Voet D., Voet Ju.G. Biochemistry

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Porter, D.J.T. and Bright, H.J., 3-Carbanionic substrate analogues

bind very tightly to fumarase and aspartase, J. Biol. Chem. 255,

4772–4780 (1980).

Remington, S.J., Structure and mechanism of citrate synthase,

Curr. Top. Cell Regul. 33, 202–229 (1992); and Mechanisms of

citrate synthase and related enzymes (triose phosphate iso-

merase and mandelate racemase), Curr. Opin. Struct. Biol. 2,

730–735 (1992).

Wolodk,W.T., Fraser, M.E., James, M.N.G., and Bridger,W.A.,The

crystal structure of succinyl-CoA synthetase from Escherichia

coli at 2.5 Å resolution, J. Biol. Chem. 269, 10883–10890 (1994).

Zheng, L., Kennedy, M.C., Beinert, H., and Zalkin, H. Mutational

analysis of active site residues in pig heart aconitase, J. Biol.

Chem. 267, 7895–7903 (1992).

Metabolic Poisons

Gibble, G.W., Fluoroacetate toxicity, J. Chem. Educ. 50, 460–462

(1973).

Jones, D.E.H. and Ledingham, K.W.D., Arsenic in Napoleon’s

wallpaper, Nature 299, 626–627 (1982).

Lauble, H., Kennedy, M.C., Emptage, M.H., Beinert,H., and Stout,

C.D., The reaction of fluorocitrate with aconitase and the crys-

tal structure of the enzyme-inhibitor complex, Proc. Natl.

Acad. Sci. 93, 13699–13703 (1996).

Winslow, J.H., Darwin’s Victorian Malady, American Philosophi-

cal Society (1971).

Control Mechanisms

Hurley, J.H., Dean, A.M., Sohl, J.L., Koshland, D.E., Jr., and

Stroud, R.M., Regulation of an enzyme by phosphorylation at

the active site, Science 249, 1012–1016 (1990).

Owen, O.E., Kalhan, S.C., and Hanson, R.W., The key role of

anaplerosis and cataplerosis for citric acid cycle function, J.

Biol. Chem. 277, 30409–30412 (2002).

Reed, L.J., Damuni, Z., and Merryfield, M.L., Regulation of mam-

malian pyruvate and branched-chain ␣-keto-acid dehydroge-

nase complexes by phosphorylation and dephosphorylation,

Curr. Top. Cell. Regul. 27, 41–49 (1985).

Srere, P.A., Sherry, A.D., Malloy, C.R., and Sumegi, B., Chan-

nelling in the Krebs tricarboxylic acid cycle, in Agius, L. and

Sherratt, H.S.A. (Eds.), Channelling in Intermediary Metabo-

lism, pp. 201–217, Portland Press (1997).

Stroud, R.M., Mechanisms of biological control by phosphoryla-

tion, Curr. Opin. Struct. Biol. 1, 826–835 (1991). [Reviews,

among other things, the inactivation of isocitrate dehydroge-

nase by phosphorylation.]

Vélot, C., Mixon, M.B., Teige, M., and Srere, P.A., Model of a

quinary structure between Krebs TCA cycle enzymes: A model

for the metabolon, Biochemistry 36, 14271–14276 (1997).

Vélot, C. and Srere,P.A., Reversible transdominant inhibition of a

metabolic pathway. In vivo evidence of interaction between

two sequential tricarboxylic acid cycle enzymes in yeast. J.

Biol. Chem. 275, 12926–12933 (2000).

Problems 821

1. Trace the course of the radioactive label in [2-

C]glucose

through glycolysis and the citric acid cycle.At what point(s) in the

cycle will the radioactivity be released as

? How many turns

of the cycle will be required for complete conversion of the ra-

dioactivity to CO

? Repeat this problem for pyruvate that is

C-

labeled at its methyl group.

2. The reaction of glutathione reductase with an excess of

NADPH in the presence of arsenite yields a nonphysiological

four-electron reduced form of the enzyme. What is the chemical

nature of this catalytically inactive species?

3. Two-electron reduced dihydrolipoyl dehydrogenase (EH

but not the oxidized enzyme (E), reacts with iodoacetate

(ICH

COO

⫺

) to yield an inactive enzyme. Explain.

4. Given the following information, calculate the physiologi-

cal ⌬G of the isocitrate dehydrogenase reaction at 25°C and pH

7.0: [NAD

⫹

]/[NADH] ⫽ 8; [␣-ketoglutarate] ⫽ 0.1 mM; [isoci-

trate] ⫽ 0.02 mM; assume standard conditions for CO

(⌬G°¿ is

given in Table 21-2). Is this reaction a likely site for metabolic con-

trol? Explain.

5. The oxidation of acetyl-CoA to two molecules of CO

in-

volves the transfer of four electron pairs to redox coenzymes. In

which of the cycle’s reactions do these electron transfers occur?

Identify the redox coenzyme in each case. For each reaction, draw

the structural formulas of the reactants, intermediates, and

products and show, using curved arrows, how the electrons are

transferred.

