Berg J.M., Tymoczko J.L., Stryer L. Biochemistry

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Figure 18.9. Sequence of Electron Carriers in the Respiratory Chain.

II. Transducing and Storing Energy 18. Oxidative Phosphorylation 18.3. The Respiratory Chain Consists of Four Complexes: Three Proton Pumps and a Physical Link to the Citric Acid Cycle

Table 18.2. Components of the mitochondrial electron-transport chain

Oxidant or reductant

Enzyme complex Mass

(kd)

Subunits Prosthetic group Matrix side Membrane core Cytosolic side

NADH-Q

oxidoreductase

880 34 FMN NADH Q

Fe-S

Succinate-Q reductase 140 4 FAD Succinate Q

Fe-S

Q-cytochrome c

oxidoreductase

250 10 Heme b

Q Cytochrome c

Heme b

Heme c

Fe-S

Cytochrome c oxidase 160 10 Heme a Cytochrome c

Heme a

and Cu

Sources: J. W. DePierre and L. Ernster, Annu. Rev. Biochem. 46(1977):215; Y. Hatefi, Annu Rev. Biochem. 54(1985);1015; and J.

E. Walker, Q. Rev. Biophys. 25(1992):253.

II. Transducing and Storing Energy 18. Oxidative Phosphorylation 18.3. The Respiratory Chain Consists of Four Complexes: Three Proton Pumps and a Physical Link to the Citric Acid Cycle

Figure 18.10. Oxidation States of Quinones. The reduction of ubiquinone (Q) to ubiquinol (QH

) proceeds through a

semiquinone anion intermediate (Q

II. Transducing and Storing Energy 18. Oxidative Phosphorylation 18.3. The Respiratory Chain Consists of Four Complexes: Three Proton Pumps and a Physical Link to the Citric Acid Cycle

Figure 18.11. Structure of NADH-Q Oxidoreductase (Complex I). The structure, determined by electron microscopy

at 22-Å resolution, consists of a membrane-spanning part and a long arm that extends into the matrix. NADH is oxidized

in the arm, and the electrons are transferred to reduce Q in the membrane. [After N. Grigorieff, J. Mol. Biol. 277

(1998):1033

1048.]

II. Transducing and Storing Energy 18. Oxidative Phosphorylation 18.3. The Respiratory Chain Consists of Four Complexes: Three Proton Pumps and a Physical Link to the Citric Acid Cycle

Figure 18.12. Oxidation States of Flavins. The reduction of flavin mononucleotide (FMN) to FMNH

proceeds

through a semiquinone intermediate.

II. Transducing and Storing Energy 18. Oxidative Phosphorylation 18.3. The Respiratory Chain Consists of Four Complexes: Three Proton Pumps and a Physical Link to the Citric Acid Cycle

Figure 18.13. Iron-Sulfur Clusters. (A) A single iron ion bound by four cysteine residues. (B) 2Fe-2S cluster with iron

ions bridged by sulfide ions. (C) 4Fe-4S cluster. Each of these clusters can undergo oxidation-reduction reactions.

II. Transducing and Storing Energy 18. Oxidative Phosphorylation 18.3. The Respiratory Chain Consists of Four Complexes: Three Proton Pumps and a Physical Link to the Citric Acid Cycle

Figure 18.14. Coupled Electron-Proton Transfer Reactions. The reduction of a quinone (Q) to QH

in an appropriate

site can result in the uptake of two protons from the mitochondrial matrix.

II. Transducing and Storing Energy 18. Oxidative Phosphorylation 18.3. The Respiratory Chain Consists of Four Complexes: Three Proton Pumps and a Physical Link to the Citric Acid Cycle

Figure 18.15. Structure of Q-Cytochrome C Oxidoreductase (Cytochrome BC

). This enzyme is a homodimer with

11 distinct polypeptide chains. The major prosthetic groups, three hemes and a 2Fe-2S cluster, mediate the

electron-transfer reactions between quinones in the membrane and cytochrome c in the intermembrane space.

