How does atractyloside affect mitochondrial respiration?

(Hint: See Section 20-4C.)

17. Certain unscrupulous operators offer, for a fee, to freeze

recently deceased individuals in liquid nitrogen until medical sci-

ence can cure the disease from which they died. What is the bio-

chemical fallacy of this procedure?

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