6. The citrate synthase reaction has been proposed to proceed

via the formation of the enol(ate) form of acetyl-CoA. How, then,

would you account for the observation that

H is not incorporated

into acetyl-CoA when acetyl-CoA is incubated with citrate syn-

thase in

7. Malonate is a competitive inhibitor of succinate in the suc-

cinate dehydrogenase reaction. (a) Sketch the graphs that would

be obtained on plotting 1/v versus 1/[succinate] at three different

malonate concentrations. Label the lines for low, medium, and

high [malonate]. (b) Explain why increasing the oxaloacetate con-

centration in a cell can overcome malonate inhibition.

8. Krebs found that malonate inhibition of the citric acid cycle

could be overcome by raising the oxaloacetate concentration. Ex-

plain the mechanism of this process in light of your findings in

Problem 7.

*9. (2R,3R)-2-Fluorocitrate contains F in the pro-S carboxy-

methyl arm of citrate [note that the rules of organic nomenclature re-

quire that atom C2 in citrate (Fig. 21-20) be renumbered as C4 in

(2R,3R)-2-fluorocitrate]. This compound, but not its diastereomer, is

a potent inhibitor of aconitase. (a) Draw the aconitase-catalyzed re-

action pathway of (2R,3R)-2-fluorocitrate assuming it follows the

same reaction pathway as citrate (Fig. 21-20). (b) Aconitase, in fact,

does not catalyze the foregoing reaction with (2R,3R)-2-fluorocitrate

but, rather, yields the following tight-binding inhibitor:

Draw an alternative aconitase-catalyzed reaction that would gener-

ate this inhibitor. (c) Draw the aconitase-catalyzed reaction of

–

OOC

COO

–

COO

–

PROBLEMS

JWCL281_c21_789-822.qxd 3/17/10 11:34 AM Page 821

(2S,3R)-3-fluorocitrate,the diastereomer of (2R,3R)-2-fluorocitrate

(fluorocitrate containing F in the pro-R carboxymethyl arm of

citrate; here the atom numbering scheme is the same as that in

Fig. 21-20). Would a tight-binding inhibitor be formed?

10. Which of the following metabolites undergo net oxidation

by the citric acid cycle: (a) ␣-ketoglutarate, (b) succinate, (c) cit-

rate, and (d) acetyl-CoA?

11. Although there is no net synthesis of intermediates by the cit-

ric acid cycle, citric acid cycle intermediates are used in biosynthetic

reactions such as the synthesis of porphyrins from succinyl-CoA.

Write a reaction for the net synthesis of succinyl-CoA from pyruvate.

12. Oxaloacetate and ␣-ketoglutarate are precursors of the

amino acids aspartate and glutamate as well as being catalytic in-

termediates in the citric acid cycle. Describe the net synthesis of

␣-ketoglutarate from pyruvate in which no citric acid cycle inter-

mediates are depleted.

13. Lipoic acid is bound to enzymes that catalyze oxidative

decarboxylation of ␣-keto acids. (a) What is the chemical mode of

attachment of lipoic acid to enzymes? (b) Using chemical struc-

tures, show how lipoic acid participates in the oxidative decar-

boxylation of ␣-keto acids.

14. British anti-lewisite (BAL), which was designed to

counter the effects of the arsenical war gas lewisite, is useful in

treating arsenic poisoning. Explain.

15. The first organisms on Earth may have been chemoau-

totrophs in which the citric acid cycle operated in reverse to “fix”

atmospheric CO

in organic compounds. Complete a catalytic cy-

cle that begins with the hypothetical overall reaction succinate ⫹

2 CO

→ citrate.

16. Why is it advantageous for citrate, the product of Reaction 1

of the citric acid cycle, to inhibit phosphofructokinase, which

catalyzes the third reaction of glycolysis?

17. Anaplerotic reactions permit the citric acid cycle to supply

intermediates to biosynthetic pathways while maintaining the

proper levels of cycle intermediates.Write the equation for the net

synthesis of citrate from pyruvate.

18. Many amino acids are broken down to intermediates of

the citric acid cycle. (a) Why can’t these amino acid “remnants” be

directly oxidized to CO

by the citric acid cycle? (b) Explain why

amino acids that are broken down to pyruvate can be completely

oxidized by the citric acid cycle.

CH CH CHCl AsCl

LewisiteBritish anti-lewisite

(BAL)

822 Chapter 21. Citric Acid Cycle

JWCL281_c21_789-822.qxd 3/17/10 11:34 AM Page 822

823

]Low

ATP

ADP

+ P

–

––

–

]High

–

I III IV

CHAPTER 22

Electron Transport

and Oxidative

Phosphorylation

1 The Mitochondrion

A. Mitochondrial Anatomy

B. Mitochondrial Transport Systems

2 Electron Transport

A. Thermodynamics of Electron Transport

B. The Sequence of Electron Transport

C. Components of the Electron-Transport Chain

3 Oxidative Phosphorylation

A. Energy Coupling Hypotheses

B. Proton Gradient Generation

C. Mechanism of ATP Synthesis

D. Uncoupling of Oxidative Phosphorylation

4 Control of ATP Production

A. Control of Oxidative Phosphorylation

B. Coordinated Control of ATP Production

C. Physiological Implications of Aerobic versus

Anaerobic Metabolism

In 1789,Armand Séguin and Antoine Lavoisier (the father

of modern chemistry) wrote:

. . . in general, respiration is nothing but a slow combustion of

carbon and hydrogen, which is entirely similar to that which

occurs in a lamp or lighted candle, and that, from this point of

view, animals that respire are true combustible bodies that burn

and consume themselves.