II. Transducing and Storing Energy 18. Oxidative Phosphorylation 18.3. The Respiratory Chain Consists of Four Complexes: Three Proton Pumps and a Physical Link to the Citric Acid Cycle

Figure 18.16. Attachment of C -Type Cytochromes. A heme group is covalently attached to a protein through

thioether linkages formed by the addition of sulfhydryl groups of cysteine residues to vinyl groups on protoporphyrin.

II. Transducing and Storing Energy 18. Oxidative Phosphorylation 18.3. The Respiratory Chain Consists of Four Complexes: Three Proton Pumps and a Physical Link to the Citric Acid Cycle

Figure 18.17. Q Cycle. The two electrons of a bound QH

are transferred, one to cytochrome c and the other to a bound

Q to form the semiquinone Q

. The newly formed Q dissociates and is replaced by a second QH

, which also gives up

its electrons, one to a second molecule of cytochrome c and the other to reduce Q

to QH

. This second electron

transfer results in the uptake of two protons from the matrix. Prosthetic groups are shown in their oxidized forms in blue

and in their reduced forms in red.

II. Transducing and Storing Energy 18. Oxidative Phosphorylation 18.3. The Respiratory Chain Consists of Four Complexes: Three Proton Pumps and a Physical Link to the Citric Acid Cycle

Figure 18.18. Structure of Cytochrome C Oxidase.

This enzyme consists of 13 polypeptide chains. The major

prosthetic groups include Cu

/Cu

, heme a, and heme a

-Cu

. Heme a

-Cu

is the site of the reduction of

oxygen to water. CO(bb) is a carbonyl group of the peptide backbone.

II. Transducing and Storing Energy 18. Oxidative Phosphorylation 18.3. The Respiratory Chain Consists of Four Complexes: Three Proton Pumps and a Physical Link to the Citric Acid Cycle

Figure 18.19. Cytochrome Oxidase Mechanism. The cycle begins with all prosthetic groups in their oxidized forms

(shown in blue). Reduced cytochrome c introduces an electron that reduces Cu

. A second reduced cytochrome c then

reduces the iron in heme a

. This Fe

center then binds oxygen. Two electrons are transferred to the bound oxygen to

form peroxide, which bridges between the iron and Cu

. The introduction of an additional electron by a third molecule

of reduced cytochrome c cleaves the O-O bond and results in the uptake of a proton from the matrix. The introduction of

a final electron and three more protons generates two molecules of H

O, which are released from the enzyme to

regenerate the initial state. The four protons found in the water molecules come from the matrix.

II. Transducing and Storing Energy 18. Oxidative Phosphorylation 18.3. The Respiratory Chain Consists of Four Complexes: Three Proton Pumps and a Physical Link to the Citric Acid Cycle

Figure 18.20. Peroxide Bridge. The oxygen bound to heme a

is reduced to peroxide by the presence of Cu

II. Transducing and Storing Energy 18. Oxidative Phosphorylation 18.3. The Respiratory Chain Consists of Four Complexes: Three Proton Pumps and a Physical Link to the Citric Acid Cycle

Figure 18.21. Proton Transport by Cytochrome C Oxidase. Four "chemical" protons are taken up from the matrix

side to reduce one molecule of O

to two molecules of H

O. Four additional "pumped" protons are transported out of the

matrix and released on the cytosolic side in the course of the reaction. The pumped protons double the efficiency of free-

energy storage in the form of a proton gradient for this final step in the electron-transport chain.

II. Transducing and Storing Energy 18. Oxidative Phosphorylation 18.3. The Respiratory Chain Consists of Four Complexes: Three Proton Pumps and a Physical Link to the Citric Acid Cycle

Figure 18.22. Superoxide Dismutase Mechanism. The oxidized form of superoxide dismutase (M

) reacts with one

superoxide ion to form O

and generate the reduced form of the enzyme (M

red

). The reduced form then reacts with a

second superoxide and two protons to form hydrogen peroxide and regenerate the oxidized form of the enzyme.

