19.2 ATP Synthesis in Chapter 19 Oxidative Phosphorylation

In the Chemiosmotic Model, Oxidation and Phosphorylation Are Obligately Coupled

The chemiosmotic model, proposed by Peter Mitchell, is the paradigm for energy coupling. According to the model (Fig. 19-19), the electrochemical energy inherent in the difference in proton concentration and the separation of charge across the inner mitochondrial membrane — the proton-motive force — drives the synthesis of ATP as protons flow passively back into the matrix through a proton pore in ATP synthase. To emphasize this crucial role of the proton-motive force, the equation for ATP synthesis is sometimes written

ADP + P_{i} + n H_{P}^{+} \to ATP + H_{2} O + n H_{N}^{+}

$ADP plus upper P Subscript i Baseline plus n upper H Subscript upper P Superscript plus Baseline right-arrow ATP plus upper H Subscript 2 Baseline upper O plus n upper H Subscript upper N Superscript plus$

(19-10)

A figure shows a representation of chemiosmotic theory using the respiratory chain on an inner mitochondrial membrane. — FIGURE 19-19 Chemiosmotic model. In this simple representation of the chemiosmotic theory applied to mitochondria, electrons from NADH and other oxidizable substrates pass through a chain of carriers arranged asymmetrically in the inner membrane. Electron flow is accompanied by proton transfer across the membrane, producing both a chemical gradient $(Δ pH)$ $left-parenthesis normal upper Delta pH right-parenthesis$ and an electrical gradient $(Δ ψ)$ $left-parenthesis normal upper Delta psi right-parenthesis$ , which, combined, create the proton-motive force. The inner mitochondrial membrane is impermeable to protons; protons can reenter the matrix only through proton-specific channels $(F_{o})$ $left-parenthesis upper F Subscript o Baseline right-parenthesis$ . The proton-motive force that drives protons back into the matrix provides the energy for ATP synthesis, catalyzed by the $F_{1}$ $upper F Subscript 1$ complex associated with $F_{o}$ $upper F Subscript o$ .

FIGURE 19-19 Chemiosmotic model. In this simple representation of the chemiosmotic theory applied to mitochondria, electrons from NADH and other oxidizable substrates pass through a chain of carriers arranged asymmetrically in the inner membrane. Electron flow is accompanied by proton transfer across the membrane, producing both a chemical gradient $(Δ pH)$ $left-parenthesis normal upper Delta pH right-parenthesis$ and an electrical gradient $(Δ ψ)$ $left-parenthesis normal upper Delta psi right-parenthesis$ , which, combined, create the proton-motive force. The inner mitochondrial membrane is impermeable to protons; protons can reenter the matrix only through proton-specific channels $(F_{o})$ $left-parenthesis upper F Subscript o Baseline right-parenthesis$ . The proton-motive force that drives protons back into the matrix provides the energy for ATP synthesis, catalyzed by the $F_{1}$ $upper F Subscript 1$ complex associated with $F_{o}$ $upper F Subscript o$ .

A horizontal piece of phospholipid membrane is shown with the region above labeled intermembrane space (P side) and the region below labeled matrix (N side). There are red highlighted plus symbols across the top boundary of the membrane and red highlighted minus symbols across the bottom boundary of the membrane. Four complexes of the respiratory chain are shown embedded in the membrane. Complex Roman numeral 1 is shown as a pink structure with a horizontal left side that bends down to a rounded portion below the membrane to the lower right. To the right, complex Roman numeral 2 is shown with a narrow vertical piece across the membrane above a rounded portion below the membrane. To the right, complex Roman numeral 3 is shown as an irregular blue shape that is wider on top and narrows as it passes through the membrane, then has protrusions to the left and right beneath the membrane, then widens to a rounded portion with a small invagination at the bottom center. To its right, complex Roman numeral 4 is green and almost rectangular but extends down farther to the lower left and right and has a purple circle on its upper left. A red vertical arrow begins below the membrane and points through the left side of the horizontal portion of complex Roman numeral 1 to end at red highlighted 4 H plus above the membrane. An arrow curves from N A D H plus H plus to the bottom left of complex Roman numeral 1, then curves down to end at N A D plus. At the point where this curved arrow meets complex Roman numeral 1, a blue arrow points up to a yellow square labeled Q. A blue arrow from this yellow square labeled Q meets a red arrow from red highlighted 2 H plus to the left of the complex and continues right to end at a yellow box labeled Q H 2 to the right of the complex. A dashed arrow points from the yellow box labeled Q H 2 across the top of complex Roman numeral 2 to a similar yellow box labeled Q H 2 to the right of the second complex. A curved arrow beneath complex Roman numeral 2 begins at succinate, curves through the bottom of the complex, and ends at fumarate. A blue arrow points from the top of this curve from succinate to fumarate to the center of complex Roman numeral 2, from which a second blue arrow points to the yellow box labeled Q H 2 in the membrane to the right. A blue arrow points from this yellow box to the top center of complex Roman numeral 3. A red arrow points from red highlighted 2 H plus beneath complex Roman numeral 3 to the top center of the complex, where it meets the blue arrow from the yellow box labeled Q H 2. A second red arrow continues out of the complex to red highlighted 4 H plus above the complex. A second blue arrow curves up to a purple circle labeled cyt italicized c end italics at top of complex Roman numeral 3, just to the left of the center. A dashed arrow points from this purple circle to a similar purple circle on top of complex Roman numeral 4, just to the right of the enter. A curved arrow points from 2 H plus plus one-half O 2 to the center of complex Roman numeral 4, then out to H 2 O below the complex. A blue arrow points down from the purple circle representing c y t italicized c end italics to the inflection point of the arrow from 2 H plus plus one-half O 2 to H 2 O. A red arrow enters complex Roman numeral 4 at the bottom left and runs up it to exit at the upper right at red highlighted 2 H plus. An orange structure to the right has a square portion in the membrane labeled F subscript 0 end subscript with a rectangular piece to its left. A cylindrical piece that is wider at its top extends down from this square to a rounded structure below that has three roughly oval pieces and is labeled F subscript 1 end subscript. An arrow runs through the right side of this structure showing the addition of A D P plus P I and the production of A T P. A curved thin piece exits the bottom of this rounded structure and curves up along its left side to end next to the base of the structure at the top of the membrane. A red arrow begins above the membrane, runs through the rectangular part of the base, and bends left to cross the thin curved piece to end at italicized n end italics H plus. Text reads, proton-motive force above two light red boxes with a plus sign between them. The left-hand box reads, chemical potential, delta p H (inside alkaline). The right-hand box reads, electrical potential, delta psi (inside negative). An arrow points right to a light red box reading, A T P synthesis driven by proton-motive force.

A photo of Peter Mitchell shows a smiling white-haired man in glasses. — Peter Mitchell, 1920–1992

Mitchell used the term “chemiosmotic” to describe enzymatic reactions that involve, simultaneously, a chemical reaction and a transport process, and the overall process is sometimes referred to as “chemiosmotic coupling.” Here, “coupling” refers to the obligate connection between mitochondrial ATP synthesis and electron flow through the respiratory chain; neither of the two processes can proceed without the other. The operational definition of coupling is shown in Figure 19-20. When isolated mitochondria are suspended in a buffer containing ADP, $P_{i}$ $upper P Subscript i$ , and an oxidizable substrate such as succinate, three easily measured processes occur: (1) the substrate is oxidized (succinate yields fumarate), (2) $O_{2}$ $upper O Subscript 2$ is consumed, and (3) ATP is synthesized. Oxygen consumption and ATP synthesis depend on the presence of an oxidizable substrate (succinate in this case) as well as ADP and $P_{i}$ $upper P Subscript i$ .

A two-part figure shows graphs plotting O 2 consumption and A T P synthesis over time with the addition of A D P and P i followed by the addition of succinate and then the addition of cyanide in part a and with the addition of succinate, then A D P plus pi, then venturicidin or oligomycin, then D N P in part b. — FIGURE 19-20 Coupling of electron transfer and ATP synthesis in mitochondria. In experiments to demonstrate coupling, mitochondria are suspended in a buffered medium, and an $O_{2}$ $upper O Subscript 2$ electrode monitors $O_{2}$ $upper O Subscript 2$ consumption. At intervals, samples are removed and assayed for the presence of ATP. (a) Addition of ADP and $P_{i}$ $upper P Subscript i$ alone results in little or no increase in either respiration ( $O_{2}$ $upper O Subscript 2$ consumption; black) or ATP synthesis (red). When succinate is added, respiration begins immediately, and ATP is synthesized. Addition of cyanide $({CN}^{-})$ $left-parenthesis CN Superscript minus Baseline right-parenthesis$ , which blocks electron transfer between cytochrome oxidase (Complex IV) and $O_{2}$ $upper O Subscript 2$ , inhibits both respiration and ATP synthesis. (b) Mitochondria provided with succinate respire and synthesize ATP only when ADP and $P_{i}$ $upper P Subscript i$ are added. Subsequent addition of venturicidin or oligomycin, inhibitors of ATP synthase, blocks both ATP synthesis and respiration. Dinitrophenol (DNP) is an uncoupler, allowing respiration to continue without ATP synthesis.

FIGURE 19-20 Coupling of electron transfer and ATP synthesis in mitochondria. In experiments to demonstrate coupling, mitochondria are suspended in a buffered medium, and an $O_{2}$ $upper O Subscript 2$ electrode monitors $O_{2}$ $upper O Subscript 2$ consumption. At intervals, samples are removed and assayed for the presence of ATP. (a) Addition of ADP and $P_{i}$ $upper P Subscript i$ alone results in little or no increase in either respiration ( $O_{2}$ $upper O Subscript 2$ consumption; black) or ATP synthesis (red). When succinate is added, respiration begins immediately, and ATP is synthesized. Addition of cyanide $({CN}^{-})$ $left-parenthesis CN Superscript minus Baseline right-parenthesis$ , which blocks electron transfer between cytochrome oxidase (Complex IV) and $O_{2}$ $upper O Subscript 2$ , inhibits both respiration and ATP synthesis. (b) Mitochondria provided with succinate respire and synthesize ATP only when ADP and $P_{i}$ $upper P Subscript i$ are added. Subsequent addition of venturicidin or oligomycin, inhibitors of ATP synthase, blocks both ATP synthesis and respiration. Dinitrophenol (DNP) is an uncoupler, allowing respiration to continue without ATP synthesis.

In both graphs, the horizontal axis is labeled time. In part a, a black curve labeled O 2 consumption begins on the left just above the horizontal axis. Just to the right, an arrow shows that A D P and P i are added. The curve increases slowly to a point almost halfway across the horizontal axis and just under one-quarter of the height of the vertical axis, where an arrow indicates that succinate is added. The curve increases rapidly to a point just past halfway across the vertical axis at two-thirds of the height of the vertical axis, where an arrow indicates that C N minus is added. After this point, the curve runs horizontally to the right end of the graph. A red curve labeled A T P synthesis begins just below the black curve on the left side of the horizontal axis, runs horizontally past the point where A D P and P I are added, and then begins to increase rapidly once succinate is added at almost halfway across the horizontal axis. The curve levels off at just past halfway across the horizontal axis at half the height of the vertical axis at the point labeled add C N minus, then runs horizontally to the right side of the graph. In part b, a black curve labeled O 2 consumption begins on the left just above the horizontal axis. Just to the right, an arrow shows that succinate is added. The curve increases slightly to a point at one-quarter of the length across the horizontal axis and just under one-quarter of the height of the vertical axis, at which point an arrow indicates that A D P and P I are added. The curve increases rapidly to a point one-third of the way across the horizontal axis and two-thirds of the height of the vertical axis, where an arrow indicates that venturicidin or oligomycin is added. The curve increases slowly to a point two-thirds of the way across the horizontal axis and only slightly higher, when D N P is added. The curve then increases rapidly to meet the top of the graph at three-quarters of the length of the horizontal axis. A red curve labeled A T P synthesis begins on the left just below the black curve. Just to the right, an arrow shows that succinate is added. The curve runs horizontally to a point at one-quarter of the length across the horizontal axis, at which point an arrow indicates that A D P and P I are added. The curve increases rapidly to a point one-third of the way across the horizontal axis and half the height of the vertical axis, where an arrow indicates that venturicidin or oligomycin is added. The curve runs horizontally to the right end of the graph with no change at the point two-thirds of the way across the horizontal axis when D N P is added. Lines point to the upward end of the black curve and the horizontal end of the red curve, showing how much they have diverged, are labeled, uncoupled. All data are approximate.