Lavoisier had by this time demonstrated that living ani-

mals consume oxygen and generate carbon dioxide. It was

not until the early twentieth century, however,after the rise

of enzymology, that it was established, largely through the

work of Otto Warburg, that biological oxidations are cat-

alyzed by intracellular enzymes.As we have seen,glucose is

completely oxidized to CO

through the enzymatic reac-

tions of glycolysis and the citric acid cycle. In this chapter

we shall examine the fate of the electrons that are removed

from glucose by this oxidation process.

The complete oxidation of glucose by molecular oxygen

is described by the following redox equation:

To see more clearly the transfer of electrons, let us break

this equation down into two half-reactions. In the first half-

reaction the glucose carbon atoms are oxidized:

¢G°¿ ⫽⫺2823 kJ ⴢ mol

⫺1

⫹ 6O

6CO

⫹ 6H

and in the second, molecular oxygen is reduced:

In living systems, the electron-transfer process connecting

these half-reactions occurs through a multistep pathway that

harnesses the liberated free energy to form ATP.

The 12 electron pairs involved in glucose oxidation are

not transferred directly to O

. Rather, as we have seen, they

are transferred to the coenzymes NAD

⫹

and FAD to form 10

NADH and 2 FADH

(Fig. 22-1) in the reactions catalyzed

by the glycolytic enzyme glyceraldehyde-3-phosphate de-

hydrogenase (Section 17-2F), pyruvate dehydrogenase

(Section 21-2A), and the citric acid cycle enzymes isocitrate

dehydrogenase, ␣-ketoglutarate dehydrogenase, succinate

dehydrogenase (the only FAD reduction), and malate de-

hydrogenase (Section 21-3). The electrons then pass into the

electron-transport chain (alternatively, the respiratory

chain), where, through reoxidation of NADH and FADH

they participate in the sequential oxidation–reduction of over

10 redox centers before reducing O

to H

O. In this process,

protons are expelled from the mitochondrion. The free en-

ergy stored in the resulting pH gradient drives the synthesis

of ATP from ADP and P

through oxidative phosphoryla-

tion. Reoxidation of each NADH results in the synthesis of

⬃2.5 ATP,and reoxidation of FADH

yields ⬃1.5 ATP for a

total of ⬃32 ATP for each glucose completely oxidized to

and H

O (including the 2 ATP made in glycolysis and

the 2 ATP made in the citric acid cycle).

In this chapter we explore the mechanisms of electron

transport and oxidative phosphorylation and their regula-

tion.We begin with a discussion of mitochondrial structure

and transport systems.

1 THE MITOCHONDRION

The mitochondrion (Section 1-2Ac) is the site of eukaryotic

oxidative metabolism. It contains, as Albert Lehninger and

Eugene Kennedy demonstrated in 1948, the enzymes that

mediate this process, including pyruvate dehydrogenase,

the citric acid cycle enzymes, the enzymes catalyzing fatty

acid oxidation (Section 25-2C), and the enzymes and redox

proteins involved in electron transport and oxidative phos-

phorylation. It is therefore with good reason that the mito-

chondrion is described as the cell’s “power plant.”

⫹ 24H

⫹

⫹ 24e

⫺

12H

⫹ 6H

6CO

⫹ 24H

⫹

⫹ 24e

⫺

JWCL281_c22_823-870.qxd 6/8/10 9:18 AM Page 823

A. Mitochondrial Anatomy

Mitochondria vary considerably in size and shape depend-

ing on their source and metabolic state. They are typically

ellipsoids ⬃0.5 ␮m in diameter and 1 ␮m in length (about

the size of a bacterium; Fig. 22-2). The mitochondrion is

bounded by a smooth outer membrane and contains an ex-

tensively invaginated inner membrane. The number of in-

vaginations, called cristae, varies with the respiratory activ-

ity of the particular type of cell.This is because the proteins

mediating electron transport and oxidative phosphoryla-

tion are bound to the inner mitochondrial membrane, so

that the respiration rate varies with membrane surface

area. Liver, for example, which has a relatively low respira-

tion rate, contains mitochondria with relatively few cristae,

whereas those of heart muscle contain many. Nevertheless,

the aggregate area of the inner mitochondrial membranes

in a liver cell is ⬃15-fold greater than that of its plasma

membrane.

The inner mitochondrial compartment consists of a gel-

like substance of ⬍50% water, named the matrix, which

contains remarkably high concentrations of the soluble en-

zymes of oxidative metabolism (e.g., citric acid cycle en-

zymes), as well as substrates, nucleotide cofactors, and inor-

ganic ions. The matrix also contains the mitochondrial

genetic machinery—DNA, RNA, and ribosomes—that in

mammals expresses only 13 mitochondrial inner mem-

brane proteins together with 22 tRNAs and two ribosomal

RNAs.

a. The Inner Mitochondrial Membrane and Cristae

Compartmentalize Metabolic Functions

The outer mitochondrial membrane contains porin, a

protein that forms nonspecific pores that permit free diffu-

sion of up to 10-kD molecules (the X-ray structures of bac-

terial porins are discussed in Sections 12-3Ad and 20-2D).

