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FAD[2H] in α-ketoglutarate dehydrogenase donates its electrons to NAD+, whereas the FAD[2H]

in succinate dehydrogenase donates its electrons directly to the electron-transfer chain). Answer D

is incorrect because both succinate dehydrogenase and aconitase have Fe–S centers. Answer E is

incorrect because succinate dehydrogenase is not regulated by a kinase. Kinases regulate enzymes

by phosphorylation (e.g., the regulation of pyruvate dehydrogenase occurs through reversible

phosphorylation).

4.The answer is E. NADH decreases during exercise in order to generate energy for the exercise (if

it were increased, it would inhibit the cycle and slow it down); thus, the NADH/NAD+ ratio is

decreased, and the lack of NADH activates flux through isocitrate dehydrogenase, α-ketoglutarate

dehydrogenase, and malate dehydrogenase. Isocitrate dehydrogenase is inhibited by NADH, so

answer A is not correct. Fumarase is not regulated; thus, answer B is incorrect. The four-carbon

intermediates of the cycle are regenerated during each turn of the cycle, so their concentrations do

not decrease (thus, C is incorrect). Product inhibition of citrate synthase would slow the cycle and

not generate more energy (hence, D is incorrect).

5.The answer is D. Pantothenate is the vitamin precursor of coenzyme A. Niacin is the vitamin

precursor of NAD, and riboflavin is the vitamin precursor of FAD and FMN. Vitamins A and C

are used with only minor modifications, if any, and are not involved in any TCA cycle reactions.

6.The answer is B. The net equation of the TCA cycle, in terms of carbon atoms, is that acetyl-CoA

is converted to two molecules of CO2. The eight electrons associated with the two carbon atoms

of acetyl-CoA are removed and placed in three molecules of NADH and one molecule of FAD(2H). The TCA cycle does not generate reduced cofactors from pyruvate, lactate, oxaloacetate, or phosphoenolpyruvate. Those compounds would need to be converted to acetylCoA in order for the cycle to generate the reduced cofactors.

7.The answer is C. With hypoxia, the TCA cycle would slow down because of the accumulation of

NADH (which cannot donate electrons to oxygen) caused by the lack of oxygen. The high NADH

inhibits pyruvate dehydrogenase, so pyruvate will accumulate, and the high levels of pyruvate will

block lactate from being converted to pyruvate (the lactate dehydrogenase reaction), leading to

lactate accumulation. Because the operation of the TCA cycle is greatly reduced, citrate and

succinyl-CoA will not be produced, so they will not accumulate. The enzymes of the TCA cycle

are located in the mitochondria, not in the cytoplasm.

8.The answer is D. GTP is generated from substrate-level phosphorylation during the TCA cycle

(not ATP). Mitochondrial ATP is generated by oxidative phosphorylation, using the electrons from

the electron carriers NADH and FAD(2H). The TCA cycle requires some B vitamins but not

vitamins C or D. One molecule of acetyl-CoA (two carbons) produces 2 CO2, 3 NADH, 1FAD(2H), and 1 GTP (not ATP).

9.The answer is A. A deficiency of the E1 subunit of pyruvate dehydrogenase would decrease

conversion of pyruvate to acetyl-CoA, leading to an accumulation of pyruvate. Pyruvate is

converted to lactate to allow glycolysis to continue to generate ATP from substrate-level

phosphorylation. The pyruvate to lactate conversion regenerates the NAD+, which is required for

glycolysis to proceed. Cells of the brain have a high ATP requirement and are highly dependent on

glycolysis and pyruvate oxidation in the TCA cycle to meet this demand for ATP. Without pyruvate

oxidation in the TCA cycle, glycolysis will try to produce ATP as fast as possible (because of an

increase of AMP levels, which activates PFK-1); however, the amount of ATP produced by

glycolysis alone is not sufficient to meet the brain’s needs. Thus, the ATP/ADP ratio actually

decreases. Even though the brain cells are low in ATP levels, the decreased production of acetylCoA from pyruvate will not provide sufficient substrate to substantially increase the activity of the

electron-transfer chain in brain cells. Fatty acids do not cross the blood–brain barrier, so ketone

body oxidation would be required to increase acetyl-CoA levels within the mitochondria to allow

rapid functioning of the TCA cycle. Acetyl-CoA is not produced from glucose when pyruvate