II. Transducing and Storing Energy 18. Oxidative Phosphorylation 18.3. The Respiratory Chain Consists of Four Complexes: Three Proton Pumps and a Physical Link to the Citric Acid Cycle

Table 18.3. Pathological and other conditions that may entail free-radical injury

Atherogenesis

Emphysema; bronchitis

Parkinson disease

Duchenne muscular dystrophy

Cervical cancer

Alcoholic liver disease

Diabetes

Acute renal failure

Down syndrome

Retrolental fibroplasia

Cerebrovascular disorders

Ischemia; reperfusion injury

Source: After D.B. Marks, A.D. Marks, and C.M. Smith, Basic Medical Biochemistry: A Clinical Approach (Williams & Wilkins,

1996, p. 331).

II. Transducing and Storing Energy 18. Oxidative Phosphorylation 18.3. The Respiratory Chain Consists of Four Complexes: Three Proton Pumps and a Physical Link to the Citric Acid Cycle

Figure 18.23. Conservation of the Three-Dimensional Structure of Cytochrome C.

The side chains are shown for the

21 conserved amino acids and the heme.

II. Transducing and Storing Energy 18. Oxidative Phosphorylation 18.3. The Respiratory Chain Consists of Four Complexes: Three Proton Pumps and a Physical Link to the Citric Acid Cycle

Figure 18.24. Evolutionary Tree Constructed From Sequences of Cytochrome C. Branch lengths are proportional to

the number of amino acid changes that are believed to have occurred. This drawing is an adaptation of the work of

Walter M. Fitch and Emanuel Margoliash.

II. Transducing and Storing Energy 18. Oxidative Phosphorylation

18.4. A Proton Gradient Powers the Synthesis of ATP

Conceptual Insights, Energy Transformations in Oxidative

Phosphorylation. View this media module for an animated, interactive

summary of how electron transfer potential is converted into proton-motive

force and, finally, phosphoryl transfer potential in oxidative phosphorylation.

Thus far, we have considered the flow of electrons from NADH to O

, an exergonic process.

Next, we consider how this process is coupled to the synthesis of ATP, an endergonic process.

A molecular assembly in the inner mitochondrial membrane carries out the synthesis of ATP. This enzyme complex was

originally called the mitochon-drial ATPase or F

ATPase because it was discovered through its catalysis of the

reverse reaction, the hydrolysis of ATP. ATP synthase, its preferred name, emphasizes its actual role in the

mitochondrion. It is also called Complex V.

How is the oxidation of NADH coupled to the phosphorylation of ADP? It was first suggested that electron transfer leads

to the formation of a covalent high-energy intermediate that serves as a high phosphoryl transfer potential compound or

to the formation of an activated protein conformation, which then drives ATP synthesis. The search for such

intermediates for several decades proved fruitless.

In 1961, Peter Mitchell proposed that electron transport and ATP synthesis are coupled by a proton gradient across the

inner mitochondrial membrane rather than by a covalent high-energy intermediate or an activated protein conformation.

In his model, the transfer of electrons through the respiratory chain leads to the pumping of protons from the matrix to

the cytosolic side of the inner mitochondrial membrane. The H

concentration becomes lower in the matrix, and an

electrical field with the matrix side negative is generated (Figure 18.25). Mitchell's idea, called the chemiosmotic

hypothesis, was that this proton-motive force drives the synthesis of ATP by ATP synthase. Mitchell's highly innovative

hypothesis that oxidation and phosphorylation are coupled by a proton gradient is now supported by a wealth of

evidence. Indeed, electron transport does generate a proton gradient across the inner mitochondrial membrane. The pH

outside is 1.4 units lower than inside, and the membrane potential is 0.14 V, the outside being positive. As we calculated

in Section 18.2.2, this membrane potential corresponds to a free energy of 5.2 kcal (21.8 kJ) per mole of protons.

An artificial system was created to elegantly demonstrate the basic principle of the chemiosmotic hypothesis. Synthetic

vesicles containing bacteriorhodopsin, a purple-membrane protein from halobacteria that pumps protons when

illuminated, and mitochondrial ATP synthase purified from beef heart were created (Figure 18.26). When the vesicles

were exposed to light, ATP was formed. This key experiment clearly showed that the respiratory chain and ATP

synthase are biochemically separate systems, linked only by a proton-motive force.