The inner membrane, which is ⬃75% protein by mass, is

considerably richer in proteins than the outer membrane

(Fig. 22-3). It is freely permeable only to O

,CO

, and H

and contains, in addition to respiratory chain proteins, nu-

merous transport proteins that control the passage of

metabolites such as ATP, ADP, pyruvate, Ca

2⫹

, and phos-

phate (see below). This controlled impermeability of the in-

ner mitochondrial membrane to most ions, metabolites, and

low molecular mass compounds permits the generation of

ionic gradients across this barrier and results in the compart-

mentalization of metabolic functions between cytosol and

mitochondria.

Two-dimensional electron micrographs of mitochondria

such as Fig. 22-2a suggest that cristae resemble baffles and

that the intercristal spaces communicate freely with the mi-

tochondrion’s intermembrane space, as Fig. 22-2b implies.

However, electron microscopy–based three-dimensional

image reconstruction methods have revealed that cristae

can vary in shape from simple tubular entities to more

complicated lamellar assemblies that merge with the inner

membrane via narrow tubular structures (Fig. 22-4). Evi-

dently, cristae form microcompartments that restrict the

diffusion of substrates and ions between the intercristal

and intermembrane spaces. This has important functional

implications because it would result in a locally greater pH

gradient across cristal membranes than across inner mem-

branes that are not part of cristae, thereby significantly

influencing the rate of oxidative phosphorylation (Sec-

tion 22-3).

B. Mitochondrial Transport Systems

The inner mitochondrial membrane is impermeable to

most hydrophilic substances. It must therefore contain spe-

cific transport systems to permit the following processes:

824 Chapter 22. Electron Transport and Oxidative Phosphorylation

Figure 22-1 The sites of electron transfer that form NADH

and FADH

in glycolysis and the citric acid cycle.

2 NAD

2 NADH

2 NAD

2 FAD

2 FADH

2 NADH

2 NAD

2 NADH

2 NAD

Glucose-6-phosphate

2 Glyceraldehyde-3-phosphate

2 NAD

2 NADH

glyceraldehyde-

3-phosphate

dehydrogenase

2 Pyruvate

2 1,3-Bisphosphoglycerate

pyruvate

dehydrogenase

2 Acetyl-CoA

2 Oxaloacetate

2 Malate

2 Fumarate

2 Succinate

2 Succinyl-CoA

2 α-Ketoglutarate

2 Isocitrate

2 Citrate

malate

dehydrogenase

succinate

dehydrogenase

isocitrate

dehydrogenase

α-ketoglutarate

dehydrogenase

Glucose

Citric acid

cycle

Glycolysis

JWCL281_c22_823-870.qxd 3/19/10 10:59 AM Page 824

Outer membrane

Inner membrane

Cristae

Matrix

Intermembrane space

(b)(a)

Rough endoplasmic reticulum

1. Glycolytically produced cytosolic NADH must gain

access to the electron-transport chain for aerobic oxida-

tion.

2. Mitochondrially produced metabolites such as ox-

aloacetate and acetyl-CoA, the respective precursors for

cytosolic glucose and fatty acid biosynthesis, must reach

their metabolic destinations.

3. Mitochondrially produced ATP must reach the cy-

tosol, where most ATP-utilizing reactions take place,

whereas ADP and P

, the substrates for oxidative phospho-

rylation, must enter the mitochondrion.

Section 22-1. The Mitochondrion 825

Figure 22-2 Mitochondria. (a) An electron micrograph of an animal mitochondrion.

[K.R. Porter/Photo Researchers, Inc.] (b) Cutaway diagram of a mitochondrion.

Figure 22-4 Electron microscopy–based three-dimensional

image reconstruction of a rat liver mitochondrion. The outer

membrane (OM) is red, the inner membrane (IM) is yellow, and

the cristae (C) are green.The arrowheads point to tubular

regions of the cristae that connect them to the inner membrane

and to each other. [Courtesy of Carmen Mannella,Wadsworth

Center,Albany, New York.]

Figure 22-3 Freeze-fracture and freeze-etch electron

micrographs of the inner and outer mitochondrial membranes.

The inner membrane contains about twice the density of

embedded particles as does the outer membrane. [Courtesy of

Lester Packer, University of California at Berkeley.]