dehydrogenase is defective, and acetyl-CoA cannot be exported to the circulation. 10. The answer is A. When pyruvate carboxylase is deficient, pyruvate cannot be converted to

oxaloacetate, thereby reducing the ability to replenish TCA cycle intermediates as they are being

used for other pathways. As oxaloacetate levels decrease, acetyl-CoA cannot be converted to

citrate, and acetyl-CoA will accumulate within the mitochondria. The elevated acetyl-CoA

inhibits pyruvate dehydrogenase, which, coupled with the reduced activity of pyruvate

carboxylase, leads to pyruvate accumulation in the cytoplasm. The increased pyruvate is then

converted to lactic acid, leading to lactic acidemia. Pyruvate is not an allosteric activator of

lactate dehydrogenase. Because the TCA cycle is slowed owing to lack of oxaloacetate, NADH is

not accumulating in the mitochondrial matrix, nor is ATP. Pyruvate is not an activator of the

pyruvate dehydrogenase complex (NAD+ and free coenzyme A are the primary activators, along

with ADP).24

Oxidative Phosphorylation and Mitochondrial Function

For additional ancillary materials related to this chapter, please visit thePoint. Energy from fuel oxidation is converted to the high-energy phosphate bonds of adenosine triphosphate

(ATP) by the process of oxidative phosphorylation. Most of the energy from oxidation of fuels in the

tricarboxylic acid (TCA) cycle and other pathways is conserved in the form of the reduced electronaccepting coenzymes, nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide

(FAD[2H]). The electron-transport chain (ETC) oxidizes NADH and FAD(2H) and donates the

electrons to O2, which is reduced to H2O (Fig. 24.1). Energy from reduction of O2 is used for

phosphorylation of adenosine diphosphate (ADP) to ATP by ATP synthase (F0F1-ATPase). The net yield

of oxidative phosphorylation is approximately 2.5 mol of ATP per mole of NADH oxidized, or 1.5 mol of

ATP per mole of FAD(2H) oxidized.

Chemiosmotic Model of Adenosine Triphosphate Synthesis. The chemiosmotic model

explains howenergy from transport of electrons to O2 is transformed into the high-energy phosphate bond of ATP (see

Fig. 24.1). Basically, the ETC contains three large protein complexes (I, III, and IV) that span the inner

mitochondrial membrane. As electrons pass through these complexes in a series of oxidation–reduction

reactions, protons are transferred from the mitochondrial matrix to the cytosolic side of the inner

mitochondrial membrane. The pumping of protons generates an electrochemical gradient (Δp) across the

membrane composed of the membrane potential and the proton gradient. ATP synthase contains a proton

pore that spans the inner mitochondrial membrane and a catalytic headpiece that protrudes into the matrix.

As protons are driven into the matrix through the pore, they change the conformation of the headpiece,

which releases ATP from one site and catalyzes formation of ATP from ADP and inorganic phosphate (Pi)

at another site.

Deficiencies of Electron Transport. In cells, complete transfer of electrons from NADH and FAD(2H)

through the chain to O2 is necessary for ATP generation. Impaired transfer through any complex can have

pathologic consequences. Fatigue can result from iron-deficiency anemia, which decreases Fe for Fe–S

centers and cytochromes. Cytochrome c1 oxidase, which contains the O2-binding site, is inhibited by

cyanide. Mitochondrial DNA (mtDNA), which is maternally inherited, encodes some of the subunits of

the ETC complexes and ATP synthase. OXPHOS diseases are caused by mutations in nuclear DNA or

mtDNA that decrease mitochondrial capacity for oxidative phosphorylation. Regulation of Oxidative Phosphorylation. The rate of the ETC is coupled to the rate of ATP synthesis

by the transmembrane electrochemical gradient. As ATP is used for energy-requiring processes and ADP

levels increase, proton influx through the ATP synthase pore generates more ATP, and the ETC responds

to restore Δp. In uncoupling, protons return to the matrix by a mechanism that bypasses the ATP synthase

pore, and the energy is released as heat. Proton leakage, chemical uncouplers, and regulated uncoupling

proteins increase our metabolic rate and heat generation.

Mitochondria and Cell Death. Although oxidative phosphorylation is a mitochondrial process, most ATP

use occurs outside of the mitochondrion. ATP synthesized from oxidative phosphorylation is actively

transported from the matrix to the intermembrane space by adenine nucleotide translocase (ANT).