Because substrate oxidation drives ATP synthesis, inhibitors of electron transfer block ATP synthesis (Fig. 19-20a). The converse is also true: inhibition of ATP synthesis blocks electron transfer in intact mitochondria. When isolated mitochondria are given $O_{2}$ $upper O Subscript 2$ and oxidizable substrates, but not ADP (Fig. 19-20b), no ATP synthesis can occur and electron transfer to $O_{2}$ $upper O Subscript 2$ does not proceed. Henry Lardy, who pioneered the use of antibiotics to explore mitochondrial function, showed coupling of oxidation and phosphorylation by using oligomycin and venturicidin. These toxic antibiotics bind to the ATP synthase in mitochondria, inhibiting both ATP synthesis and the transfer of electrons through the chain of carriers to $O_{2}$ $upper O Subscript 2$ (Fig. 19-20b). As oligomycin does not interact with the electron carriers, it follows that electron transfer and ATP synthesis are obligately coupled: neither reaction occurs without the other.

A photo shows Henry Lardy in a white lab coat holding a notebook and pen while leaning on a table containing a large cylindrical piece of equipment with vertical bars on all sides. — Henry Lardy, 1917–2010

Chemiosmotic theory readily explains the dependence of electron transfer on ATP synthesis in mitochondria. When the flow of protons into the matrix through the proton channel of ATP synthase is blocked (with oligomycin, for example), no path exists for the return of protons to the matrix, and the continued extrusion of protons driven by the activity of the respiratory chain generates a large proton gradient. The proton-motive force builds up until the cost (free energy) of pumping protons out of the matrix against this gradient equals or exceeds the energy released by the transfer of electrons from NADH to $O_{2}$ $upper O Subscript 2$ . At this point electron flow must stop; the free energy for the overall process of electron flow coupled to proton pumping becomes zero, and the system is at equilibrium.

Certain conditions and reagents, however, can uncouple oxidation from phosphorylation. When intact mitochondria are disrupted by treatment with detergent or by physical shear, the resulting membrane fragments can still catalyze electron transfer from succinate or NADH to $O_{2}$ $upper O Subscript 2$ , but no ATP synthesis is coupled to this respiration. Certain chemical compounds cause uncoupling without physically disrupting mitochondrial structure. Chemical uncouplers include 2,4-dinitrophenol (DNP) and carbonylcyanide-p-trifluoromethoxyphenylhydrazone (FCCP) (Table 19-4; Fig. 19-21), weak acids with hydrophobic properties that permit them to diffuse readily across mitochondrial membranes. After entering the matrix in the protonated form, they can release a proton, thus dissipating the proton gradient. Resonance stabilization delocalizes the charge on the anionic forms, making them sufficiently hydrophobic to diffuse back across the membrane, where they can pick up a proton and repeat the process. Ionophores such as valinomycin (see Fig. 11-43) allow inorganic ions to pass easily through membranes. Ionophores uncouple electron transfer from oxidative phosphorylation by dissipating the electrical contribution to the electrochemical gradient across the mitochondrial membrane.

A figure shows the structures of two chemical uncouplers of oxidative phosphorylation, 2, 4-dinitrophenol (D N P) and carbonylcyanide-italicized p end italics-trifluoromethoxyphenylhydrazone (F C C P). — FIGURE 19-21 Two chemical uncouplers of oxidative phosphorylation. Both DNP and FCCP have a dissociable proton (red) and are very hydrophobic. They carry protons across the inner mitochondrial membrane, dissipating the proton gradient. Both also uncouple photophosphorylation.

A prediction of the chemiosmotic theory is that, because the role of electron transfer in mitochondrial ATP synthesis is simply to pump protons to create the electrochemical potential of the proton-motive force, an artificially created proton gradient should be able to replace electron transfer in driving ATP synthesis. This has been experimentally confirmed (Fig. 19-22). In the absence of an oxidizable substrate, the proton-motive force alone suffices to drive ATP synthesis.

A two-part figure shows evidence for the role of a proton gradient in A T P synthesis by showing isolated mitochondria incubated in a p H 9 buffer containing 0.1 M K C l in part a and then mitochondria in a p H 7 buffer containing valinomycin with no K C l in part b. — FIGURE 19-22 Evidence for the role of a proton gradient in ATP synthesis. An artificially imposed electrochemical gradient can drive ATP synthesis in the absence of an oxidizable substrate as electron donor. In this two-step experiment, (a) isolated mitochondria are first incubated in a pH 9 buffer containing 0.1 m KCl. Slow leakage of buffer and KCl into the mitochondria eventually brings the matrix into equilibrium with the surrounding medium. No oxidizable substrates are present. (b) Mitochondria are now removed from the pH 9 buffer and resuspended in pH 7 buffer containing valinomycin but no KCl. The change in buffer creates a difference of two pH units across the inner mitochondrial membrane. The outward flow of $K^{+}$ $upper K Superscript plus$ , carried by valinomycin down the $K^{+}$ $upper K Superscript plus$ ion concentration gradient without a counterion, creates a charge imbalance across the membrane (matrix negative). The sum of the chemical potential provided by the pH difference and the electrical potential provided by the separation of charges is a proton-motive force large enough to support ATP synthesis in the absence of an oxidizable substrate.

FIGURE 19-22 Evidence for the role of a proton gradient in ATP synthesis. An artificially imposed electrochemical gradient can drive ATP synthesis in the absence of an oxidizable substrate as electron donor. In this two-step experiment, (a) isolated mitochondria are first incubated in a pH 9 buffer containing 0.1 m KCl. Slow leakage of buffer and KCl into the mitochondria eventually brings the matrix into equilibrium with the surrounding medium. No oxidizable substrates are present. (b) Mitochondria are now removed from the pH 9 buffer and resuspended in pH 7 buffer containing valinomycin but no KCl. The change in buffer creates a difference of two pH units across the inner mitochondrial membrane. The outward flow of $K^{+}$ $upper K Superscript plus$ , carried by valinomycin down the $K^{+}$ $upper K Superscript plus$ ion concentration gradient without a counterion, creates a charge imbalance across the membrane (matrix negative). The sum of the chemical potential provided by the pH difference and the electrical potential provided by the separation of charges is a proton-motive force large enough to support ATP synthesis in the absence of an oxidizable substrate.

Part a shows a beaker three-quarters filled with light beige fluid containing mitochondria. An arrow points to a close-up to the right. A wavy membrane extends from the left to right. The region above the membrane is labeled matrix and the region below the membrane is labeled intermembrane space. A small piece of membrane made up of small pieces is shown at the lower right. Text in the matrix reads [K plus] equals [C l minus] equals 0.1 M and [H plus] equals 10 superscript negative 9 end superscript M. A T P synthase is shown with a rectangular part in the membrane and a rounded part extending into the matrix, where it is labeled F subscript 0 end subscript F subscript 1 end subscript. A thin strand begins at the top of the rounded part and runs down along the right side of the rectangular part. Text in the intermembrane space reads [H plus] equals 10 superscript negative 9 end superscript M. An arrow points downward to a similar illustration in part b accompanied by text reading, p H lowered from 9 to 7; valinomycin added; no added K plus. To the left of the arrow, a rectangle is light beige on top, where it is labeled p H 9 and darker beige on the bottom, where it is labeled p H 7. Part b shows a similar beaker three-quarters filled with dark beige fluid containing mitochondria. An arrow points to a close-up to the right. The intermembrane space is dark beige. One of the folds in the membrane has minus symbols all along the matrix side and plus signs all along the intermembrane space side. A yellow circle containing a green circle labeled K plus is next to arrows pointing diagonally to and from a similar yellow circle in the intermembrane space. The downward-pointing arrow has a branched arrow showing that the green circle labeled K plus separates from the larger yellow circle, which is now empty. A T P synthase is shown with a curved arrow next to the rounded top portion showing that A D P plus P I is added and A T P is produced. Text in the matrix reads, [K plus] less than symbol less than symbol [C l minus]. Red highlighted text in the intermembrane space beneath A T P synthase reads, [H plus] equals 10 superscript negative 7 end superscript M. An arrow points from this red text through the right side of A T P synthase to red text in the matrix reading, [H plus] equals 10 superscript negative 9 end superscript M.

ATP Synthase Has Two Functional Domains, $F_{0}$ $bold upper F Subscript bold 0$ and $F_{1}$ $bold upper F Subscript 1$

Mitochondrial ATP synthase is an F-type ATPase (see Fig. 11-40b) similar in structure and mechanism to the ATP synthases of bacteria and (as we will see in Chapter 20) chloroplasts. This large enzyme complex of the inner mitochondrial membrane catalyzes the formation of ATP from ADP and $P_{i}$ $upper P Subscript i$ , driven by the flow of protons from the p to the n side of the membrane (Eqn 19-10). ATP synthase, also called Complex V to relate it to the electron-transfer complexes described in the last section, has two distinct components. These are $F_{1}$ $upper F Subscript 1$ , a peripheral membrane protein, and $F_{o}$ $upper F Subscript o$ (o denoting oligomycin-sensitive), which is integral to the membrane. $F_{1}$ $upper F Subscript 1$ , the first factor recognized as essential for oxidative phosphorylation, was identified and purified by Efraim Racker and his colleagues in the early 1960s.

In the laboratory, small membrane vesicles formed from inner mitochondrial membranes carry out ATP synthesis coupled to electron transfer. When $F_{1}$ $upper F Subscript 1$ is gently extracted, the “stripped” vesicles still contain intact respiratory chains and the $F_{o}$ $upper F Subscript o$ portion of ATP synthase. The vesicles can catalyze electron transfer from NADH to $O_{2}$ $upper O Subscript 2$ but cannot produce a proton gradient: $F_{o}$ $upper F Subscript o$ has a proton pore through which protons leak as fast as they are pumped by electron transfer, and without a proton gradient the $F_{1}$ $upper F Subscript 1$ -depleted vesicles cannot make ATP. Isolated $F_{1}$ $upper F Subscript 1$ catalyzes ATP hydrolysis (the reversal of synthesis) and was therefore originally called $bold upper F bold 1$ $bold upper F bold 1$ ATPase. When purified $F_{1}$ $upper F Subscript 1$ is added back to the depleted vesicles, it reassociates with $F_{o}$ $upper F Subscript o$ , plugging its proton pore and restoring the membrane’s capacity to couple electron transfer and ATP synthesis.

ATP Is Stabilized Relative to ADP on the Surface of $F_{1}$ $bold upper F Subscript bold 1$

Isotope exchange experiments using purified $F_{1}$ $upper F Subscript 1$ reveal an extraordinary fact about the enzyme’s catalytic mechanism: on the enzyme surface, the reaction $ADP + P_{i} ⇌ ATP + H_{2} O$ $ADP plus upper P Subscript i Baseline right harpoon over left harpoon ATP plus upper H Subscript 2 Baseline upper O$ is readily reversible — the free-energy change for ATP synthesis is close to zero. When ATP is hydrolyzed by $F_{1}$ $upper F Subscript 1$ in the presence of $^{18} O$ $Superscript 18 Baseline upper O$ -labeled water, the $P_{i}$ $upper P Subscript i$ released contains an $^{18} O$ $Superscript 18 Baseline upper O$ atom. Careful measurement of the $^{18} O$ $Superscript 18 Baseline upper O$ content of $P_{i}$ $upper P Subscript i$ formed in vitro by $F_{1}$ $upper F Subscript 1$ -catalyzed hydrolysis of ATP reveals that the $P_{i}$ $upper P Subscript i$ has not one but three or four $^{18} O$ $Superscript 18 Baseline upper O$ atoms (Fig. 19-23). This indicates that the terminal pyrophosphate bond in ATP is cleaved and re-formed repeatedly before $P_{i}$ $upper P Subscript i$ leaves the enzyme surface. This exchange reaction occurs in unenergized $F_{o} F_{1}$ $upper F Subscript o Baseline upper F Subscript 1$ complexes (with no proton gradient) and with isolated $F_{1}$ $upper F Subscript 1$ — the exchange does not require the input of energy.

