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Berg JM, Tymoczko JL, Stryer L. Biochemistry. 5th edition. New York: W H Freeman; 2002.

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Biochemistry. 5th edition.

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Section 18.4A Proton Gradient Powers the Synthesis of ATP

Conceptual Insights, Energy Transformations in Oxidative Phosphorylation

Image mouse.jpg 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 O2, an exergonic process.

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

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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 1 F 0 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.

Figure 18.25. Chemiosmotic Hypothesis.

Figure 18.25

Chemiosmotic Hypothesis. Electron transfer through the respiratory chain leads to the pumping of protons from the matrix to the cytosolic side of the inner mitochondrial membrane. The pH gradient and membrane potential constitute a proton-motive force (more...)

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.

Figure 18.26. Testing the Chemiosmotic Hypothesis.

Figure 18.26

Testing the Chemiosmotic Hypothesis. ATP is synthesized when reconstituted membrane vesicles containing bacteriorhodopsin (a light-driven proton pump) and ATP synthase are illuminated. The orientation of ATP synthase in this reconstituted membrane is (more...)

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 F1 subunit, protrudes into the mitochondrial matrix and contains the catalytic activity of the synthase. In fact, isolated F1 subunits display ATPase activity. The F1 subunit consists of five types of polypeptide chains (α3, β3, γ, δ, and ϵ) with the indicated stoichiometry. The α and β subunits, which make up the bulk of the F1, 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 α3β3 hexamer. The γ subunit breaks the symmetry of the α 3 β 3 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.

Figure 18.27. Structure of ATP Synthase.

Figure 18.27

Structure of ATP Synthase. Image mouse.jpg A schematic structure is shown along with detailed structures of the components for which structures have been determined to high resolution. The P-loop NTPase domains of the α and β subunits are indicated (more...)

The F0 subunit is a hydrophobic segment that spans the inner mitochondrial membrane. F 0 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 F0 and F1 subunits are connected in two ways, by the central γϵ 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 γϵ 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

Image mouse.jpg looks further into the chemistry and mechanics of ATP synthase rotation.

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

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The actual substrates are Mg2+ 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 Pi to form a pentacovalent intermediate, which then dissociates into ATP and H2O (Figure 18.28). The attacking oxygen atom of ADP and the departing oxygen atom of Pi occupy the apices of a trigonal bipyramid.

Figure 18.28. ATP Synthesis Mechanism.

Figure 18.28

ATP Synthesis Mechanism. One of the oxygen atoms of ADP attacks the phosphorus atom of Pi to form a pentacovalent intermediate, which then forms ATP and releases a molecule of H2O.

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 Pi were added to ATP synthase in H218O, 18O became incorporated into Pi through the synthesis of ATP and its subsequent hydrolysis (Figure 18.29). The rate of incorporation of 18O into Pi 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.

Figure 18.29. ATP Forms Without a Proton-Motive Force But Is Not Released.

Figure 18.29

ATP Forms Without a Proton-Motive Force But Is Not Released. The results of isotope-exchange experiments indicate that enzyme-bound ATP is formed from ADP and Pi in the absence of a proton-motive force.

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 Pi 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 Pi 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 L, or loose, conformation. This conformation binds ADP and Pi. It, too, is sufficiently constrained that it cannot release bound nucleotides. The final subunit will be in the O, or open, form. This form can exist with a bound nucleotide in a structure that is similar to those of the T and L forms, but it can also convert to form a more open conformation and release a bound nucleotide (Figure 18.31). This structure, with one of the three β subunits in an open, nucleotide-free state, as well as one with one of the β subunits in a nucleotide-bound O conformation, have been observed crystallographically.

Figure 18.30. ATP Synthase Nucleotide-Binding Sites Are Not Equivalent.

Figure 18.30

ATP Synthase Nucleotide-Binding Sites Are Not Equivalent. The γ subunit passes through the center of the α3β3 hexamer and makes the nucleotide-binding sites in the β subunits distinct from one another.

Figure 18.31. ATP Release From the β subunit in the open form.

Figure 18.31

ATP Release From the β subunit in the open form. Unlike the tight and loose forms, the open form of the β subunit can change conformation sufficiently to release bound nucleotides.