18.4.1. ATP Synthase Is Composed of a Proton-Conducting Unit and a Catalytic Unit

Biochemical, electron microscopic, and crystallographic studies of ATP synthase have revealed many details of its

structure (Figure 18.27). It is a large, complex membrane-embedded enzyme that looks like a ball on a stick. The 85-Å-

diameter ball, called the F

subunit, protrudes into the mitochondrial matrix and contains the catalytic activity of the

synthase. In fact, isolated F

subunits display ATPase activity. The F

subunit consists of five types of polypeptide

chains (α

, β

, γ, δ, and ε) with the indicated stoichiometry. The α and β subunits, which make up the bulk of the F

are arranged alternately in a hexameric ring; they are homologous to one another and are members of the P-loop NTPase

family (Section 9.4.1). Both bind nucleotides but only the β subunits participate directly in catalysis. The central stalk

consists of two proteins: γ and ε. The γ subunit includes a long α-helical coiled coil that extends into the center of the α

hexamer. The γ subunit breaks the symmetry of the α

hexamer: each of the β subunits is distinct by virtue of its

interaction with a different face of γ. Distinguishing the three β subunits is crucial for the mechanism of ATP synthesis.

The F

subunit is a hydrophobic segment that spans the inner mitochondrial membrane. F

contains the proton channel

of the complex. This channel consists of a ring comprising from 10 to 14 c subunits that are embedded in the membrane.

A single a subunit binds to the outside of this ring. The proton channel depends on both the a subunit and the c ring. The

and F

subunits are connected in two ways, by the central γ ε stalk and by an exterior column. The exterior column

consists of one a subunit, two b subunits, and the δ subunit. As will be discussed shortly, we can think of the enzyme as

consisting of two functional components: (1) a moving unit, or rotor, consisting of the c ring and the γ ε stalk, and (2) a

stationary unit, or stator, composed of the remainder of the molecule.

18.4.2. Proton Flow Through ATP Synthase Leads to the Release of Tightly Bound

ATP: The Binding-Change Mechanism

Conceptual Insights, ATP Synthase as Motor Protein, looks further into the

chemistry and mechanics of ATP synthase rotation.

ATP synthase catalyzes the formation of ATP from ADP and orthophosphate.

The actual substrates are Mg

complexes of ADP and ATP, as in all known phosphoryl transfer reactions with these

nucleotides. A terminal oxygen atom of ADP attacks the phosphorus atom of P

to form a pentacovalent intermediate,

which then dissociates into ATP and H

O (Figure 18.28). The attacking oxygen atom of ADP and the departing oxygen

atom of P

occupy the apices of a trigonal bipyramid.

How does the flow of protons drive the synthesis of ATP? The results of isotopic-exchange experiments unexpectedly

revealed that enzyme-bound ATP forms readily in the absence of a proton-motive force. When ADP and P

were added to

ATP synthase in H

O became incorporated into P

through the synthesis of ATP and its subsequent hydrolysis

(Figure 18.29). The rate of incorporation of

O into P

showed that about equal amounts of bound ATP and ADP are in

equilibrium at the catalytic site, even in the absence of a proton gradient. However, ATP does not leave the catalytic site

unless protons flow through the enzyme. Thus, the role of the proton gradient is not to form ATP but to release it from

the synthase.

On the basis of these and other observations, Paul Boyer proposed a binding-change mechanism for proton-driven ATP

synthesis. This proposal states that changes in the properties of the three β subunits allow sequential ADP and P

binding,

ATP synthesis, and ATP release. The concepts of this initial proposal refined by more recent crystallographic and other

data yield a satisfying mechanism for ATP synthesis. As already noted, interactions with the γ subunit make the three β

subunits inequivalent (Figure 18.30). One β subunit can be in the T, or tight, conformation. This conformation binds

ATP with great avidity. Indeed, its affinity for ATP is so high that it will convert bound ADP and P

into ATP with an

equilibrium constant near 1, as indicated by the aforediscussed isotopic-exchange experiments. However, the

conformation of this subunit is sufficiently constrained that it cannot release ATP. A second subunit will then be in the