Porins form voltage-dependent anion channels (VDACs) through the outer mitochondrial membrane for

the diffusion of H2O, ATP metabolites, and other ions. Under certain types of stress, ANT, VDACs, and

other proteins form a nonspecific open channel known as the mitochondrial permeability transition

pore. This pore is associated with events that lead rapidly to necrotic cell death. THE WAITING ROOM

Cora N. was recovering uneventfully from her heart attack 1 month earlier (see Chapter 20), when

she won the Georgia state lottery. When she heard her number announced over the television, she

experienced crushing chest pain and grew short of breath. Her family called 911 and she was rushed to

the hospital emergency department.

On initial examination, her blood pressure was extremely high and her heart rhythm

irregular. Cora is

experiencing yet another myocardial infarction. An electrocardiogram showed unequivocal evidence of

severe lack of oxygen (ischemia) in the muscles of the anterior and lateral walls of her heart. Life-supportmeasures including nasal oxygen were initiated. An intravenous drip of nitroglycerin, a vasodilating

agent, was started in an effort to reduce her hypertension (it will also help to decrease her “preload” by

vasodilating the vessels going to the heart). She was also given a β-blocker, which will also help

decrease her blood pressure as well as decrease the work of her heart by slowing her heart rate. She also

required a small amount of intravenous nitroprusside to help lower her blood pressure. After her blood

pressure was well controlled, and because the hospital did not have a cardiac catheterization laboratory

and a transfer to a hospital with a cath lab was not possible, a decision was made to administer

intravenous tissue plasminogen activator (TPA) in an attempt to break up any intracoronary artery blood

clots in vessels supplying the ischemic myocardium (thrombolytic therapy).

Cora N. is experiencing a second myocardial infarction. Ischemia (low blood flow) has

caused hypoxia (low levels of oxygen) in the threatened area of her heart muscle, resulting

in inadequate generation of ATP for the maintenance of low intracellular Na+ and Ca2+ levels (see

Chapter 20). As a consequence, the myocardial cells in that specific location have become

swollen and the cytosolic proteins creatine kinase (MB isoform) and troponin (heart isoform)

have leaked into the blood. (See Ann J., Chapters 6 and 7).

Stanley T. A 123I thyroid uptake and scan performed on Stanley T. confirmed that his hyperthyroidism was the result of Graves disease (see Chapter 20). Graves disease, also known as

diffuse toxic goiter, is an autoimmune genetic disorder caused by the generation of human thyroidstimulating immunoglobulins. These immunoglobulins stimulate growth of the thyroid gland (goiter) and

excess secretion of the thyroid hormones triiodothyronine (T3) and tetraiodothyronine (T4). Because heat

production is increased under these circumstances, Mr. T.’s heat intolerance and sweating were growing

worse with time.

Isabel S., an intravenous drug user, appeared to be responding well to her multidrug regimens to

treat pulmonary tuberculosis and HIV (see Chapters 12, 13, 14, and 17). In the past 6 weeks,

however, she has developed increasing weakness in her extremities to the point that she has difficulty

carrying light objects or walking. Physical examination indicates a diffuse proximal and distal muscle

weakness associated with muscle atrophy. The muscles are not painful on motion but are mildly tender to

palpation. The blood level of the muscle enzymes creatine phosphokinase and aldolase are elevated. An

electromyogram revealed a generalized reduction in the muscle action potentials, suggestive of a primary

myopathic process. Proton spectroscopy of her brain and upper spinal cord showed no anatomic or

biochemical abnormalities. The diffuse and progressive skeletal muscle weakness was out of proportion

to that expected from her HIV or tuberculosis. This information led her physicians to consider other

etiologies.

An electromyogram measures the electrical potential of muscle cells both at rest and

while

contracting. Electrodes are inserted through the skin and into the muscle, and baselinerecordings (no contraction) are obtained, followed by measurements of electrical activity when

the muscle contracts. The electrode is retracted a small amount, and the measurements are

repeated. This occurs for up to 10 to 20 measurements, thereby sampling, many distinct areas of

the muscle. Under normal conditions, muscles at rest will have minimal electrical activity, which

increases significantly as the muscle contracts. Electromyograms that deviate from the norm

suggest an underlying pathology interfering with membrane polarization–depolarization as the

nerve cells instruct the muscle cells to contract. I. Oxidative Phosphorylation

Generation of ATP from oxidative phosphorylation requires an electron donor (NADH or FAD[2H]), an

electron acceptor (O2), and an intact inner mitochondrial membrane that is impermeable to protons, all the

components of the ETC and ATP synthase. It is regulated by the rate of ATP use. Most cells are dependent on oxidative phosphorylation for ATP homeostasis. During oxygen

deprivation from ischemia (low blood flow), an inability to generate energy from the ETC results in

increased permeability of this membrane and mitochondrial swelling. Mitochondrial swelling is a key

element in the pathogenesis of irreversible cell injury, leading to cell lysis and death (necrosis).