The interconversion of these three forms can be driven by rotation of the γ subunit (Figure 18.32). Suppose the γ subunit is rotated 120 degrees in a counterclockwise direction (as viewed from the top). This rotation will change the subunit in the T conformation into the O conformation, allowing the subunit to release the ATP that has been formed within it. The subunit in the L conformation will be converted into the T conformation, allowing the transition of bound ADP + Pi into ATP. Finally, the subunit in the O conformation will be converted into the L conformation, trapping the bound ADP and Pi so that they cannot escape. The binding of ADP and Pi to the subunit now in the O conformation completes the cycle. This mechanism suggests that ATP can be synthesized by driving the rotation of the γ subunit in the appropriate direction. Likewise, this mechanism suggests that the hydrolysis of ATP by the enzyme should drive the rotation of the γ subunit in the opposite direction.

Figure 18.32. Binding-Change Mechanism for ATP Synthase.

Figure 18.32

Binding-Change Mechanism for ATP Synthase. The rotation of the γ subunit interconverts the three β subunits. The subunit in the T (tight) form, which contains newly synthesized ATP that cannot be released, is converted into the O (open) (more...)

18.4.3. The World's Smallest Molecular Motor: Rotational Catalysis

Is it possible to observe the proposed rotation directly? Elegant experiments were performed with the use of a simple experimental system consisting of cloned α3β3γ subunits only (Figure 18.33). The β subunits were engineered to contain amino-terminal polyhistidine tags, which have a high affinity for nickel ions. This property of the tags allowed the α3β3 assembly to be immobilized on a glass surface that had been coated with nickel ions. The γ subunit was linked to a fluorescently labeled actin filament to provide a long segment that could be observed under a fluorescence microscope. Remarkably, the addition of ATP caused the actin filament to rotate unidirectionally in a counterclockwise direction. The γ subunit was rotating, being driven by the hydrolysis of ATP. Thus, the catalytic activity of an individual molecule could be observed. The counterclockwise rotation is consistent with the predicted mechanism for hydrolysis because the molecule was viewed from below relative to the view shown in Figure 18.32.

Figure 18.33. Direct Observation of ATP-Driven Rotation in ATP Synthase.

Figure 18.33

Direct Observation of ATP-Driven Rotation in ATP Synthase. The α3β3 hexamer of ATP synthase is fixed to a surface, with the γ subunit projecting upward and linked to a fluorescently labeled actin filament. The addition and subsequent hydrolysis (more...)

More detailed analysis in the presence of lower concentrations of ATP revealed that the γ subunit rotates in 120-degree increments, with each step corresponding to the hydrolysis of a single ATP molecule. In addition, from the results obtained by varying the length of the actin filament and mea-suring the rate of rotation, the enzyme appears to operate near 100% efficiency; that is, essentially all of the energy released by ATP hydrolysis is converted into rotational motion.

18.4.4. Proton Flow Around the c Ring Powers ATP Synthesis

The direct observation of rotary motion of the γ subunit is strong evidence for the rotational mechanism for ATP synthesis. The last remaining question is: How does proton flow through F0 drive the rotation of the γ subunit? Howard Berg and George Oster proposed an elegant mechanism that provides a clear answer to this question. The mechanism depends on the structures of the a and c subunits of F0 (Figure 18.34). The structure of the c subunit was determined both by NMR methods and by x-ray crystallography. Each polypeptide chain forms a pair of α helices that span the membrane. An aspartic acid residue (Asp 61) is found in the middle of the second helix. When Asp 61 is in contact with the hydrophobic part of the membrane, the residue must be in the neutral aspartic acid form, rather than in the charged, aspartate form. From 9 to 12 c subunits assemble into a symmetric membrane-spanning ring. Although the structure of the a subunit has not yet been experimentally determined, a variety of evidence is consistent with a structure that includes two proton half-channels that do not span the membrane (see Figure 18.34). Thus, protons can pass into either of these channels, but they cannot move completely across the membrane. The a subunit directly abuts the ring comprising the c subunits, with each half-channel directly interacting with one c subunit.

Figure 18.34. Components of the Proton-Conducting Unit of ATP Synthase.

Figure 18.34

Components of the Proton-Conducting Unit of ATP Synthase. The c subunit consists of two α helices that span the membrane. An aspartic acid residue in the second helix lies on the center of the membrane. The structure of the a subunit has not yet (more...)