JWCL281_c22_823-870.qxd 3/19/10 10:59 AM Page 825

We have already studied the ADP–ATP translocator and

its dependence on ⌬⌿, the electric potential difference

across the mitochondrial membrane (Section 20-4C). The

export mechanisms of oxaloacetate and acetyl-CoA from

the mitochondrion are, respectively, discussed in Sections

23-1Ag and 25-4D. In the remainder of this section we ex-

amine the mitochondrial transport systems for P

and Ca

2⫹

and the shuttle systems for NADH.

a. P

Transport

ATP is generated from ADP ⫹ P

in the mitochondrion

but is utilized in the cytosol.The P

produced is returned to

the mitochondrion by the phosphate carrier, an electroneu-

tral P

–H

⫹

symport that is driven by ⌬pH. The proton that

accompanies the P

into the mitochondrion had, in effect,

been previously expelled from the mitochondrion by the

redox-driven pumps of the electron-transport chain (Sec-

tion 22-3B). The electrochemical potential gradient gener-

ated by these proton pumps is therefore responsible for

maintaining high mitochondrial ADP and P

concentra-

tions in addition to providing the free energy for ATP

synthesis.

b. Ca

2⫹

Transport

Since Ca

2⫹

, like cAMP, functions as a second messenger

(Section 18-3Ce), its concentrations in the various cellular

compartments must be precisely controlled.The mitochon-

drion, endoplasmic reticulum, and extracellular spaces act

as Ca

2⫹

storage tanks.We studied the Ca

2⫹

–ATPases of the

plasma membrane, endoplasmic reticulum, and sarcoplas-

mic reticulum in Section 20-3B. Here we consider the mito-

chondrial Ca

2⫹

transport systems.

Mitochondrial inner membrane systems separately me-

diate the influx and the efflux of Ca

2⫹

(Fig. 22-5). The Ca

2⫹

influx is driven by the inner mitochondrial membrane’s

membrane potential (⌬°, inside negative), which attracts

positively charged ions. The rate of influx varies with the

external [Ca

2⫹

] because the K

for Ca

2⫹

transport by this

system is greater than the cytosolic Ca

2⫹

concentration.

In heart, brain, and skeletal muscle mitochondria espe-

cially, Ca

2⫹

efflux is independently driven by the Na

⫹

gra-

dient across the inner mitochondrial membrane. Ca

2⫹

exits

the matrix only in exchange for Na

⫹

, so that this system is an

antiport. This exchange process normally operates at its

maximal velocity. Mitochondria (as well as the endoplasmic

and sarcoplasmic reticulum) therefore can act as a “buffer”

for cytosolic Ca

2⫹

(Fig. 22-6): If cytosolic [Ca

2⫹

] rises, the

rate of mitochondrial Ca

2⫹

influx increases while that of

2⫹

efflux remains constant, causing the mitochondrial

[Ca

2⫹

] to increase while the cytosolic [Ca

2⫹

] decreases to

its original level (its set point). Conversely, a decrease in cy-

tosolic [Ca

2⫹

] reduces the influx rate, causing net efflux of

[Ca

2⫹

] and an increase of cytosolic [Ca

2⫹

] back to the set

point.

Oxidation carried out by the citric acid cycle in the mito-

chondrial matrix is controlled by the matrix [Ca

2⫹

] (Section

21-4c). It is interesting to note, therefore, that in response

to increases in cytosolic [Ca

2⫹

] caused by increased muscle

activity, the matrix [Ca

2⫹

] increases, thereby activating

the enzymes of the citric acid cycle. This leads to an in-

crease in [NADH], whose reoxidation by oxidative phos-

phorylation (as we study in this chapter) generates the

ATP needed for this increased muscle activity.

826 Chapter 22. Electron Transport and Oxidative Phosphorylation

Figure 22-5 The two mitochondrial Ca

2ⴙ

transport systems.

System 1 mediates Ca

2⫹

influx to the matrix in response to the

membrane potential (negative inside). System 2 mediates Ca

2⫹

efflux in exchange for Na

⫹

(efflux)

(influx)

Matrix

–

2 1

Electron transport

Intermembrane

space

0.5 1.0 2.0

Cytosolic concentration of Ca

(μM)

Activity of uptake or efflux pathway

(nmol Ca

•

min

–1

•

–1

)

Set point

Efflux pathway

Uptake pathway

Net uptake

Net efflux

Figure 22-6 The regulation of cytosolic [Ca

2ⴙ

]. The efflux

pathway operates at a constant rate independent of [Ca

2⫹

whereas the activity of the influx pathway varies with [Ca

2⫹

].At

the set point, the activities of the two pathways are equal and

there is no net Ca

2⫹

flux.An increase in cytosolic [Ca

2⫹

] results

in net mitochondrial influx, and a decrease in cytosolic [Ca

2⫹

]

results in net mitochondrial efflux. Both effects lead to the

restoration of the cytosolic [Ca

2⫹

]. [After Nicholls, D., Trends

Biochem. Sci. 6, 37 (1981).]

JWCL281_c22_823-870.qxd 3/19/10 10:59 AM Page 826

c. Cytoplasmic Shuttle Systems “Transport” NADH

Across the Inner Mitochondrial Membrane

Although most of the NADH generated by glucose oxi-

dation is formed in the mitochondrial matrix via the citric

acid cycle, that generated by glycolysis occurs in the cy-

tosol. Yet the inner mitochondrial membrane lacks an

NADH transport protein. Only the electrons from cytosolic

NADH are transported into the mitochondrion by one of

several ingenious “shuttle” systems. In the malate–aspartate

shuttle (Fig. 22-7), which functions in heart, liver, and kid-

ney, mitochondrial NAD

⫹

is reduced by cytosolic NADH

through the intermediate reduction and subsequent regen-

eration of oxaloacetate. This process occurs in two phases

of three reactions each:

Phase A (transport of electrons into the matrix):

1. In the cytosol, NADH reduces oxaloacetate to yield

NAD

⫹

and malate in a reaction catalyzed by cytosolic

malate dehydrogenase.