Charles F., who has a follicular-type non-Hodgkin lymphoma, was being treated with the

anthracycline drug doxorubicin (see Chapter 16). During the course of his treatment, he

developed biventricular heart failure. Although doxorubicin is a highly effective anticancer agent

against a wide variety of human tumors, its clinical use is limited by a specific, cumulative, dosedependent cardiotoxicity. Impairment of mitochondrial function may play a major role in this

toxicity. Doxorubicin binds to cardiolipin, a lipid component of the inner membrane of

mitochondria, where it might directly affect components of oxidative phosphorylation.

Doxorubicin inhibits succinate oxidation, inactivates cytochrome oxidase, interacts with CoQ,

adversely affects ion pumps, and inhibits ATP synthase, resulting in decreased ATP levels and

mildly swollen mitochondria. It decreases the ability of the mitochondria to sequester Ca2+ and

increases free radicals (highly reactive single-electron forms), leading to damage of the

mitochondrial membrane (see Chapter 25). It also might affect heart function indirectly through

other mechanisms.

A. Overview of Oxidative Phosphorylation

Our understanding of oxidative phosphorylation is based on the chemiosmotic hypothesis, which

proposes that the energy for ATP synthesis is provided by an electrochemical gradient across the inner

mitochondrial membrane. This electrochemical gradient is generated by the components of the ETC,

which pump protons across the inner mitochondrial membrane as they sequentially accept and donate

electrons (see Fig. 24.1). The final acceptor is O2, which is reduced to H2O.

1. Electron Transfer from NADH to O2In the ETC, electrons donated by NADH or FAD(2H) are passed sequentially through a series of electron

carriers embedded in the inner mitochondrial membrane. Each of the components of the ETC is reduced

as it accepts an electron and then oxidized as it passes the electrons to the next member of the chain. From

NADH, electrons are transferred sequentially through NADH:CoQ oxidoreductase (complex I, also

known as NADH dehydrogenase), coenzyme Q (CoQ), the cytochrome b–c1 complex (complex III),

cytochrome c, and finally, cytochrome c oxidase (complex IV). NADH:CoQ oxidoreductase, the

cytochrome b–c1 complex, and cytochrome c oxidase are multisubunit protein complexes that span the

inner mitochondrial membrane. CoQ is a lipid-soluble quinone that is not protein-bound and is free to

diffuse in the lipid membrane. It transports electrons from complex I to complex III and is an intrinsic part

of the proton pump for each of these complexes. Cytochrome c is a small protein in the intermembrane

space that transfers electrons from the b–c1 complex to cytochrome oxidase. The terminal complex,

cytochrome c oxidase, contains the binding site for O2. As O2 accepts electrons from the chain, it is

reduced to H2O.

2. The Electrochemical Potential Gradient

At each of the three large membrane-spanning complexes in the chain, electron transfer is accompanied by

proton pumping across the membrane. There is an energy drop of approximately 16 kilocalories (kcal) in

reduction potential as electrons pass through each of these complexes, which provides the energy required

to move protons against a concentration gradient. The membrane is impermeable to protons, so they

cannot diffuse through the lipid bilayer back into the matrix. Thus, in actively respiring mitochondria, the

intermembrane space and cytosol may be approximately 0.75 pH unit lower than the matrix.