A two-part figure shows the catalytic mechanism of F subscript 1 end subscript by showing how labeled oxygen is incorporated into phosphate produced by hydrolysis in part a and by showing the likely transition state complex for A T P hydrolysis and synthesis by A T P synthase in part b. — FIGURE 19-23 Catalytic mechanism of $F_{1}$ $upper F Subscript 1$ . (a) An $^{18} O$ $Superscript 18 Baseline upper O$ -exchange experiment. $F_{1}$ $upper F Subscript 1$ solubilized from mitochondrial membranes is incubated with ATP in the presence of $^{18} O$ $Superscript 18 Baseline upper O$ -labeled water. At intervals, a sample of the solution is withdrawn and analyzed for the incorporation of $^{18} O$ $Superscript 18 Baseline upper O$ into the $P_{i}$ $upper P Subscript i$ produced from ATP hydrolysis. In minutes, the $P_{i}$ $upper P Subscript i$ contains three or four $^{18} O$ $Superscript 18 Baseline upper O$ atoms, indicating that both ATP hydrolysis and ATP synthesis have occurred several times during the incubation. (b) The likely transition state complex for ATP hydrolysis and synthesis by ATP synthase. The α subunit is shown in gray, β in purple. The positively charged residues $β {-Arg}^{182}$ $beta hyphen Arg Superscript 182$ and $α {-Arg}^{376}$ $alpha hyphen Arg Superscript 376$ coordinate two oxygens of the pentavalent phosphate intermediate; $β {-Lys}^{155}$ $beta hyphen Lys Superscript 155$ interacts with a third oxygen, and the ${Mg}^{2 +}$ $Mg Superscript 2 plus$ ion further stabilizes the intermediate. The blue sphere represents the leaving group $(H_{2} O)$ $left-parenthesis upper H Subscript 2 Baseline upper O right-parenthesis$ . These interactions result in ready equilibration of ATP and $ADP + P_{i}$ $ADP plus upper P Subscript i Baseline$ in the active site. [(b) Data from PDB ID 1BMF, J. P. Abrahams et al., *Nature* 370:621, 1994.]

Part a shows A T P synthase on the right with a square base labeled F subscript 0 end subscript with a cylindrical piece extending up that is narrower at its base, where it joins with the rectangle. This cylinder extends up into a rounded portion consisting of vertical ovals. The front oval is gray and is labeled F subscript 1 end subscript alpha and the ovals to the left and right are each purple and labeled beta. A thin piece extends from the top of this circular part and curves along the left side to end by running along a thin rectangular piece that joins with the square base. An arrow points left to a purple crescent that opens to the left and that is labeled enzyme (F subscript 1 end subscript). Within the opening, an equation is shown as A T P italicized plus end italics H 2 superscript 18 end superscript O. Four sets of upward- and downward-pointing arrows are shown lined up vertically beneath this equation. A T P is beneath these sets of arrows above an italicized plus symbol above a labeled phosphate. The phosphate has a central P hashed wedge bonded to superscript 18 end superscript O above, solid wedge bonded to superscript 18 end superscript O to the right, hashed wedge bonded to superscript 18 end superscript O below, and solid wedge bonded to superscript 18 end superscript O to the left. Each pair of O atoms has a dashed curve connecting them. A second arrow from A T P synthase points to a structure in part b below. A purple helix extends diagonally from the left side to the top center. Beta-G l u superscript 181 end superscript is shown as a wireframe extending from near the top end of this purple helix. It has a fork with two red ends representing O and one of these has a dashed blue line to a blue sphere labeled H 2 O to the lower right. Behind this blue sphere, there is a purple wireframe running up to the helix above and down to a blue bend that connects to a purple fork with two blue ends. This is labeled beta-Arg superscript 182 end superscript. An orange structure beneath the blue sphere has three projecting ends that are all red. There are dashed blue lines between the red ends to the rear left and right and to the blue ends of the purple fork to the right. The right rear red end also has a dashed blue line down to the blue end of a fork with two blue ends attached to a gray chain below and labeled alpha-Arg superscript 376 end superscript. The front red end has two dashed blue lines, one to a blue end to the left in front of a green sphere labeled M g 2 plus and further bonded to a purple wireframe labeled beta-Lys superscript 155 end superscript that is further connected to a purple ribbon and the second to a red end of an orange piece with three red ends that is further connected to the rest of A D P, with the other phosphates, ring, and double-ring adenine all shown below.

Kinetic studies of the initial rates of ATP synthesis and hydrolysis confirm the conclusion that $Δ G^{'} °$ $normal upper Delta upper G prime degree$ for ATP synthesis on the enzyme is near zero. From the measured rates of hydrolysis $(k_{1} = 10 s^{- 1})$ $left-parenthesis k 1 equals 10 s Superscript negative 1 Baseline right-parenthesis$ and synthesis $(k_{-}_{1} = 24 s^{- 1})$ $left-parenthesis k Subscript minus Subscript 1 Baseline equals 24 s Superscript negative 1 Baseline right-parenthesis$ , the calculated equilibrium constant for the reaction

Enz-ATP ⇌ {Enz-(ADP + P}_{i})

$Enz hyphen ATP right harpoon over left harpoon Enz hyphen left-parenthesis ADP plus upper P Subscript i Baseline right-parenthesis$

is

{K^{'}}_{eq} = \frac{k_{- 1}}{k_{1}} = \frac{24 s^{- 1}}{10 s^{- 1}} = 2.4

$upper K prime Subscript eq Baseline equals StartFraction k Subscript negative 1 Baseline Over k 1 EndFraction equals StartFraction 24 s Superscript negative 1 Baseline Over 10 s Superscript negative 1 Baseline EndFraction equals 2.4$

From this ${K^{'}}_{eq}$ $upper K prime Subscript eq$ , the calculated apparent $Δ G^{'} °$ $normal upper Delta upper G prime degree$ is close to zero. This is much different from the ${K^{'}}_{eq}$ $upper K prime Subscript eq$ of about $10^{5} (Δ G^{'} ° = - 30.5 kJ/mol)$ $10 Superscript 5 Baseline left-parenthesis normal upper Delta upper G Superscript prime Baseline degree equals negative 30.5 kJ slash mol right-parenthesis$ for the hydrolysis of ATP free in solution (i.e., not on the enzyme surface).

What accounts for the huge difference? ATP synthase stabilizes ATP relative to $ADP + P_{i}$ $ADP plus upper P Subscript i Baseline$ by binding ATP more tightly, releasing enough energy to counterbalance the cost of making ATP. Careful measurements of the binding constants show that $F_{o} F_{1}$ $upper F Subscript o Baseline upper F Subscript 1$ binds ATP with very high affinity $(K_{d} \leq 10^{- 12} M)$ $left-parenthesis upper K Subscript d Baseline less-than-or-equal-to 10 Superscript negative 12 Baseline upper M right-parenthesis$ and ADP with much lower affinity $(K_{d} \approx 10^{- 5} M)$ $left-parenthesis upper K Subscript d Baseline almost-equals 10 Superscript negative 5 Baseline upper M right-parenthesis$ . The difference in $K_{d}$ $upper K Subscript d$ corresponds to a difference of about 40 kJ/mol in binding energy, and this binding energy drives the equilibrium toward formation of the product ATP.

The Proton Gradient Drives the Release of ATP from the Enzyme Surface

Although ATP synthase equilibrates ATP with $ADP + P_{i}$ $ADP plus upper P Subscript i Baseline$ , in the absence of a proton gradient the newly synthesized ATP does not leave the surface of the enzyme. Effectively, the enzyme cannot turn over and synthesize a second molecule of ATP. It is the proton gradient that causes the enzyme to release the ATP formed on its surface. The reaction coordinate diagram of the process (Fig. 19-24) illustrates the difference between the mechanism of ATP synthase and that of many other enzymes that catalyze endergonic reactions.

A figure shows two reaction coordinate diagrams, one for a typical enzyme and one for A T P synthase. — FIGURE 19-24 Reaction coordinate diagrams for ATP synthase and for a more typical enzyme. In a typical enzyme-catalyzed reaction (left), reaching the transition state $(‡)$ $left-parenthesis double-dagger right-parenthesis$ between substrate and product is the major energy barrier to overcome. In the reaction catalyzed by ATP synthase (right), release of ATP from the enzyme, not formation of ATP, is the major energy barrier. The free-energy change for the formation of ATP from ADP and $P_{i}$ $upper P Subscript i$ in aqueous solution is large and positive, but on the enzyme surface, the very tight binding of ATP provides sufficient binding energy to bring the free energy of the enzyme-bound ATP close to that of $ADP + P_{i}$ $ADP plus upper P Subscript i Baseline$ , so the reaction is readily reversible. The equilibrium constant is near 1. The free energy required for the release of ATP is provided by the proton-motive force.

FIGURE 19-24 Reaction coordinate diagrams for ATP synthase and for a more typical enzyme. In a typical enzyme-catalyzed reaction (left), reaching the transition state $(‡)$ $left-parenthesis double-dagger right-parenthesis$ between substrate and product is the major energy barrier to overcome. In the reaction catalyzed by ATP synthase (right), release of ATP from the enzyme, not formation of ATP, is the major energy barrier. The free-energy change for the formation of ATP from ADP and $P_{i}$ $upper P Subscript i$ in aqueous solution is large and positive, but on the enzyme surface, the very tight binding of ATP provides sufficient binding energy to bring the free energy of the enzyme-bound ATP close to that of $ADP + P_{i}$ $ADP plus upper P Subscript i Baseline$ , so the reaction is readily reversible. The equilibrium constant is near 1. The free energy required for the release of ATP is provided by the proton-motive force.

On both graphs, the horizontal axis is labeled reaction coordinate and the vertical axis is labeled G (k J / mol), ranging from below 0 to 80 and labeled in increments of 20. In the graph labeled typical enzyme, a blue curve begins horizontally at the left side of the horizontal axis at 0 on the vertical axis above text reading, E plus S. The curve begins to rise at one-quarter of the length of the horizontal axis and peaks at halfway across the horizontal axis and 59 on the vertical axis. This peak is labeled with a double dagger above text reading, E S. The curve decreases slightly to a point at just past halfway across the horizontal axis and 45 on the vertical axis, then runs horizontally to end at a point labeled P at the right end of the graph. In the graph labeled A T P synthase, a red curve begins at the left side of the horizontal axis at 0 on the vertical axis and is labeled A D P plus P i. At one-eighth of the length of the horizontal axis, the curve rises slightly to reach 10 on the vertical axis slightly farther to the right, then decreases to a point at one-quarter of the length of the horizontal axis and negative 2 on the vertical axis. The curve runs horizontally from this point to just before halfway across the horizontal axis and this horizontal region is labeled E dot A D P plus P i. At almost halfway across the horizontal axis, the curve then runs up to 9 on the vertical axis and down to negative 4 on the vertical axis. Beginning at this point, just past halfway across the vertical axis and at negative 4 on the vertical axis, the line runs horizontally and is labeled E dot A T P. Slightly to the right, the curve increases rapidly to peak at just past three-quarters of the length of the horizontal axis at 58 on height of the vertical axis, then curves down slightly to level off near the right end of the horizontal axis at 45 on the vertical axis. It runs horizontally to the right end of the graph and accompanying text reads, A T P (in solution).

For the continued synthesis of ATP, the enzyme must cycle between a form that binds ATP very tightly and a form that releases ATP. Chemical and crystallographic studies of the ATP synthase have revealed the structural basis for this alternation in function.

Each β Subunit of ATP Synthase Can Assume Three Different Conformations

Mitochondrial $F_{1}$ $upper F Subscript 1$ has nine subunits of five different types, with the composition $α_{3} β_{3} γ δ ε$ $alpha 3 beta 3 gamma delta epsilon$ . Each of the three β subunits has one catalytic site for ATP synthesis. The crystallographic determination of the $F_{1}$ $upper F Subscript 1$ structure by John E. Walker and colleagues revealed structural details that help explain the catalytic mechanism of the enzyme. The knoblike portion of $F_{1}$ $upper F Subscript 1$ is a flattened sphere, 8 nm by 10 nm, consisting of alternating α and β subunits arranged like the sections of an orange (Fig. 19-25a–d). Although the amino acid sequences of the three β subunits are identical, their conformations differ. The conformational differences extend to differences in their ATP/ADP-binding sites. When the protein is crystallized in the presence of ADP and App(NH)p, a close structural analog of ATP that cannot be hydrolyzed by the ATPase activity of $F_{1}$ $upper F Subscript 1$ , the binding site of one of the three β subunits is filled with App(NH)p, the second is filled with ADP, and the third is empty. The corresponding β subunit conformations are designated β-ATP, β-ADP, and β-empty (Fig. 19-25b). This difference in nucleotide binding among the three subunits is critical to the mechanism of the complex. The polypeptides that make up the stalk in the $F_{1}$ $upper F Subscript 1$ crystal structure are asymmetrically arranged. One domain of the single γ subunit makes up a central shaft that passes through $F_{1}$ $upper F Subscript 1$ . Another globular domain of γ helps to stabilize the β-empty conformation in a β subunit it is transiently associated with (Fig. 19-25c).