2. The malate–␣-ketoglutarate carrier transports

malate from the cytosol to the mitochondrial matrix in ex-

change for ␣-ketoglutarate from the matrix.

3. In the matrix, NAD

⫹

reoxidizes malate to yield

NADH and oxaloacetate in a reaction catalyzed by mito-

chondrial malate dehydrogenase (Section 21-3H).

Phase B (regeneration of cytosolic oxaloacetate):

4. In the matrix, a transaminase (Section 26-1A) con-

verts oxaloacetate to aspartate with the concomitant con-

version of glutamate to ␣-ketoglutarate.

5. The glutamate–aspartate carrier transports aspartate

from the matrix to the cytosol in exchange for cytosolic

glutamate.

6. In the cytosol, a transaminase converts aspartate

to oxaloacetate with the concomitant conversion of

␣-ketoglutarate to glutamate.

The electrons of cytosolic NADH are thereby transferred

to mitochondrial NAD

⫹

to form NADH, which is subject

to reoxidation via the electron-transport chain. The

malate–aspartate shuttle yields ⬃2.5 ATPs for every cytoso-

lic NADH. Note, however, that every NADH that enters

the matrix is accompanied by a proton which, as we shall

Section 22-1. The Mitochondrion 827

Aspartate

COO

–

COO

–

α -Ketoglutarate

COO

–

COO

–

Cytosol MatrixInner

mitochondrial

membrane

malate–

-ketoglutarate

carrier

NADH + H

NAD

malate dehydrogenase

COO

–

COO

–

HHO

Malate

COO

–

COO

–

Oxaloacetate

Aspartate

COO

–

COO

–

Glutamate

COO

–

N CH

COO

–

aspartate

aminotransferase

glutamate–

aspartate carrier

α -Ketoglutarate

COO

–

COO

–

NAD

malate dehydrogenase

COO

–

COO

–

HHO

Malate

COO

–

COO

–

Oxaloacetate

Glutamate

COO

–

N CH

COO

–

aspartate

aminotransferase

+ NADH

Figure 22-7 The malate–aspartate shuttle. The electrons of

cytosolic NADH are transported to mitochondrial NADH

(shown in red as hydride transfers) in Steps 1 to 3. Steps 4 to 6

then serve to regenerate cytosolic oxaloacetate.

JWCL281_c22_823-870.qxd 3/19/10 10:59 AM Page 827

see (Section 22-3C), would otherwise be used to generate

⬃0.3 ATP. Consequently, every cytosolic NADH that is

translocated to the matrix by the malate–aspartate shuttle

yields ⬃2.2 ATPs.

The glycerophosphate shuttle (Fig. 22-8), which is sim-

pler but less energy efficient than the malate–aspartate

shuttle, occurs in brain and skeletal muscle and is particu-

larly prominent in insect flight muscle (the tissue with the

largest known sustained power output—about the same

power-to-weight ratio as a small automobile engine). In it,

glycerol-3-phosphate dehydrogenase catalyzes the oxida-

tion of cytosolic NADH by dihydroxyacetone phosphate to

yield NAD

⫹

, which reenters glycolysis.The electrons of the

resulting glycerol-3-phosphate are transferred to flavopro-

tein dehydrogenase to form FADH

.This enzyme, which is

situated on the inner mitochondrial membrane’s outer sur-

face, supplies electrons to the electron-transport chain in a

manner similar to that of succinate dehydrogenase (Sec-

tion 22–2C2). The glycerophosphate shuttle therefore results

in the synthesis of ⬃1.5 ATPs for every cytoplasmic NADH

reoxidized, ⬃0.7 ATP less than the malate–aspartate shuttle.

However, the advantage of the glycerophosphate shuttle is

that, being essentially irreversible, it operates efficiently

even when the cytoplasmic NADH concentration is low

relative to that of NAD

⫹

, as occurs in rapidly metabolizing

tissues. In contrast, the malate–aspartate shuttle is re-

versible and hence is driven by concentration gradients.

2 ELECTRON TRANSPORT

In the electron-transport process, the free energy of electron

transfer from NADH and FADH

to O

via protein-bound

redox centers is coupled to ATP synthesis. We begin our

study of this process by considering its thermodynamics.

We then examine the path of electrons through the redox

centers of the system and discuss the experiments used to

unravel this pathway. Finally, we study the four complexes

that make up the electron-transport chain. In the next sec-

tion we discuss how the free energy harvested by the

electron-transport process is coupled to ATP synthesis.