The transmembrane movement of protons generates an electrochemical gradient with two components:

the membrane potential (the external face of the membrane is charged positive relative to the matrix side)

and the proton gradient (the intermembrane space has a higher proton concentration and is, therefore,

more acidic than the matrix) (Fig. 24.2). The electrochemical gradient is sometimes called the proton

motive force because it is the energy that pushes the protons to reenter the matrix to equilibrate on both

sides of the membrane. The protons are attracted to the more negatively charged matrix side of the

membrane, where the pH is more alkaline.3. Adenosine Triphosphate Synthase

ATP synthase (F0F1-ATPase), the enzyme that generates ATP, is a multisubunit enzyme that contains an

inner membrane portion (F0) and a stalk and headpiece (F1) that project into the matrix (Fig. 24.3). The 12

c-subunits in the membrane form a rotor that is attached to a central asymmetric shaft composed of the ε-

and γ-subunits. The headpiece is composed of three αβ-subunit pairs. Each β-subunit contains a catalytic

site for ATP synthesis. The headpiece is held stationary by a δ-subunit attached to a long b-subunit

connected to subunit a in the membrane.

The influx of protons through the proton channel turns the rotor. The proton channel is formed by the csubunits on one side and the a-subunit on the other side. Although the channel is continuous, it has two

offset portions, one portion open directly to the intermembrane space and one portion open directly to the

matrix. In the current model, each c-subunit contains a glutamyl carboxyl group that extends into the proton

channel. Because this carboxyl group accepts a proton from the intermembrane space, the c-subunit

rotates into the hydrophobic lipid membrane. The rotation exposes a different proton-containing c-subunitto the portion of the channel that is open directly to the matrix side. Because the matrix has a lower proton

concentration, the glutamyl carboxylic acid group releases a proton into the matrix portion of the channel.

Rotation is completed by an attraction between the negatively charged glutamyl residue and a positively

charged arginyl group on the a-subunit.

According to the binding-change mechanism, as the asymmetric shaft rotates to a new position, it

forms different binding associations with the αβ-subunits (Fig. 24.4). The new position of the shaft alters

the conformation of one β-subunit so that it releases a molecule of ATP and another subunit spontaneously

catalyzes synthesis of ATP from Pi, one proton, and ADP. Thus, energy from the electrochemical gradient

is used to change the conformation of the ATP synthase subunits so that the newly synthesized ATP is

released.B. Oxidation–Reduction Components of the Electron-Transport Chain Electron transport to O2 occurs via a series of oxidation–reduction steps in which each successive

component of the chain is reduced as it accepts electrons and oxidized as it passes electrons to the next

component of the chain. The oxidation–reduction components of the chain include flavin mononucleotide

(FMN), Fe–S centers, CoQ, and Fe in cytochromes b, c1, c, a, and a3. Copper (Cu) is also a component of

cytochromes a and a3 (Fig. 24.5). With the exception of CoQ, all of these electron acceptors are tightly

bound to the protein subunits of the carriers. FMN, like FAD, is synthesized from the vitamin riboflavin

(see Fig. 20.10).

The reduction potential of each complex of the chain is at a lower energy level than the previous

complex, so energy is released as electrons pass through each complex. This energy is used to move

protons against their concentration gradient, so they become concentrated on the cytosolic side of the

inner membrane.

1. NADH:CoQ Oxidoreductase

NADH:CoQ oxidoreductase (also named NADH dehydrogenase) is an enormous 42-subunit complex that

contains a binding site for NADH, several FMN and iron–sulfur (Fe–S) center binding proteins, and

binding sites for CoQ (see Fig. 24.5). An FMN accepts two electrons from NADH and is able to pass

single electrons to the Fe–S centers. Fe–S centers, which are able to delocalize single electrons into large

orbitals, transfer electrons to and from CoQ. Fe–S centers are also present in other enzyme systems—such

as proteins within the cytochrome b–c1 complex, which transfer electrons to CoQ—and in aconitase in the

TCA cycle.

Although iron-deficiency anemia is characterized by decreased levels of hemoglobin and

other iron-containing proteins in the blood, the iron-containing cytochromes and Fe–S

centers of the ETC in tissues such as skeletal muscle are affected as rapidly. Fatigue in irondeficiency anemia, in patients such as Ann R. (see Chapter 16), results in part from the lack of

electron transport for ATP production.2. Succinate Dehydrogenase and Other

Flavoproteins

In addition to NADH:CoQ oxidoreductase, succinate dehydrogenase and other flavoproteins in the inner

mitochondrial membrane also pass electrons to CoQ (see Fig. 24.5). Succinate dehydrogenase is part of

the TCA cycle and also a component of complex II of the ETC. Electron-transferring flavoprotein

(ETF):CoQ oxidoreductase accepts electrons from ETF, which acquires them from fatty acid oxidation

and other pathways. Both of these flavoproteins have Fe–S centers. Glycerol 3-phosphate dehydrogenase

is a flavoprotein that is part of a shuttle for reoxidizing cytosolic NADH (see Section I.E).