A figure shows the structure of App (N H) p (beta, gamma-imidoadenosine 5 prime-triphosphate.

A five-membered ring has O at its top vertex. C 1 prime at the right vertex is bonded to N of a five-membered ring above and to H below; C 2 prime and C 3 prime are each bonded to H above and O H below; C 4 prime is bonded to C 5 above and H below, and C 5 prime is bonded to 2 H and to O further bonded to P double bonded to O above, bonded to O minus below, and bonded to O to the left further bonded to P double bonded to O above, bonded to O minus below, and bonded to red highlighted N to the left bonded to H above and further connected by a nonhydrolyzable beta to gamma bond to P to the left further double bonded to O above, bonded to O minus to the left, and bonded to O minus below. From right to left, the phosphate groups are labeled alpha for the group next to the ring, beta for the central group, and gamma for the group on the left. The five-membered ring shares its right side with six-membered ring. The five-membered ring has double bonds at its upper left side between C 7 and C 8 and at its right side shared with the six-membered ring between C 4 and C 5. The five-membered ring also has N substituted for C at the upper left vertex at position 7 and at the bottom left vertex at position 9, where it is further bonded to C 1 prime of the five-membered ring below. The six-membered ring has a double bond at its left side shared with the five-membered ring and double bonds at its upper right side between C 1 and C 6 and at its lower right side between C 2 and C 3. The six-membered ring also has N substituted for C at the upper right vertex at position 1 and at the bottom vertex at position 3 and has a top vertex at position 6 bonded to N H 2.

A figure shows views of the mitochondrial A T P synthase complex with a cartoon representation of the F subscript 0 end subscript F subscript 1 end subscript complex in part a, F subscript 1 end subscript viewed from above in part b, the entire enzyme viewed from the side in part c, and the bottom view of F subscript 0 end subscript in part d. — FIGURE 19-25 Mitochondrial ATP synthase complex. (a) A cartoon representation of the $F_{o} F_{1}$ $upper F Subscript o Baseline upper F Subscript 1$ complex. The dimeric form is found in eukaryotic mitochondria. The monomeric form is observed in bacteria. (b) $F_{1}$ $upper F Subscript 1$ viewed from above (that is, from the n side of the membrane), showing the three β (shades of purple) and three α (shades of gray) subunits and the central shaft (γ subunit, green). Each β subunit, near its interface with the neighboring α subunit, has a nucleotide-binding site critical to the catalytic activity. The single γ subunit associates primarily with one of the three $α β$ $alpha beta$ pairs, forcing each of the three β subunits into slightly different conformations, with different nucleotide-binding sites. In the crystalline enzyme, one subunit, β-ADP, has ADP (yellow) in its binding site; the next, β-ATP, has ATP (red); and the third, β-empty, has no bound nucleotide. (c) The entire enzyme viewed from the side (in the plane of the membrane). The $F_{1}$ $upper F Subscript 1$ portion has three α subunits and three β subunits arranged like the segments of an orange around a central shaft, the γ subunit (green). (Two α subunits and one β subunit have been omitted to reveal the γ subunit and the binding sites for ATP and ADP on the β subunits.) The δ subunit confers oligomycin sensitivity on the ATP synthase, and the ε subunit may serve to inhibit the enzyme’s ATPase activity under some circumstances. The $F_{o}$ $upper F Subscript o$ subunit consists of one a subunit and two b subunits, which anchor the $F_{o} F_{1}$ $upper F Subscript o Baseline upper F Subscript 1$ complex in the membrane and act as a stator (the stationary part of a rotary system), holding the α and β subunits in place. $F_{o}$ $upper F Subscript o$ also includes the c ring, made up of a number (8 to 17, depending on the species) of identical c subunits, small, hydrophobic proteins. The c ring and the a subunit interact to provide a transmembrane path for protons. Each of the c subunits in $F_{o}$ $upper F Subscript o$ has a critical Asp residue near the middle of the membrane, which undergoes protonation/deprotonation during the catalytic cycle of the ATP synthase. Shown here is the homologous $c_{11}$ $c Subscript 11$ ring of the ${Na}^{+} -ATPase$ $Na Superscript plus Baseline zero width space hyphen ATPase$ of *Ilyobacter tartaricus*, for which the structure is well established. The ${Na}^{+}$ $Na Superscript plus$ -binding sites, which correspond to the proton-binding sites of the $F_{o} F_{1}$ $upper F Subscript o Baseline upper F Subscript 1$ complex, are shown with their bound ${Na}^{+}$ $Na Superscript plus$ ions (red spheres). (d) A view of $F_{o}$ $upper F Subscript o$ perpendicular to the membrane. As in (c), red spheres represent the ${Na}^{+} -$ $Na Superscript plus Baseline zero width space hyphen$ or proton-binding sites in Asp residues. [(a) Information from W. Kühlbrandt and K. M. Davies, *Trends Biochem. Sci.* 41:106, 2016. (b, c, d) Data from $F_{1}$ $upper F Subscript 1$ : PDB ID 1BMF, J. P. Abrahams et al., *Nature* 370:621, 1994; PDB ID 1JNV, A. C. Hausrath et al., *J. Biol. Chem.* 276:47,227, 2001; PDB ID 2A7U, S. Wilkens et al., *Biochemistry* 44:11,786, 2005; PDB ID 2CLY, V. Kane Dickson et al., *EMBO J.* 25:2911, 2006; $F_{o}$ $upper F Subscript o$ : PDB ID 1B9U, O. Dmitriev et al., *J. Biol. Chem.* 274:15,598, 1999; c ring: PDB ID 1YCE, T. Meier et al., *Science* 308:659, 2005.]

FIGURE 19-25 Mitochondrial ATP synthase complex. (a) A cartoon representation of the $F_{o} F_{1}$ $upper F Subscript o Baseline upper F Subscript 1$ complex. The dimeric form is found in eukaryotic mitochondria. The monomeric form is observed in bacteria. (b) $F_{1}$ $upper F Subscript 1$ viewed from above (that is, from the n side of the membrane), showing the three β (shades of purple) and three α (shades of gray) subunits and the central shaft (γ subunit, green). Each β subunit, near its interface with the neighboring α subunit, has a nucleotide-binding site critical to the catalytic activity. The single γ subunit associates primarily with one of the three $α β$ $alpha beta$ pairs, forcing each of the three β subunits into slightly different conformations, with different nucleotide-binding sites. In the crystalline enzyme, one subunit, β-ADP, has ADP (yellow) in its binding site; the next, β-ATP, has ATP (red); and the third, β-empty, has no bound nucleotide. (c) The entire enzyme viewed from the side (in the plane of the membrane). The $F_{1}$ $upper F Subscript 1$ portion has three α subunits and three β subunits arranged like the segments of an orange around a central shaft, the γ subunit (green). (Two α subunits and one β subunit have been omitted to reveal the γ subunit and the binding sites for ATP and ADP on the β subunits.) The δ subunit confers oligomycin sensitivity on the ATP synthase, and the ε subunit may serve to inhibit the enzyme’s ATPase activity under some circumstances. The $F_{o}$ $upper F Subscript o$ subunit consists of one a subunit and two b subunits, which anchor the $F_{o} F_{1}$ $upper F Subscript o Baseline upper F Subscript 1$ complex in the membrane and act as a stator (the stationary part of a rotary system), holding the α and β subunits in place. $F_{o}$ $upper F Subscript o$ also includes the c ring, made up of a number (8 to 17, depending on the species) of identical c subunits, small, hydrophobic proteins. The c ring and the a subunit interact to provide a transmembrane path for protons. Each of the c subunits in $F_{o}$ $upper F Subscript o$ has a critical Asp residue near the middle of the membrane, which undergoes protonation/deprotonation during the catalytic cycle of the ATP synthase. Shown here is the homologous $c_{11}$ $c Subscript 11$ ring of the ${Na}^{+} -ATPase$ $Na Superscript plus Baseline zero width space hyphen ATPase$ of *Ilyobacter tartaricus*, for which the structure is well established. The ${Na}^{+}$ $Na Superscript plus$ -binding sites, which correspond to the proton-binding sites of the $F_{o} F_{1}$ $upper F Subscript o Baseline upper F Subscript 1$ complex, are shown with their bound ${Na}^{+}$ $Na Superscript plus$ ions (red spheres). (d) A view of $F_{o}$ $upper F Subscript o$ perpendicular to the membrane. As in (c), red spheres represent the ${Na}^{+} -$ $Na Superscript plus Baseline zero width space hyphen$ or proton-binding sites in Asp residues. [(a) Information from W. Kühlbrandt and K. M. Davies, *Trends Biochem. Sci.* 41:106, 2016. (b, c, d) Data from $F_{1}$ $upper F Subscript 1$ : PDB ID 1BMF, J. P. Abrahams et al., *Nature* 370:621, 1994; PDB ID 1JNV, A. C. Hausrath et al., *J. Biol. Chem.* 276:47,227, 2001; PDB ID 2A7U, S. Wilkens et al., *Biochemistry* 44:11,786, 2005; PDB ID 2CLY, V. Kane Dickson et al., *EMBO J.* 25:2911, 2006; $F_{o}$ $upper F Subscript o$ : PDB ID 1B9U, O. Dmitriev et al., *J. Biol. Chem.* 274:15,598, 1999; c ring: PDB ID 1YCE, T. Meier et al., *Science* 308:659, 2005.]