A. Thermodynamics of Electron Transport

We can estimate the thermodynamic efficiency of electron

transport through knowledge of standard reduction poten-

tials. As we have seen in our thermodynamic considera-

tions of oxidation–reduction reactions (Section 16-5), an

oxidized substrate’s affinity for electrons increases with its

standard reduction potential, [the voltage generated by

the reaction of the half-cell under standard biochemical

conditions (1M reactants and products with [H

⫹

] defined

as 1 at pH 7) relative to the standard hydrogen electrode;

Table 16-4 lists the standard reduction potentials of several

half-reactions of biochemical interest].The standard reduc-

tion potential difference, for a redox reaction involv-

ing any two half-reactions is therefore expressed:

a. NADH Oxidation Is a Highly Exergonic Reaction

The half-reactions for O

oxidation of NADH are

(Table 16-4)

and

Since the O

O half-reaction has the greater standard

reduction potential and therefore the higher electron affin-

ity, the NADH half-reaction is reversed, so that NADH is

the electron donor in this couple and O

the electron ac-

ceptor.The overall reaction is

so that

The standard free energy change for the reaction can then

be calculated from Eq. [16.7]:

where f, the Faraday constant, is 96,485 C ⴢ mol

–1

of elec-

trons and n is the number of electrons transferred per

¢G°¿ ⫽⫺nf ¢e°¿

¢e°¿ ⫽ 0.815 ⫺ (⫺0.315) ⫽ 1.130 V

⫹ NADH ⫹ H

⫹

Δ H

O ⫹ NAD

⫹

⫹ 2H

⫹

⫹ 2e

⫺

Δ H

e°¿ ⫽ 0.815 V

NAD

⫹

⫹ H

⫹

⫹ 2e

⫺

Δ NADH

e°¿ ⫽⫺0.315 V

¢e°¿ ⫽ e°¿

⫺

acceptor)

⫺ e°¿

⫺

donor)

¢e°¿,

e°¿

828 Chapter 22. Electron Transport and Oxidative Phosphorylation

Figure 22-8 The glycerophosphate shuttle. The electrons of

cytosolic NADH are transported to the mitochondrial electron-

transport chain in three steps (shown in red as hydride transfers):

(1) Cytosolic oxidation of NADH by dihydroxyacetone

phosphate catalyzed by glycerol-3-phosphate dehydrogenase.

(2) Oxidation of glycerol-3-phosphate by flavoprotein

dehydrogenase with the reduction of FAD to FADH

(3) Reoxidation of FADH

with the passage of electrons into

the electron-transport chain. Note that the glycerophosphate

shuttle is not a membrane transport system.

OPO

–

OPO

2–

Inner

mitochondrial

membrane

Electron-

transport

chain

f lavoprotein

dehydrogenase

Matrix

Cytosol

FAD

FADH

–

Dihydroxyacetone

phosphate

glycerol-3-phosphate

dehydrogenase

NAD

NADH

CHO

Glycerol-3-phosphate

JWCL281_c22_823-870.qxd 6/8/10 9:18 AM Page 828

mole of reactants. Thus, since 1 V ⫽ 1 J ⴢ C

–1

, for NADH

oxidation:

In other words, the oxidation of 1 mol of NADH by O

(the

transfer of 2e

⫺

) under standard biochemical conditions is

associated with the release of 218 kJ of free energy.

b. Electron Transport Is Thermodynamically Efficient

The standard free energy required to synthesize 1 mol

of ATP from ADP ⫹ P

is 30.5 kJ. The standard free energy

of oxidation of NADH by O

, if coupled to ATP synthesis,

is therefore sufficient to drive the formation of several

moles of ATP. This coupling, as we shall see, is achieved by

an electron-transport chain in which electrons are passed

through three protein complexes containing redox centers

with progressively greater affinity for electrons (increasing

standard reduction potentials) instead of directly to O

This allows the large overall free energy change to be broken

up into three smaller packets, each of which is coupled with

ATP synthesis in a process called oxidative phosphoryla-

tion. Oxidation of 1 NADH therefore results in the synthe-

sis of ⬃2.5 ATP. (Oxidation of FADH

, whose entrance

⫽⫺218 kJ ⴢ mol

⫺1

¢G°¿ ⫽⫺2

mol e

⫺

mol reactant

⫻ 96,485

mol e

⫺

⫻ 1.13 J ⴢ C

⫺1

into the electron-transport chain is regulated by a fourth

protein complex, is similarly coupled to the synthesis of

⬃1.5 ATP.) The thermodynamic efficiency of oxidative

phosphorylation is therefore 2.5 ⫻ 30.5 kJ ⴢ mol

⫺1

⫻

100/218 kJ ⴢ mol

⫺1

⫽ 35% under standard biochemical

conditions. However, under physiological conditions in

active mitochondria (where the reactant and product con-

centrations as well as the pH deviate from standard condi-

tions), this thermodynamic efficiency is thought to be

⬃70%. In comparison, the energy efficiency of a typical

automobile engine is ⬍30%.

B. The Sequence of Electron Transport

See Guided Exploration 19: Electron transport and oxidative phos-

phorylation overview

The free energy necessary to generate ATP

is extracted from the oxidation of NADH and FADH

by the

electron-transport chain, a series of four protein complexes

through which electrons pass from lower to higher standard

reduction potentials (Fig. 22-9). Electrons are carried from

Complexes I and II to Complex III by coenzyme Q (CoQ

or ubiquinone; so named because of its ubiquity in respir-

ing organisms), and from Complex III to Complex IV by

the peripheral membrane protein cytochrome c (Sections

7-3B and 9-6A).