The free-energy drop in electron transfer between NADH and CoQ of approximately −13 to −14 kcal

is able to support movement of four protons. However, the FAD in succinate dehydrogenase (as well as

ETF:CoQ oxidoreductase and glycerol 3-phosphate dehydrogenase) is at roughly the same redox potential

as CoQ, and no energy is released as they transfer electrons to CoQ. These proteins do not span the

membrane and consequently do not have a proton pumping mechanism. 3. Coenzyme Q

CoQ is the only component of the ETC that is not protein-bound. The large hydrophobic side chain of 10

isoprenoid units (50 carbons) confers lipid solubility, and CoQ is able to diffuse freely through the lipids

of the inner mitochondrial membrane (Fig. 24.6). When the oxidized quinone form accepts a single

electron (to form the semiquinone), it forms a free radical (a compound with a single electron in an

orbital). The transfer of single electrons makes it the major site for generation of toxic oxygen free

radicals in the body (see Chapter 25).

The semiquinone can accept a second electron and two protons from the matrix side of the membrane

to form the fully reduced quinone. The mobility of CoQ in the membrane, its ability to accept one or two

electrons, and its ability to accept and donate protons enable it to participate in the proton pumps for both

complexes I and III as it shuttles electrons between them (see Section I.C). CoQ is also called ubiquinone

(the ubiquitous quinone), because quinones with similar structures are found in all plants and animals.

4. Cytochromes

The remaining components in the ETC are cytochromes (see Fig. 24.5). Each cytochrome is a protein that

contains a bound heme (i.e., an Fe atom bound to a porphyrin nucleus similar in structure to the heme in

hemoglobin) (Fig. 24.7).Because of differences in the protein component of the cytochromes and small differences in the heme

structure, each heme has a different reduction potential. The cytochromes of the b–c1 complex have a

higher energy level than those of cytochrome oxidase (a and a3). Thus, energy is released by electron

transfer between complexes III and IV. The iron atoms in the cytochromes are in the Fe3+ state. As they

accept an electron, they are reduced to Fe2+. As they are reoxidized to Fe3+, the electrons pass to the next

component of the ETC.

The iron in the heme in hemoglobin, unlike the iron in the heme of cytochromes, never

changes its oxidation state (it is Fe2+ in hemoglobin). If the iron in hemoglobin were to

become oxidized (Fe3+), the oxygen-binding capacity of the molecule would be lost.

What

accounts for this difference in iron oxidation states between hemoglobin and cytochromes?

Normally, the protein structures binding the heme either protect the iron from oxidation

(such as the globin proteins) or allow oxidation to occur (such as happens in the cytochromes). However, in hemoglobin M, a rare hemoglobin variant found in the human population, a tyrosine is substituted for the histidine at position F8 in the normal hemoglobin A.

This tyrosine stabilizes the Fe3+ form of heme, and these subunits cannot bind oxygen. This is a

lethal condition if it is homozygous. 5. Copper and the Reduction of Oxygen

The last cytochrome complex is cytochrome oxidase, which passes electrons from cytochrome c to O2

(see Fig. 24.5). It contains cytochromes a and a3 and the oxygen-binding site. A whole oxygen molecule,

O2, must accept four electrons to be reduced to two H2O molecules. Bound copper (Cu+) ions in the

cytochrome oxidase complex facilitate the collection of the four electrons and the reduction of O2.

Cytochrome oxidase has a much lower Km for O2 than myoglobin (the heme-containing intracellularoxygen carrier) or hemoglobin (the heme-containing oxygen transporter in the blood). Thus, O2 is

“pulled” from the erythrocyte to myoglobin, and from myoglobin to cytochrome oxidase, where it is

reduced to H2O.

C. Pumping of Protons

One of the tenets of the chemiosmotic theory is that energy from the oxidation–reduction reactions of the

ETC is used to transport protons from the matrix to the intermembrane space. This proton pumping is

generally facilitated by the vectorial arrangement of the membrane-spanning complexes. Their structure

allows them to pick up electrons and protons on one side of the membrane and release protons on the

other side of the membrane as they transfer an electron to the next component of the chain. The direct

physical link between proton movement and electron transfer can be illustrated by an examination of the Q

cycle for the b–c1 complex (Fig. 24.8). The Q cycle involves a double cycle of CoQ reduction and

oxidation. CoQ accepts two protons at the matrix side together with two electrons; it then releases protons

into the intermembrane space while donating one electron back to another component of the cytochrome

b–c1 complex and one to cytochrome c.