Part a shows two A T P synthase in a piece of membrane and a single view of A T P synthase in a membrane. The top piece of membrane is bent so that it peaks in the center with one A T P synthase on each side. The top of the membrane is labeled N side and the bottom half is labeled P side. The left-hand A T P synthase has an orange rounded base made up of many cylinders lined up vertically as repeating units. Red arrows in each cylinder point to the right. A black counterclockwise arrow on the top of this entire structure, on the N side, indicates the direction of rotation. There is an opening in the center of the cylinder with a thin light green cylinder labeled gamma extending from it perpendicular to the membrane. A small blue oval labeled epsilon is on top of the orange rounded base and slightly to the right of center. The thin light green cylinder extends up through the center of a ring of vertical ovals. A blue oval labeled beta is in front. Clockwise from the front, the remaining ovals are a gray oval labeled alpha is to its left, a dark purple oval labeled beta to the rear left, a dark gray oval labeled alpha at the rear, a light purple oval labeled beta to the rear right, and a light gray oval labeled alpha at the right front. A small orange oval labeled delta is on top of the light green cylinder in the center and joins to a thin piece labeled b subscript 2 end subscript that runs horizontally to the end of the circle of ovals, then curves down to run along the side through the membrane, where there is a thin rectangle labeled a between it and the rounded base to its left. The thin rectangle labeled a is curved to fit against the rounded base. Red highlighted H plus is shown below the membrane. An arrow points up from this H plus through the thin rectangle, then to the left. Counterclockwise arrows are present along the outside of the rounded base. An arrow enters the thin rectangle labeled a higher up and emerges to point upward. A D P plus P I is shown to the left of the ring of ovals with a curved arrow that meets the ovals and then points to A T P. A similar A T P synthase is shown to the right in the reverse orientation, with the long thin piece to the left, then the rectangular piece labeled a, then the base. Below, text pointing to both A T P synthases reads, C subscript 8 end subscript to C subscript 17 end subscript (C subscript 10 end subscript in yeast mitochondria). A single A T P synthase is shown below in a horizontal piece of membrane. Part b is labeled top view of F subscript 1 end subscript. It has a green ribbon in the center surrounded by four even lobes from the left to right sides with two lobes below that each have a more triangular shape with an outer vertex. The left-hand lobe, beta-A D P, contains bright purple ribbon with a clump of orange spheres labeled A D P near the middle of its right side. Clockwise, the next lobe contains gray ribbon and is labeled alpha. The next lobe, beta-A T P, contains purple ribbon with a small red molecule labeled A T P near its bottom, close to the green center. The next lobe contains dark gray ribbon and is labeled alpha. The next lobe, labeled beta-empty, has a vertex on its outer edge and contains light purple ribbon. The final lobe, labeled alpha, has a vertex on its outer edge and contains light gray ribbon. Part c is labeled side view of F subscript 0 end subscript F subscript 1 end subscript. It has an orange piece at the base labeled a that is roughly rectangular, but curved on the left side and somewhat crescent-shaped on the right side to make space for a cylinder of alternating orange and yellow vertical helices. Just above the middle of these helices, there is a row of red spheres labeled proton-binding sites on A s p residues. The bottom of one of the orange ribbons is labeled c subscript 11 end subscript. This entire bottom portion is labeled F subscript 0 end subscript. A small, square structure consisting of blue ribbons is labeled epsilon and is at the upper left of this cylinder of helices. Two green vertical helices extend up from the center of the cylinder of helices and run upward through most of the structure. There are some additional helices to the right just above the base of helices below. There is an oval region of purple helices to the right of the two long green helices and a clump of spheres in the middle of it is labeled A T P. A single blue helix runs diagonally across its base. Gray helices are visible in the back. A similar structure of purple helices is to the left and contains a clump of yellow spheres labeled A D P. A small sphere of orange helices at the top center is labeled delta. Gold helices extend from the top center and down along the side. There are two almost vertical helices along the side labeled b subscript 2 end subscript. These join dashed lines just above the roughly rectangular structure labeled a, then form two helices again that run vertically to the left of the structure labeled a. The entire top portion of the structure, including the green, purple, and gray helices, is labeled F subscript 1 end subscript. Part d is labeled bottom view of F subscript 0 end subscript. An orange crescent-shaped piece is labeled a. To its left, there is a small linear structure made up of helices and labeled b subscript 2 end subscript. To the right, fitting into the curve of structure a, there is a circular structure that is open in the center with a symmetrical shape. There are greenish helices extending away from the observer along the center, all lined up evenly, with orange helices to their outside that curve around to produce circular openings. Most of these openings contain purple spheres labeled, N a plus – or proton-binding sites on A s p residues. The overall structure to the right of structure a is a symmetrical ring of open circles surrounded by helices.

The $F_{o}$ $upper F Subscript o$ complex, with its proton pore, is composed of three subunits, a, b, and c, in the proportion ${ab}_{2} c_{n}$ $ab Subscript 2 Baseline c Subscript n$ , where n ranges from 8 to 17, depending on the species. Subunit c is a small $(M_{r} 8,000)$ $left-parenthesis upper M Subscript r Baseline 8,000 right-parenthesis$ , very hydrophobic polypeptide, consisting almost entirely of two transmembrane helices, with a small loop extending from the matrix side of the membrane. The crystal structure of the yeast $F_{o} F_{1}$ $upper F Subscript o Baseline upper F Subscript 1$ shows 10 c subunits, each with two transmembrane helices roughly perpendicular to the plane of the membrane and arranged in two concentric circles to create the c ring. The inner circle is made up of the amino-terminal helices of each c subunit; the outer circle, about 55 Å in diameter, is made up of the carboxyl-terminal helices. The c subunits in the c ring rotate together as a unit around an axis perpendicular to the membrane. The $ε$ $epsilon$ and γ subunits of $F_{1}$ $upper F Subscript 1$ form a leg-and-foot that projects from the bottom (membrane) side of $F_{1}$ $upper F Subscript 1$ and stands firmly on the ring of c subunits. The a subunit consists of several hydrophobic helices that span the membrane in close association with one of the c subunits in the c ring.

Rotational Catalysis Is Key to the Binding-Change Mechanism for ATP Synthesis

On the basis of detailed kinetic and binding studies of the reactions catalyzed by $F_{o} F_{1}$ $upper F Subscript o Baseline upper F Subscript 1$ , Paul Boyer proposed a rotational catalysis mechanism in which the three active sites of $F_{1}$ $upper F Subscript 1$ take turns catalyzing ATP synthesis (Fig. 19-26). A given β subunit starts in the β-ADP conformation, which binds ADP and $P_{i}$ $upper P Subscript i$ from the surrounding medium. The subunit now changes conformation, assuming the β-ATP form that tightly binds and stabilizes ATP, bringing about the ready equilibration of $ADP + P_{i}$ $ADP plus upper P Subscript i Baseline$ with ATP on the enzyme surface. Finally, the subunit changes to the β-empty conformation, which has very low affinity for ATP, and the newly synthesized ATP leaves the enzyme surface. Another round of catalysis begins when this subunit again assumes the β-ADP form and binds ADP and $P_{i}$ $upper P Subscript i$ .

A figure shows the binding-change model for A D P synthase. — FIGURE 19-26 Binding-change model for ATP synthase. The $F_{1}$ $upper F Subscript 1$ complex has three nonequivalent adenine nucleotide–binding sites, one for each pair of α and β subunits. At any given moment, one of these sites is in the β-ATP conformation (which binds ATP tightly), a second is in the β-ADP (loose-binding) conformation, and a third is in the β-empty (very-loose-binding) conformation. In this view from the n side, the proton-motive force causes rotation of the central shaft — the γ subunit, shown as a green arrowhead — which comes into contact with each $α β$ $alpha beta$ subunit pair in succession. This produces a cooperative conformational change in which the β-ATP site is converted to the β-empty conformation, and ATP dissociates; the β-ADP site is converted to the β-ATP conformation, which promotes condensation of bound $ADP + P_{i}$ $ADP plus upper P Subscript i Baseline$ to form ATP; and the β-empty site becomes a β-ADP site, which loosely binds $ADP + P_{i}$ $ADP plus upper P Subscript i Baseline$ entering from the solvent. Note that the direction of rotation reverses when the ATP synthase is acting as an ATPase, as in the experiment depicted in Figure 19-27.

A figure shows three rounded structures, one at the top and others at the lower left and right sides. A rounded structure has two red-outlined lobes on the left that resemble rounded triangles that narrow toward the center. The left side lobe is labeled beta next to a yellow circle labeled A D P plus P i. The upper left lobe is similar and labeled alpha. The upper right and right lobes are gray and more rounded. The upper right lobe is labeled beta and contains an orange oval labeled A T P. The right side lobe is labeled alpha. The bottom two lobes have outer vertices instead of being rounded. A green arrow points from the center to the lower right lobe. The lower right lobe is labeled beta, has a dashed circle inside, and has an arrow from this dashed circle to an orange oval labeled A T P outside of the entire structure. The lower left lobe is labeled alpha. An arrow points down to the lower left accompanied by a curved arrow showing the addition of 3 H subscript P end subscript plus and loss of 3 H subscript N end subscript plus. This yields a similar structure that has rotated so that the upper left lobe is now at the lower right. The left side and upper left lobes, which had previously been at the upper right and right, are now outlined in red. The rounded left side lobe is labeled beta next to an orange oval labeled A T P and the upper left lobe is labeled alpha. The upper right and right side lobes have vertices. A green arrow points from the center to the upper right lobe. The upper right lobe is labeled beta and has a dashed circle from which an arrow points to an orange oval labeled A T P outside of the structure. The right side lobe is labeled alpha. The lower left and right lobes resemble rounded triangles. The lower right lobe is labeled beta next to a yellow circle labeled A D P plus P i. The lower left lobe is labeled alpha. An arrow points right accompanied by a curved arrow showing the addition of 3 H subscript P end subscript plus and loss of 3 H subscript N end subscript plus. This yields a similar structure that has rotated so that the two lobes with vertices are at the left and upper left sides and highlighted in red. A green arrow points to the left side vertex, which is labeled beta next to a dashed circle with an arrow pointing to an orange oval labeled A T P outside of the structure. The upper right lobe is labeled alpha. The upper right and right lobes are rounded triangles. The upper right lobe is labeled beta next to a yellow circle labeled A D P plus P i. The right side lobe is labeled alpha. The lower right and left lobes are rounded. The lower right lobe is labeled beta next to an orange oval labeled A T P and the lower left lobe is labeled alpha. An arrow points up to the starting structure at the top center and is accompanied by a curved arrow showing the addition of 3 H subscript P end subscript plus and loss of 3 H subscript N end subscript plus.

The conformational changes central to this mechanism are driven by the passage of protons through the $F_{o}$ $upper F Subscript o$ portion of ATP synthase. The streaming of protons through the $F_{o}$ $upper F Subscript o$ pore causes the c ring and the attached γ subunit to rotate about the long axis of γ, which is perpendicular to the plane of the membrane. The γ subunit passes through the center of the $α_{3} β_{3}$ $alpha 3 beta 3$ spheroid, which is held stationary relative to the membrane surface by the $b_{2}$ $b Subscript 2$ and $δ$ $delta$ subunits (Fig. 19-25a). With each rotation of 120°, γ comes into contact with a different β subunit, and the contact forces that β subunit into the β-empty conformation.

The three β subunits interact in such a way that when one assumes the β-empty conformation, its neighbor to one side must assume the β-ADP form, and the other neighbor the β-ATP form. Thus, one complete rotation of the γ subunit causes each β subunit to cycle through all three of its possible conformations, and for each rotation, three ATP are synthesized and released from the enzyme surface.

One strong prediction of this binding-change model is that the γ subunit should rotate in one direction when $F_{o} F_{1}$ $upper F Subscript o Baseline upper F Subscript 1$ is synthesizing ATP and in the opposite direction when the enzyme is hydrolyzing ATP. This prediction of rotation with ATP hydrolysis was confirmed in elegant experiments in the laboratories of Masasuke Yoshida and Kazuhiko Kinosita, Jr. The rotation of γ in a single $F_{1}$ $upper F Subscript 1$ molecule was observed microscopically by attaching a long, thin, fluorescent actin polymer to γ and watching it move relative to $α_{3} β_{3}$ $alpha 3 beta 3$ immobilized on a microscope slide as ATP was hydrolyzed. (The expected reversal of the rotation when ATP is being synthesized could not be tested in this experiment; there is no proton gradient to drive ATP synthesis.) When the entire $F_{o} F_{1}$ $upper F Subscript o Baseline upper F Subscript 1$ complex (not just $F_{1}$ $upper F Subscript 1$ ) was used in a similar experiment, the entire ring of c subunits rotated with γ (Fig. 19-27). The “shaft” rotated in the predicted direction through 360°. The rotation was not smooth but occurred in three discrete steps of 120°. As calculated from the known rate of ATP hydrolysis by one $F_{1}$ $upper F Subscript 1$ molecule and from the frictional drag on the long actin polymer, the efficiency of this mechanism in converting chemical energy into motion is close to 100%. It is, in Boyer’s words, “a splendid molecular machine!”

A two-part figure shows an experimental demonstration of the rotation of F subscript 0 end subscript and gamma in a cartoon and in fluorescent micrographs showing fluorescently labeled actin attached to the gamma subunit. — FIGURE 19-27 Experimental demonstration of rotation of $F_{o}$ $upper F Subscript o$ and $γ$ $bold-italic gamma$ . This fundamental property of the ATP synthase reaction was demonstrated in several creative ways. (a) In one experiment, illustrated in this cartoon, $F_{1}$ $upper F Subscript 1$ , genetically engineered to contain a run of His residues, was tightly adhered to a microscope slide coated with a Ni complex; biotin was covalently attached to a c subunit of $F_{o}$ $upper F Subscript o$ . The protein avidin, which binds biotin very tightly, was covalently attached to long filaments of actin labeled with a fluorescent probe. Biotin-avidin binding then attached the actin filaments to the c subunit. When ATP was provided as substrate for the ATPase activity of $F_{1}$ $upper F Subscript 1$ , the labeled filament rotated in one direction, proving that the $F_{o}$ $upper F Subscript o$ cylinder of c subunits rotates. (b) In another experiment, a fluorescent actin filament was attached directly to the γ subunit. The series of fluorescence micrographs (read left to right) shows the position of the actin filament at intervals of 133 ms. Note that as the filament rotated, it made discrete jumps rather than smooth rotation about the circle. The cylinder and shaft move as one unit. [(a) Information from Y. Sambongi et al., *Science* 286:1722, 1999.]