With this structure in mind, we can see how a proton gradient can drive rotation of the c ring. Suppose that the Asp 61 residues of the two c subunits that are in contact with a half-channel have given up their protons so that they are in the charged aspartate form (Figure 18.35), which is possible because they are in relatively hydrophilic environments inside the half-channel. The c ring cannot rotate in either direction, because such a rotation would move a charged aspartate residue into the hydrophobic part of the membrane. A proton can move through either half-channel to protonate one of the aspartate residues. However, it is much more likely to pass through the channel that is connected to the cytosolic side of the membrane because the proton concentration is more than 25 times as high on this side as on the matrix side, owing to the action of the electron-transport-chain proteins. The entry of protons into the cytosolic half-channel is further facilitated by the membrane potential of +0.14 V (positive on the cytoplasmic side), which increases the concentration of protons near the mouth of the cytosolic half-channel. If the aspartate residue is protonated to its neutral form, the c ring can now rotate, but only in a clockwise direction. Such a rotation moves the newly protonated aspartic acid residue into contact with the membrane, moves the charged aspartate residue from contact with the matrix half-channel to the cytosolic half-channel, and moves a different protonated aspartic acid residue from contact with the membrane to the matrix half-channel. The proton can then dissociate from aspartic acid and move through the half-channel into the proton-poor matrix to restore the initial state. This dissociation is favored by the positive charge on a conserved arginine residue (Arg 210) in the a subunit. Thus, the difference in proton concentration and potential on the two sides of the membrane leads to different probabilities of protonation through the two half-channels, which yields directional rotational motion. Each proton moves through the membrane by riding around on the rotating c ring to exit through the matrix half-channel (Figure 18.36).

Figure 18.35. Proton Motion Across the Membrane Drives Rotation of the C Ring.

Figure 18.35

Proton Motion Across the Membrane Drives Rotation of the C Ring. A proton enters from the intermembrane space into the cytosolic half-channel to neutralize the charge on an aspartate residue in a c subunit. With this charge neutralized, the c ring can (more...)

Figure 18.36. Proton Path Through the Membrane.

Figure 18.36

Proton Path Through the Membrane. Each proton enters the cytosolic half-channel, follows a complete rotation of the c ring, and exits through the other half-channel into the matrix.

The c ring is tightly linked to the γ and ϵ subunits. Thus, as the c ring turns, these subunits are turned inside the α3β3 hexamer unit of F1. The exterior column formed by the two b chains and the δ subunit prevent the α3β3 hexamer from rotating. Thus, the proton-gradient-driven rotation of the c ring drives the rotation of the γ subunit, which in turn promotes the synthesis of ATP through the binding-change mechanism. Recall that the number of c subunits in the c ring appears to range between 10 and 14. This number is significant because it determines the number of protons that must be transported to generate a molecule of ATP. Each 360-degree rotation of the γ subunit leads to the synthesis and release of three molecules of ATP. Thus, if there are 10 c subunits in the ring (as was observed in a crystal structure of yeast mitochondrial ATP synthase), each ATP generated requires the transport of 10/3 = 3.33 protons. For simplicity, we will assume that 3 protons must flow into the matrix for each ATP formed, but we must keep in mind that the true value may differ.

18.4.5. ATP Synthase and G Proteins Have Several Common Features

Image tree.jpg The α and β subunits of ATP synthase are members of the P-loop NTPase family of proteins. In Chapter 15, we learned that the signaling properties of other members of this family, the G proteins, depend on their ability to bind nucleoside triphosphates and nucleoside diphosphates with great kinetic tenacity. They do not exchange nucleotides unless they are stimulated to do so by interaction with other proteins. The binding-change mechanism of ATP synthase is a variation on this theme. The three different faces of the γ subunit of ATP synthase interact with the P-loop regions of the β subunits to favor the structures of either the NDP- or NTP-binding forms or to facilitate nucleotide release. The conformational changes take place in an orderly way, driven by the rotation of the γ subunit.

By agreement with the publisher, this book is accessible by the search feature, but cannot be browsed.

Copyright © 2002, W. H. Freeman and Company.
Bookshelf ID: NBK22388