Section 22-2. Electron Transport 829

O2H

+ O

NADH NAD

(–0.315 V)

–

Succinate

Fumarate

Complex

FADH

CoQ

(+0.031 V)

(+0.045 V)

Cytochrome c (+0.235 V)

(+0.815 V)

–

+0.8

+0.6

+0.4

+0.2

–0.2

–0.4

°′(V)

Complex I

Δ °′ = 0.360 V

(ΔG°′ = –69.5 kJ · mol

–1

)

Complex III

Δ °′ = 0.190 V

(ΔG°′ = –36.7 kJ · mol

–1

)

Complex IV

Δ °′ = 0.580 V

(ΔG°′ = –112 kJ · mol

–1

)

ADP + P

–

ATP

ADP + P

Antimycin

ATP

ADP + P

Rotenone or

Amytal

ATP

Figure 22-9 The mitochondrial electron-transport chain. The

standard reduction potentials of its most mobile components

(green) are indicated, as are the points where sufficient free

energy is harvested to synthesize ATP (blue) and the sites of action

of several respiratory inhibitors (red). (Note that Complexes I,

III, and IV do not directly synthesize ATP but, rather, sequester

the free energy necessary to do so by pumping protons outside

the mitochondrion to form a proton gradient; Section 22-3.)

JWCL281_c22_823-870.qxd 3/19/10 10:59 AM Page 829

Top view

Platinum cathode

–

Silver anode

Bubble

escape

slit

Glass

sample

chamber

Sample

KCl

Bubble slit

Magnetic stirrer

-Permeable Teflon membrane

Lucite holder

Epoxy plug

Side view

Complex I catalyzes oxidation of NADH by CoQ:

Complex III catalyzes oxidation of CoQ (reduced) by cy-

tochrome c:

Complex IV catalyzes oxidation of cytochrome c (reduced)

by O

, the terminal electron acceptor of the electron-

transport process:

The changes in standard reduction potential of an electron

pair as it successively traverses Complexes I, III, and IV

correspond,at each stage,to sufficient free energy to power

the synthesis of nearly one ATP molecule.

Complex II catalyzes the oxidation of FADH

by CoQ.

This redox reaction does not release sufficient free energy to

synthesize ATP; it functions only to inject the electrons from

FADH

into the electron-transport chain.

¢G°¿ ⫽⫺16.4 kJ ⴢ mol

⫺1

¢e°¿ ⫽ 0.085 V

FADH

⫹ CoQ (oxidized)

FAD ⫹ CoQ (reduced)

¢G°¿ ⫽⫺112 kJ ⴢ mol

⫺1

¢e°¿ ⫽ 0.580 V

2 cytochrome c (oxidized) ⫹ H

2 cytochrome c (reduced)

⫹

¢G°¿ ⫽⫺36.7 kJ ⴢ mol

⫺1

¢e°¿ ⫽ 0.190 V

CoQ (oxidized) ⫹ 2 cytochrome c (reduced)

CoQ (reduced) ⫹ 2 cytochrome c (oxidized)

¢G°¿ ⫽⫺69.5 kJ ⴢ mol

⫺1

¢e°¿ ⫽ 0.360 V

NAD

⫹

⫹ CoQ (reduced)

NADH ⫹ CoQ (oxidized)

a. The Workings of the Electron-Transport Chain

Have Been Elucidated through the Use of Inhibitors

Our understanding of the sequence of events in elec-

tron transport is largely based on the use of specific in-

hibitors. This sequence has been corroborated by meas-

urements of the standard reduction potentials of the redox

components of each of the complexes as well as by deter-

mining the stoichiometry of electron transport and the

coupled ATP synthesis.

The rate at which O

is consumed by a suspension of mi-

tochondria is a sensitive measure of the functioning of the

electron-transport chain. It is conveniently measured with

an oxygen electrode (Fig. 22-10). Compounds that inhibit

electron transport, as judged by their effect on O

disap-

pearance in such an experimental system, have been

invaluable experimental probes in tracing the path of

electrons through the electron-transport chain and in

determining the points of entry of electrons from various

substrates. Among the most useful such substances are

rotenone (a plant toxin used by Amazonian Indians to poi-

son fish and which is also used as an insecticide), amytal (a

barbiturate), antimycin (an antibiotic), and cyanide:

The following experiment illustrates the use of these

inhibitors:

A buffered solution containing excess ADP and P

equilibrated in the reaction vessel of an oxygen electrode.

OCH

(CH

)

CH(CH

)

CH(CH

)

Rotenone

–

Amytal Cyanide

CHO

Antimycin

830 Chapter 22. Electron Transport and Oxidative Phosphorylation

Figure 22-10 The oxygen electrode. This electrode consists of

an Ag/AgCl reference electrode and a Pt electrode, both

immersed in a KCl solution and in contact with the sample

chamber through an O

-permeable Teflon membrane. O

reduced to H

O at the Pt electrode, thereby generating a voltage

with respect to the Ag/AgCl electrode that is proportional to the

concentration in the sealed sample chamber. [After Cooper,

T.G., The Tools of Biochemistry, p. 69, Wiley (1977).]

JWCL281_c22_823-870.qxd 6/8/10 9:19 AM Page 830