Part a shows A T P synthase above a circle in a horizontal square labeled N i complex. A T P synthase is inverted so that the ring of ovals is just above the horizontal square. The front oval is gray alpha and has a bond to H i s below, the left-hand oval is light purple beta and has a bond to H i s below, the left rear oval is light gray, the rear oval is purple, the right rear oval is gray, and the right front oval is dark purple beta with a bond to H i s below and a curved arrow showing the entry of A T P and departure of A D P plus P i. A light green cylinder labeled gamma extends up from the center of this ring of ovals and a black arrow wraps around from the back across the front toward the right. A rounded orange cylinder with an open center labeled c subscript 10 end subscript is above with a small blue oval labeled epsilon below to the right and behind the light green central cylinder. Red arrows point clockwise around the top of this orange cylinder. At the one o’clock position of this orange cylinder, there is a small yellow sphere labeled biotin beneath a roughly rectangular piece labeled avidin attached to a long structure of two intertwined strands of spheres labeled actin. A roughly rectangular orange piece on the left side of the orange cylinder is labeled a and forms a crescent to fit against the cylinder, Two long strands labeled b subscript 2 end subscript run along the orange cylinder labeled a all the way down to curve to the bottom center of the ring of ovals. The bottom portion of the structure, below the orange cylinder, is labeled F subscript 1 end subscript and the top portion including the orange cylinder is labeled F subscript 0 end subscript. Part b shows a series of six micrographs that all have a black background. In the first micrograph, a small white piece extends diagonally from the center toward the lower left. This piece is darker in the center and an arrow points along it to the lower left. In each of the remaining micrographs, this white piece has moved. In the second micrograph, it extends almost vertically down next to an arrow pointing down. In the third, it extends to the right above an arrow that points right. In the fourth, it extends diagonally to the upper right along with an arrow pointing to the upper right. In the fifth, it extends to the upper left along with an arrow that points to the upper left. The sixth micrograph resembles the first micrograph. All data are approximate.

A model that illustrates how proton flow and rotary motion are coupled in the $F_{o}$ $upper F Subscript o$ complex is shown in Figure 19-28. The a subunit is stationary, while the c ring rotates. Critical interactions occur between conserved amino acids in the a and c subunits. The individual subunits in the c ring are arranged in a circle with only a few in contact with the a subunit at any moment. Protons diffuse across the membrane through a path made up of both a and c subunits. Transient protonation of a key Glu residue in each c subunit elicits conformation changes that drive rotation and transmit protons between hydrophilic half channels positioned on each side of the membrane. The rotary movement of the c ring is made unidirectional by the large difference in proton concentration across the membrane. The number of protons that must be transferred to produce one complete rotation of the c ring is equal to the number of c subunits in the ring. Structural studies of the c ring have shown that the number of c subunits differs in different organisms (Fig. 19-29). In bovine mitochondria the number is 8, in yeast mitochondria and in Escherichia coli it is 10, and the number of c subunits can range as high as 17, as is seen in the soil bacterium Burkholderia pseudomallei. The rate of rotation in intact mitochondria has been estimated at about 6,000 rpm — 100 rotations per second.

A figure shows a model for proton-driven rotation of the c ring. — FIGURE 19-28 A model for proton-driven rotation of the c ring. The a subunit of the $F_{o}$ $upper F Subscript o$ complex of the ATP synthase (see Fig. 19-25a) has two hydrophilic half-channels for protons, one leading from the p side to the middle of the membrane, the other leading from the middle of the membrane to the n side (matrix). The function of the stationary a subunit is to conduct protons to and from the c ring subunits to drive c ring rotational motion. Individual c subunits in $F_{o}$ $upper F Subscript o$ (the total number varies from 8 to 17 in different species) are arranged in a circle about a central core. Each c subunit has a critical Glu residue (an Asp in some species) about midway across the membrane, with a perturbed $p K_{a}$ $p upper K Subscript a Baseline$ allowing it to donate or accept a proton (red $H^{+}$ $upper H Superscript plus$ ) at pH near neutrality. The cycle that c subunits go through is illustrated. One c subunit is initially positioned so that a proton that enters the half-channel on the p side (where the proton concentration is relatively high) encounters and protonates a conserved His residue in the a subunit, transferring it to the c subunit Glu residue. This triggers a rotation-facilitating conformational change in the protonated c subunit as the Glu loses its negative charge. The now neutral Glu residue is sequestered in the hydrophobic membrane layer as it rotates as part of the c ring . As the c ring rotates, the c subunit we are following eventually makes contact with the channel to the n side of the membrane, where the environment is relatively alkaline, and the proton is released . As the Glu reacquires its negative charge, another rotation-facilitating conformational change occurs such that the Glu interacts transiently with a conserved Arg residue in the immobile a subunit . The interaction with the Arg is disrupted as the Glu is again protonated by the His residue, in motions that again facilitate rotation. The c subunits positioned near the half channels are providing the rotational driving force at any given moment, as they are the ones undergoing conformation changes associated with protonation and deprotonation. The orientation of the proton gradient dictates the direction of proton flow and makes rotation of the c ring essentially unidirectional. [Information from W. Kühlbrandt and K. M. Davies, *Trends Biochem. Sci.* 41:106, 2016.]

A figure shows a horizontal piece of membrane with the N side above and the P side below. A ring of vertical tubular structures are embedded in the membrane. These cylinders are labeled c subunits. A vertical line extending above the center of the ring is surrounded by a counterclockwise arrow showing rotation. A diagonal helix runs from the center left to lower right and is labeled a subunit. An invagination in the membrane at the upper left of the ring is labeled N-side half channel and a narrow invagination that exposes the bottom of a single cylinder labeled with the number 1 is labeled P-side half channel. Wireframe pieces are shown between the cylinders. A wireframe model ending with a ring and labeled H I s extends from the a subunit helix and ends at the edge of the cylinder labeled with the number 1. An arrow points up from this H I s to a partial ring with red highlighted H plus to its right showing from behind the same cylinder. To the left of this cylinder, a wireframe labeled A r g plus extends up and a wireframe labeled G l u minus is above it. Wireframe rings ending at red highlighted H plus extend from behind the cylinder labeled 1 and the next two cylinders to its right. To the left of the cylinder labeled 1, there is a cylinder labeled 4 and then a cylinder labeled 3. Wireframes ending at red highlighted H plus extend from cylinder 3 and the cylinder to its left. Five red H plus ions are shown below the P-side half channel and an arrow points up from one to H plus within the cylinder labeled 1. The numbers on the cylinders represent steps as follows. Step 1: A vertical cylinder is positioned in the P-side half channel. An H plus enters the channel and moves up to the H i s residue, which it protonates to yield the protonated H i s residue shown higher on the same cylinder. Step 2: The c ring rotates and the cylinder with the protonated H i s reaches the right side, where there it contacts the channel to the N side of the membrane and the environment is more alkaline, allowing H i s to release the proton. Step 3: A cylinder at the left front has a wireframe with red highlighted H plus above that extends into the N-side half channel. An arrow points up to red highlighted H plus above the cylinder. G l u gains a negative charge. Step 4: As this cylinder moves to the right, negatively charged G l u interacts with positively charged A r g, shown as a wireframe extending from the a subunit helix, and then this interaction is disrupted as the cylinder re-enters the P-side half channel and protonation occurs again.

A four-part figure shows top and side views of c subunits for bovine mitochondria in part a, a top view of c subunits for yeast mitochondria in part b, atomic force microscopy of c rings in a thermophilic bacterium in part c, and atomic force microscopy of c rings in spinach in part d. — FIGURE 19-29 Species differences in number of c subunits in the c ring of the $F_{o}$ $bold upper F Subscript bold o$ complex. The structures of the c rings from several species have been determined by x-ray crystallography. Each helix in the inner ring is half of a hairpin-shaped c subunit; the outer ring of helices forms the other half of the hairpin structure. The essential Glu residue (Asp in some species) is shown as a red dot. Views of the c ring perpendicular to the membrane show the number of c subunits for (a) bovine mitochondria (8 subunits) and (b) yeast mitochondria (10). Atomic force microscopy has been used to visualize the c rings of (c) a thermophilic bacterium, *Bacillus* species TA2.A1 (13 subunits), and (d) spinach (14). According to the model in Figure 19-28, different numbers of c subunits in the c ring should result in different ratios of ATP formed per pair of electrons passing through the respiratory chain (i.e., different $P/O$ $upper P slash upper O$ ratios). [(a) Data from PDB ID 1OHH, E. Cabezon et al., *Nat. Struct. Biol.* 10:744, 2003. (c) D. Matthies et al., *J. Mol. Biol.* 388:611, 2009. (d) H. Seelert et al., *Nature* 405:418, 2000. Republished with permission of Elsevier from *J. Mol. Biol.*, Matthies et al., Vol. 388(3), ©2009; permission conveyed through Copyright Clearance Center, Inc.]

FIGURE 19-29 Species differences in number of c subunits in the c ring of the $F_{o}$ $bold upper F Subscript bold o$ complex. The structures of the c rings from several species have been determined by x-ray crystallography. Each helix in the inner ring is half of a hairpin-shaped c subunit; the outer ring of helices forms the other half of the hairpin structure. The essential Glu residue (Asp in some species) is shown as a red dot. Views of the c ring perpendicular to the membrane show the number of c subunits for (a) bovine mitochondria (8 subunits) and (b) yeast mitochondria (10). Atomic force microscopy has been used to visualize the c rings of (c) a thermophilic bacterium, *Bacillus* species TA2.A1 (13 subunits), and (d) spinach (14). According to the model in Figure 19-28, different numbers of c subunits in the c ring should result in different ratios of ATP formed per pair of electrons passing through the respiratory chain (i.e., different $P/O$ $upper P slash upper O$ ratios). [(a) Data from PDB ID 1OHH, E. Cabezon et al., *Nat. Struct. Biol.* 10:744, 2003. (c) D. Matthies et al., *J. Mol. Biol.* 388:611, 2009. (d) H. Seelert et al., *Nature* 405:418, 2000. Republished with permission of Elsevier from *J. Mol. Biol.*, Matthies et al., Vol. 388(3), ©2009; permission conveyed through Copyright Clearance Center, Inc.]

Part a shows the top view of a structure with a central ring of eight yellow helices extending away from the viewer surrounded by an outer ring of eight orange helices extending away from the viewer. A side view of one yellow and one orange helix shows a long yellow helix connected by a white strand extending from its base to the bottom of a shorter orange helix on the right with a red dot halfway up its length. Part b shows the top view of a similar structure with ten inner yellow helices and ten orange outer helices. Part c shows an orange ring with a central protrusion and numbered white protrusions along its surface. The background is black. The numbering begins at 1 at the eight o’clock position and continues counterclockwise to 13 in the nine o’clock position. Part d is similar to part c except that there is no central protrusion, the light parts are less bright, and the numbering runs from 1 to 14. All data are approximate.

Chemiosmotic Coupling Allows Nonintegral Stoichiometries of $O_{2}$ $bold upper O Subscript bold 2$ Consumption and ATP Synthesis

The overall reaction equation for ATP synthesis has the following form:

x ADP + x P_{i} + ½ O_{2} + H^{+} + NADH \to x {ATP + H}_{2} O + {NAD}^{+}

$x ADP plus x upper P Subscript i Baseline plus one half upper O Subscript 2 Baseline plus upper H Superscript plus Baseline plus NADH right-arrow x ATP plus upper H Subscript 2 Baseline upper O plus NAD Superscript plus$

(19-11)

The value of x is sometimes called the P/O ratio or the $P / 2 e^{-} ratio$ $bold upper P slash bold 2 bold-italic e Superscript minus Baseline bold ratio$ . When a proton gradient is coupled to ATP synthesis as described above, there is no theoretical requirement for $P/O$ $upper P slash upper O$ to be integral. The relevant questions about stoichiometry become these: How many protons are pumped outward by electron transfer from one NADH to $O_{2}$ $upper O Subscript 2$ ? and How many protons must flow inward through the $F_{o} F_{1}$ $upper F Subscript o Baseline upper F Subscript 1$ complex to drive the synthesis of one ATP? The measurement of proton fluxes is technically complicated; the investigator must take into account the buffering capacity of mitochondria, nonproductive leakage of protons across the inner membrane, and use of the proton gradient for functions other than ATP synthesis, such as driving the transport of substrates across the inner mitochondrial membrane (described below). When NADH or succinate (which sends electrons into the respiratory chain at the level of ubiquinone) is the oxidizable substrate, the consensus experimental values for number of protons pumped out per pair of electrons are 10 and 6, respectively. The most widely accepted experimental value for number of protons required to drive the synthesis of an ATP molecule is 4, of which 1 is used in transporting $P_{i}$ $upper P Subscript i$ , ATP, and ADP across the mitochondrial membrane (see below). If 10 protons are pumped out per NADH and 4 must flow in to produce 1 ATP, the proton-based $P/O$ $upper P slash upper O$ ratio is 2.5 for NADH as the electron donor and $1.5 (6/4)$ $1.5 left-parenthesis Number 6 slash 4 right-parenthesis$ for succinate. However, as we will see in Worked Example 19-2, the proton stoichiometry of ATP synthesis by ATP synthase depends upon the number of c units in $F_{o}$ $upper F Subscript o$ , which ranges from 8 to 17, depending on the species.