The mechanism for pumping protons at the NADH:CoQ oxidoreductase complex is not well understood, but it involves a Q cycle in which the Fe–S centers and FMN might participate. However,

transmembrane proton movement at cytochrome c oxidase probably involves direct transport of the proton

through a series of bound water molecules or amino acid side chains in the protein complex, a mechanism

that has been described as a proton wire.

The significance of the direct link between the electron transfer and proton movement is that one

cannot occur without the other (the processes are said to be “coupled”). Thus, when protons are not being

used for ATP synthesis, the proton gradient and the membrane potential build up. This “proton

backpressure” controls the rate of proton pumping, which controls electron transport and O2 consumption.D. Energy Yield from the Electron-Transport Chain

The overall free-energy release from oxidation of NADH by O2 is approximately −53 kcal, and from

FAD(2H), it is approximately −41 kcal. This ΔG0 is so negative that the chain is never reversible; we

never synthesize oxygen from H2O. The negative ΔG0 also drives NADH and FAD(2H) formation from

the pathways of fuel oxidation, such as the TCA cycle and glycolysis, to completion. Overall, each NADH donates two electrons, equivalent to the reduction of one-half of an O2

molecule. A generally (but not universally) accepted estimate of the stoichiometry of ATP synthesis is that

four protons are pumped at complex I, four protons at complex III, and two at complex IV. With three

protons translocated for each ATP synthesized, and one proton for each phosphate transported into the

matrix (see Section IV.A of this chapter), an estimated 2.5 ATPs are formed for each NADH oxidized, and

1.5 ATPs are formed for each of the other FAD(2H)-containing flavoproteins that donate electrons to

CoQ. (This calculation neglects the basal proton leak.) Thus, only approximately 30% of the energy

available from NADH and FAD(2H) oxidation by O2 is used for ATP synthesis. Some of the remaining

energy in the electrochemical potential is used for the transport of anions and Ca2+ into the mitochondrion.

The remainder of the energy is released as heat. Consequently, the ETC is also our major source of heat.

Cora N. has a lack of oxygen in the anterior and lateral walls of her heart caused by severe

ischemia (lack of blood flow) resulting from blockage of the coronary arteries supplying

blood to this area of her heart. The arteries are blocked by a clot at the site of ruptured

atherosclerotic plaques. The limited availability of O2 to act as an electron acceptor will decrease

proton pumping and generation of an electrochemical potential gradient across the inner

mitochondrial membrane of ischemic cells. As a consequence, the rate of ATP generation in these

specific areas of her heart will decrease, thereby triggering events that lead to irreversible cell

injury.

E. Cytoplasmic NADH

There is no transport system for cytoplasmic NADH to cross the inner mitochondrial membrane, or for

mitochondrial NADH to enter the cytoplasm. However, there are two shuttle systems to transport the

electrons from NADH (cytoplasmic) to NAD+ (mitochondrial). NADH can be reoxidized to NAD+ in the

cytosol by a reaction that transfers the electrons to dihydroxyacetone phosphate (DHAP) in the glycerol 3-

phosphate (glycerol 3-P) shuttle or to oxaloacetate in the malate–aspartate shuttle. The NAD+ that is

formed in the cytosol returns to glycolysis, whereas glycerol 3-P or malate carry the reducing equivalents

that are ultimately transferred across the inner mitochondrial membrane. Thus, these shuttles transfer

electrons and not NADH per se. 1. Glycerol 3-Phosphate Shuttle

The glycerol 3-P shuttle is the major shuttle in most tissues. In this shuttle, cytosolic NAD+ is regenerated

by cytoplasmic glycerol 3-P dehydrogenase, which transfers electrons from NADH to DHAP to form

glycerol 3-P (Fig. 24.9). Glycerol 3-P then diffuses through the outer mitochondrial membrane to the innermitochondrial membrane, where the electrons are donated to a membrane-bound FAD-containing

glycerophosphate dehydrogenase. This enzyme, like succinate dehydrogenase,