WORKED EXAMPLE 19-2 Stoichiometry of ATP Production: Effect of c Ring Size

(a) If the ATP synthase of bovine mitochondria has 8 c subunits per c ring, what is the predicted ratio of ATP formed per NADH oxidized? (b) What is the predicted value for yeast mitochondria, with 10 c subunits per ATP synthase? (c) What are the comparable values for electrons entering the respiratory chain from ${FADH}_{2}$ $FADH Subscript 2$ ?

SOLUTION:

(a) Here we are asked to determine how many ATP molecules are produced per NADH. This is another way of asking us to calculate the $P/O$ $upper P slash upper O$ ratio, or x, in Equation 19-11. If the c ring has 8 c subunits, then one full rotation will transfer 8 protons to the matrix and produce 3 ATP molecules. But this synthesis also requires the transport of 3 $P_{i}$ $upper P Subscript i$ into the matrix, at a cost of 1 proton each, adding 3 more protons to the total number required. This brings the total cost to $(11 protons) / (3 ATP) = 3.7 protons/ATP$ $left-parenthesis 11 protons right-parenthesis slash left-parenthesis 3 ATP right-parenthesis equals 3.7 protons slash ATP$ . The generally agreed value for the number of protons pumped out per pair of electrons transferred from NADH is 10 (Eqn 19-7). So, oxidizing 1 NADH produces $(10 protons) / (3.7 protons/ATP) = 2.7 ATP$ $left-parenthesis 10 protons right-parenthesis slash left-parenthesis 3.7 protons slash ATP right-parenthesis equals 2.7 ATP$ .

(b) If the c ring has 10 c subunits, then one full rotation will transfer 10 protons to the matrix and produce 3 ATP molecules. Adding in the 3 protons to transport the 3 $P_{i}$ $upper P Subscript i$ into the matrix brings the total cost to $(13 protons) / (3 ATP) = 4.3 protons/ATP$ $left-parenthesis 13 protons right-parenthesis slash left-parenthesis 3 ATP right-parenthesis equals 4.3 protons slash ATP$ . Oxidizing 1 NADH produces $(10 protons) / (4.3 protons/ATP) = 2.3 ATP$ $left-parenthesis 10 protons right-parenthesis slash left-parenthesis 4.3 protons slash ATP right-parenthesis equals 2.3 ATP$ .

(c) When electrons enter the respiratory chain from ${FADH}_{2}$ $FADH Subscript 2$ (at ubiquinone), only 6 protons are available to drive ATP synthesis. This changes the calculation for bovine mitochondria to $(6 protons) / (3.7 protons/ATP) = 1.6 ATP$ $left-parenthesis 6 protons right-parenthesis slash left-parenthesis 3.7 protons slash ATP right-parenthesis equals 1.6 ATP$ per pair of electrons from ${FADH}_{2}$ $FADH Subscript 2$ . For yeast mitochondria, the calculation is $(6 protons) / (4.3 protons/ATP) = 1.4 ATP$ $left-parenthesis 6 protons right-parenthesis slash left-parenthesis 4.3 protons slash ATP right-parenthesis equals 1.4 ATP$ per pair of electrons from ${FADH}_{2}$ $FADH Subscript 2$ .

These calculated values of x, or the $P/O$ $upper P slash upper O$ ratio, define a range that includes the experimental values of $2.5 ATP/NADH$ $2.5 ATP slash NADH$ and $1.5 {ATP/FADH}_{2}$ $1.5 ATP slash FADH Subscript 2 Baseline$ , and we therefore use these values throughout this book.

The Proton-Motive Force Energizes Active Transport

Although the primary role of the proton gradient in mitochondria is to furnish energy for the synthesis of ATP, the proton-motive force also drives several transport processes essential to oxidative phosphorylation. The inner mitochondrial membrane is generally impermeable to charged species, but two specific systems transport ADP and $P_{i}$ $upper P Subscript i$ into the matrix and ATP out to the cytosol (Fig. 19-30).

A figure shows adenine nucleotide and phosphate translocases in an inner mitochondrial membrane. — FIGURE 19-30 Adenine nucleotide and phosphate translocases. Transport systems of the inner mitochondrial membrane carry ADP and $P_{i}$ $upper P Subscript i$ into the matrix and newly synthesized ATP into the cytosol. The adenine nucleotide translocase is an antiporter; the same protein moves ADP into the matrix and ATP out. The effect of replacing ${ATP}^{4 -}$ $ATP Superscript 4 minus$ with ${ADP}^{3 -}$ $ADP Superscript 3 minus$ in the matrix is the net efflux of one negative charge, which is favored by the charge difference across the inner membrane (outside positive). At pH 7, $P_{i}$ $upper P Subscript i$ is present as both ${HPO}_{4}^{2 -}$ $HPO Subscript 4 Superscript 2 minus$ and $H_{2} {PO}_{4}^{-}$ $upper H Subscript 2 Baseline PO Subscript 4 Superscript minus$ ; the phosphate translocase is specific for $H_{2} {PO}_{4}^{-}$ $upper H Subscript 2 Baseline PO Subscript 4 Superscript minus$ . There is no net flow of charge during symport of $H_{2} {PO}_{4}^{-}$ $upper H Subscript 2 Baseline PO Subscript 4 Superscript minus$ and $H^{+}$ $upper H Superscript plus$ , but the relatively low proton concentration in the matrix favors the inward movement of $H^{+}$ $upper H Superscript plus$ . Thus the proton-motive force is responsible both for providing the energy for ATP synthesis and for transporting substrates (ADP and $P_{i}$ $upper P Subscript i$ ) into and product (ATP) out of the mitochondrial matrix. All three of these transport systems can be isolated as a single membrane-bound complex (ATP synthasome).

FIGURE 19-30 Adenine nucleotide and phosphate translocases. Transport systems of the inner mitochondrial membrane carry ADP and $P_{i}$ $upper P Subscript i$ into the matrix and newly synthesized ATP into the cytosol. The adenine nucleotide translocase is an antiporter; the same protein moves ADP into the matrix and ATP out. The effect of replacing ${ATP}^{4 -}$ $ATP Superscript 4 minus$ with ${ADP}^{3 -}$ $ADP Superscript 3 minus$ in the matrix is the net efflux of one negative charge, which is favored by the charge difference across the inner membrane (outside positive). At pH 7, $P_{i}$ $upper P Subscript i$ is present as both ${HPO}_{4}^{2 -}$ $HPO Subscript 4 Superscript 2 minus$ and $H_{2} {PO}_{4}^{-}$ $upper H Subscript 2 Baseline PO Subscript 4 Superscript minus$ ; the phosphate translocase is specific for $H_{2} {PO}_{4}^{-}$ $upper H Subscript 2 Baseline PO Subscript 4 Superscript minus$ . There is no net flow of charge during symport of $H_{2} {PO}_{4}^{-}$ $upper H Subscript 2 Baseline PO Subscript 4 Superscript minus$ and $H^{+}$ $upper H Superscript plus$ , but the relatively low proton concentration in the matrix favors the inward movement of $H^{+}$ $upper H Superscript plus$ . Thus the proton-motive force is responsible both for providing the energy for ATP synthesis and for transporting substrates (ADP and $P_{i}$ $upper P Subscript i$ ) into and product (ATP) out of the mitochondrial matrix. All three of these transport systems can be isolated as a single membrane-bound complex (ATP synthasome).

A horizontal membrane is shown with the region above labeled intermembrane space and the region below labeled matrix. There are red highlighted positive charges along the top surface of the membrane and along the bottom surface of the membrane. A light purple channel running vertically through the membrane is labeled adenine nucleotide translocase (antiporter). Arrows show that A D P 3 minus moves through this channel from the intermembrane space to the matrix and A D P 4 minus moves from the matrix to the intermembrane space. To the right, A T P synthase is shown with a square portion in the membrane adjacent to a rectangular portion to the left with a cylindrical portion that is wider adjacent to the square portion and that extends to a rounded portion below, from which a thin portion loops back to run along the rectangular portion across the membrane. Red highlighted H plus is shown above the rectangular portion and an arrow shows that it moves through this portion, then left across the thin portion to reach red highlighted H plus below. A D P 3 minus to the left that entered through the channel above has an arrow that curves to meet the red arrow to H plus and then away to A T P 4 minus, which then travels back through the channel above to the intermembrane space. A pink channel to the right is labeled phosphate translocase (symporter) and two molecules enter the matrix through it. H 2 P O 4 minus above this channel has an arrow through the channel to H 2 P O 4 minus below, from which an arrow extends left to join the red arrow to H plus from A T P synthase. Red highlighted H plus above the phosphate translocase has a red arrow through the channel to red highlighted H plus in the matrix.

The adenine nucleotide translocase, integral to the inner membrane, binds ${ADP}^{3 -}$ $ADP Superscript 3 minus$ in the intermembrane space and transports it into the matrix in exchange for an ${ATP}^{4 -}$ $ATP Superscript 4 minus$ molecule simultaneously transported outward (see Fig. 13-11 for the ionic forms of ATP and ADP). Because this antiporter moves four negative charges out for every three moved in, its activity is favored by the transmembrane electrochemical gradient, which gives the matrix a net negative charge; the proton-motive force drives ATP-ADP exchange. Adenine nucleotide translocase is specifically inhibited by atractyloside, a toxic glycoside produced by a species of thistle. If the transport of ADP into and ATP out of mitochondria is inhibited, cytosolic ATP cannot be regenerated from ADP, explaining the toxicity of atractyloside.

A second membrane transport system essential to oxidative phosphorylation is the phosphate translocase, which promotes symport of one $H_{2} {PO}_{4}^{-}$ $upper H Subscript 2 Baseline PO Subscript 4 Superscript minus$ and one $H^{+}$ $upper H Superscript plus$ into the matrix. This transport process, too, is favored by the transmembrane proton gradient (Fig. 19-30). Notice that the process requires movement of one proton from the p side to the n side of the inner membrane, consuming some of the energy of electron transfer. A complex of the ATP synthase and both translocases, the ATP synthasome, can be isolated from mitochondria by gentle dissection with detergents, suggesting that the functions of these three proteins are very tightly integrated.

ATP and ADP cross the outer mitochondrial membrane via the voltage-dependent anion channel (VDAC), a 19-stranded β barrel with an opening about 27 Å wide, connecting the cytosol and the intermembrane space. Each VDAC, when open, can move $10^{5}$ $10 Superscript 5$ ATP molecules per second. The opening is gated by voltage, as its name indicates, and under some conditions VDAC is closed to ATP.

Shuttle Systems Indirectly Convey Cytosolic NADH into Mitochondria for Oxidation

The NADH dehydrogenase of the inner mitochondrial membrane of animal cells can accept electrons only from NADH in the matrix. Given that the inner membrane is not permeable to NADH, how can the NADH generated by glycolysis in the cytosol be reoxidized to ${NAD}^{+}$ $NAD Superscript plus$ by $O_{2}$ $upper O Subscript 2$ via the respiratory chain? Special shuttle systems carry reducing equivalents from cytosolic NADH into mitochondria by an indirect route. The most active NADH shuttle, which functions in liver, kidney, and heart mitochondria, is the malate-aspartate shuttle (Fig. 19-31). The reducing equivalents of cytosolic NADH are first transferred to cytosolic oxaloacetate to yield malate, catalyzed by cytosolic malate dehydrogenase. The malate thus formed passes through the inner membrane via the malate–α-ketoglutarate transporter. Within the matrix, the reducing equivalents are passed to ${NAD}^{+}$ $NAD Superscript plus$ by the action of matrix malate dehydrogenase, forming NADH; this NADH can pass electrons directly to the respiratory chain. About 2.5 molecules of ATP are generated as this pair of electrons passes to $O_{2}$ $upper O Subscript 2$ . Cytosolic oxaloacetate must be regenerated by transamination reactions and the activity of membrane transporters to start another cycle of the shuttle.

A figure shows six steps of the malate-aspartate shuttle. — FIGURE 19-31 Malate-aspartate shuttle. This shuttle for transporting reducing equivalents from cytosolic NADH into the mitochondrial matrix is used in liver, kidney, and heart. NADH in the cytosol enters the intermembrane space through openings in the outer membrane (porins), then passes two reducing equivalents to oxaloacetate, producing malate. Malate crosses the inner membrane via the malate–α-ketoglutarate transporter. In the matrix, malate passes two reducing equivalents to ${NAD}^{+}$ $NAD Superscript plus$ , and the resulting NADH is oxidized by the respiratory chain; the oxaloacetate formed from malate cannot pass directly into the cytosol. Oxaloacetate is first transaminated to aspartate, and aspartate can leave via the glutamate-aspartate transporter. Oxaloacetate is regenerated in the cytosol, completing the cycle, and glutamate produced in the same reaction enters the matrix via the glutamate-aspartate transporter.

FIGURE 19-31 Malate-aspartate shuttle. This shuttle for transporting reducing equivalents from cytosolic NADH into the mitochondrial matrix is used in liver, kidney, and heart. NADH in the cytosol enters the intermembrane space through openings in the outer membrane (porins), then passes two reducing equivalents to oxaloacetate, producing malate. Malate crosses the inner membrane via the malate–α-ketoglutarate transporter. In the matrix, malate passes two reducing equivalents to ${NAD}^{+}$ $NAD Superscript plus$ , and the resulting NADH is oxidized by the respiratory chain; the oxaloacetate formed from malate cannot pass directly into the cytosol. Oxaloacetate is first transaminated to aspartate, and aspartate can leave via the glutamate-aspartate transporter. Oxaloacetate is regenerated in the cytosol, completing the cycle, and glutamate produced in the same reaction enters the matrix via the glutamate-aspartate transporter.

The malate-aspartate shuttle is shown as a circular series of reactions with a vertical piece of membrane running across the center. The region to the left of the membrane is labeled intermembrane space (P side) and the region to the right of the membrane is labeled matrix (N side). The reaction begins with oxaloacetate, shown at the eleven o’clock position. Numbered from right to left, oxaloacetate has C 1 forming C O O minus, C 2 double bonded to O, C 3 bonded to 2 H, and C 4 forming C O O minus. Step 1: An arrow labeled malate dehydrogenase points from oxaloacetate to malate and is accompanied by a curved blue arrow showing the addition of H plus plus red highlighted N A D H and loss of N A D plus. Malate is similar to oxaloacetate except that C 2 is bonded to O H above and H below. Step 2: An arrow points from malate through a peach channel in the membrane labeled malate-alpha-ketoglutarate transporter to malate in the matrix. Step 3: An arrow labeled malate dehydrogenase points from malate to oxaloacetate and is accompanied by a curved blue arrow showing the addition of H plus plus red highlighted N A D H and loss of N A D plus. Step 4: An arrow labeled aspartate aminotransferase points from oxaloacetate to aspartate accompanied by a curved arrow from glutamate to alpha-ketoglutarate. Aspartate is similar to oxaloacetate except that C 2 is bonded to N H 3 plus above and to H below. Step 5: An arrow points from aspartate through a channel labeled glutamate-aspartate transporter to aspartate in the intermembrane space. Step 6: An arrow labeled aspartate aminotransferase points from aspartate to oxaloacetate accompanied by a curved arrow from alpha-ketoglutarate to glutamate. Numbered from right to left, glutamate is C 1 forming C O O minus, C 2 bonded to N H 3 plus above and H below, C 3 bonded to 2 H, C 4 bonded to 2 H, and C 5 forming C O O Minus. Glutamate produced in reaction 6 travels through the glutamate-aspartate transporter to participate in reaction 4, in which oxaloacetate is converted to aspartate by aspartate by aminotransferase as glutamate is converted to alpha-ketoglutarate. Alpha-ketoglutarate is similar to glutamate except that C 2 is double bonded to O above. Alpha-ketoglutarate produced by this reaction travels through the malate-alpha-ketoglutarate transporter to participate in reaction 6, in which aspartate is converted to oxaloacetate by aspartate aminotransferase as alpha-ketoglutarate is converted to glutamate. All data are approximate.

Skeletal muscle and brain use a different NADH shuttle, the glycerol 3-phosphate shuttle (Fig. 19-32). It differs from the malate-aspartate shuttle in that it delivers the reducing equivalents from NADH through FAD in glycerol 3-phosphate dehydrogenase to ubiquinone and thus into Complex III, not Complex I (Fig. 19-15), providing enough energy to synthesize only 1.5 ATP molecules per pair of electrons.

A figure shows the glycerol 3-phosphate shuttle. — FIGURE 19-32 Glycerol 3-phosphate shuttle. This alternative means of moving reducing equivalents from the cytosol to the respiratory chain operates in skeletal muscle and the brain. In the cytosol, dihydroxyacetone phosphate accepts two reducing equivalents from NADH in a reaction catalyzed by cytosolic glycerol 3-phosphate dehydrogenase. An isozyme of glycerol 3-phosphate dehydrogenase bound to the outer face of the inner membrane then transfers two reducing equivalents from glycerol 3-phosphate in the intermembrane space to ubiquinone. Note that this shuttle does not involve membrane transport systems.

A horizontal piece of plasma membrane is shown with the region above labeled intermembrane space (P side) and the region below labeled matrix (N side). The word glycolysis is shown at the upper right with an arrow pointing down to N A D H with a red highlighted H plus red highlighted H plus. A curved arrow from N A D H plus H plus to N A D plus at the upper left meets a curved arrow below from dihydroxyacetone phosphate on the right to glycerol 3-phosphate on the left. The place where these arrows meet is labeled cytosolic glycerol 3-phosphate dehydrogenase. The arrow from N A D H plus H plus is highlighted in blue until it meets the arrow from dihydroxyacetone to glycerol 3-phosphate, at which the blue highlighting switches to the second half of the arrow to glycerol 3-phospahte. Dihydroxyacetone phosphate is shown as a three-carbon vertical chain with the top carbon bonded to 2 H and O H, the middle carbon double bonded to O, and the bottom carbon bonded to 2 H and to O bonded to a circle labeled P. Glycerol 3-phospahte is similar except that the middle carbon is bonded to red highlighted H and to O bonded to red highlighted H. A blue arrow curves down from glycerol 3-phosphate into a purple irregular shape at the top of the membrane labeled mitochondrial glycerol 3-phosphate dehydrogenase, where it meets a curved arrow from F A D to F A D H 2 with red highlighted H 2 below before curving back up to dihydroxyacetone phosphate. The blue highlighting follows the arrow from glycerol 3-phosphate until it meets the second curved arrow, then the blue highlighting shifts to the second half of the arrow from F A D to F A D H 2. A blue arrow points from F A D H 2 to a yellow box labeled Q red highlighted H 2 in the membrane, from which a blue arrow points right into a large blue structure labeled complex Roman numeral 3 that extends through the membrane.

The mitochondria of plants have an externally oriented NADH dehydrogenase that can transfer electrons directly from cytosolic NADH into the respiratory chain at the level of ubiquinone. Because this pathway bypasses the NADH dehydrogenase of Complex I and the associated proton movement, the yield of ATP from cytosolic NADH is less than that from NADH generated in the matrix (Box 19-1).

BOX 19-1

Hot, Stinking Plants and Alternative Respiratory Pathways

Many flowering plants attract insect pollinators by releasing odorant molecules that mimic an insect’s natural food sources or potential egg-laying sites. Plants pollinated by flies or beetles that normally feed on or lay their eggs in dung or carrion sometimes use foul-smelling compounds to attract these insects.

One family of stinking plants is the Araceae, which includes philodendrons, arum lilies, and skunk cabbages. These plants have tiny flowers densely packed on an erect structure, the spadix, surrounded by a modified leaf, the spathe. The spadix releases odors of rotting flesh or dung. Before pollination the spadix also heats up, in some species to as much as 20 to 40 °C above ambient temperatures. Heat production (thermogenesis) helps evaporate odorant molecules for better dispersal, and because rotting flesh and dung are usually warm from the hyperactive metabolism of scavenging microbes, the heat itself might also attract insects. In the case of the eastern skunk cabbage (Fig. 1), which flowers in late winter or early spring when snow still covers the ground, thermogenesis allows the spadix to grow up through the snow.

FIGURE 1 Eastern skunk cabbage.

How does a skunk cabbage heat its spadix? The mitochondria of plants, fungi, and unicellular eukaryotes have respiratory chains that are essentially the same as those in animals, but they also have an alternative respiratory pathway. A ${QH}_{2}$ $QH Subscript 2$ oxidase transfers electrons from the ubiquinone pool directly to oxygen, bypassing the two proton-translocating steps of Complexes III and IV (Fig. 2). Energy that might have been conserved as ATP is instead released as heat. Plant mitochondria also have an alternative NADH dehydrogenase, insensitive to the Complex I inhibitor rotenone (see Table 19-4), that transfers electrons from NADH in the matrix directly to ubiquinone, bypassing Complex I and its associated proton pumping. And plant mitochondria have yet another NADH dehydrogenase, on the external face of the inner membrane, that transfers electrons from NADPH or NADH in the intermembrane space to ubiquinone, again bypassing Complex I. Thus when electrons enter the alternative respiratory pathway through the rotenone-insensitive NADH dehydrogenase, the external NADH dehydrogenase, or succinate dehydrogenase (Complex II), and pass to $O_{2}$ $upper O Subscript 2$ via the cyanide-resistant alternative oxidase, energy is not conserved as ATP but is released as heat. A skunk cabbage can use the heat to melt snow, produce a foul stench, or attract beetles or flies.

FIGURE 2 Electron carriers of the inner membrane of plant mitochondria. Electrons can flow through Complexes I, III, and IV, as in animal mitochondria, or through plant-specific alternative carriers by the paths shown with blue arrows.

A horizontal membrane is shown with the region above the membrane labeled intermembrane space (P side) and the region below the membrane labeled matrix (N side). Complex Roman numeral 1 is shown on the left as a pink structure with a horizontal piece in the membrane that bends down to a roughly oval portion below. A roughly rectangular structure extends through the membrane to the right and is wider at its top and bottom, like a spool. This rectangular structure is labeled alternative N A D H dehydrogenase. A curved arrow across its bottom shows the addition of N A D H and loss of N A D plus. A blue arrow points right to a yellow box labeled Q in the membrane. Resting on top of the membrane, a roughly rectangular structure labeled external N A D (P) H dehydrogenase has a curved arrow through its upper left showing the addition of N A D (P) H and loss of N A D (P) plus. A blue arrow points down from this structure to the same yellow box labeled Q, from which a blue arrow points right into to a yellow structure labeled alternative oxidase that is embedded in the membrane that resembles a rectangle with protrusions at each vertex. A the end of this blue arrow, a second blue arrow point down to meet the inflection point of a curved arrow that meets the bottom of the yellow structure to show that one-half O 2 is added and H 2 O is lost. A yellow arrow outlined in red shows that heat is also released from this yellow alternative oxidase into the matrix. To the right, complex Roman numeral 3 is shown as an irregular blue shape that is wider on top and narrows as it passes through the membrane, then has protrusions to the left and right beneath the membrane, then widens to a rounded portion with a small invagination at the bottom center. To its right, complex Roman numeral 4 is green and almost rectangular but extends down farther to the lower left and right and has a purple circle on top just to the right of the center and labeled C y t italicized c end italics.