A molecular assembly in the inner mitochondrial membrane carries out the synthesis of ATP. This enzyme complex was originally called the mitochondrial ATPase or F₁F₀ ATPase because it was discovered through its catalysis of the reverse reaction, the hydrolysis of ATP. ATP synthase or F₁F₀ ATP synthase are preferred names since these emphasize its actual role in the mitochondrion.

How is the oxidation of NADH coupled to the phosphorylation of ADP? This was one of the most perplexing questions biochemists ever faced, requiring decades of work to solve. Electron transfer was first suggested to lead to the formation of a covalent high-energy intermediate that serves as a compound having a high phosphoryl-transfer potential. Such a compound could, in a manner analogous to substrate-level phosphorylation in glycolysis, transfer a phosphoryl group to ADP to form ATP. An alternative proposal was that electron transfer aids the formation of an activated protein conformation, which then drives ATP synthesis. The search for such intermediates for several decades proved fruitless.

In 1961, Peter Mitchell suggested a radically different mechanism, the chemiosmotic hypothesis. He proposed that electron transport and ATP synthesis are coupled by a proton gradient across the inner mitochondrial membrane. In his model, the transfer of electrons through the respiratory chain leads to the pumping of protons from the matrix to the cytoplasmic side of the inner mitochondrial membrane. The H⁺ concentration becomes lower in the matrix, and an electric field with the matrix side negative is generated (Figure 21.1). Protons then flow back into the matrix to equalize the distribution. Mitchell’s idea was that this flow of protons drives the synthesis of ATP by ATP synthase. The energy-rich, unequal distribution of protons is called the proton-motive force, which is composed of two components: a chemical gradient and a charge gradient. The chemical gradient for protons can be represented as a pH gradient. The charge gradient is created by the positive charge on the unequally distributed protons forming the chemical gradient. Mitchell proposed that both components power the synthesis of ATP:

385

Mitchell’s highly innovative hypothesis that oxidation and phosphorylation are coupled by a proton gradient is now supported by a wealth of evidence, including, importantly, that an intact proton-impermeable membrane is required for this coupling. Indeed, electron transport does generate a proton gradient across the inner mitochondrial membrane. The pH outside is 1.4 units lower than inside, and the voltage difference, or membrane potential, is 0.14 V, the outside being positive. This membrane potential is equivalent to a free energy of 20.8 kJ (5.2 kcal) per mole of protons.

An artificial system representing the cellular respiration system was created to elegantly demonstrate the basic principle of the chemiosmotic hypothesis. The role of the electron-transport chain was played by bacteriorhodopsin, a purple membrane protein from halobacteria that pumps protons when illuminated. Synthetic vesicles containing bacteriorhodopsin and mitochondrial ATP synthase purified from beef heart were created (Figure 21.2). When the vesicles were exposed to light, ATP was formed. This key experiment clearly showed that the respiratory chain and ATP synthase are biochemically separate systems, linked only by a proton-motive force.

Two parts of the puzzle of how NADH oxidation is coupled to ATP synthesis are now evident: (1) electron transport generates a proton-motive force; (2) ATP synthesis by ATP synthase is powered by a proton-motive force. How is the proton-motive force converted into the high phosphoryl-transfer potential of ATP?

ATP synthase is a large, complex enzyme that looks like a ball on a stick, located in the inner mitochondrial membrane (Figure 21.3). Much of the “stick” part, called the F₀ subunit, is embedded in the inner mitochondrial membrane. The 85-Å-diameter ball, called the F₁ subunit, protrudes into the mitochondrial matrix. The F₁ subunit contains the catalytic activity of the synthase. In fact, isolated F₁ subunits display ATPase activity.

The F₁ subunit consists of five types of polypeptide chains (α₃, β₃, γ, δ, and ε). The three a and three β subunits, which make up the bulk of the F₁, are arranged alternately in a hexameric ring. The active sites reside on the β subunits. Beginning just above the α and β subunits is a central stalk consisting of the γ and e proteins. The γ subunit includes a long helical coiled coil that extends into the center of the α₃β₃ hexamer. Each of the β subunits is distinct because each interacts with a different face of the γ subunit. Distinguishing the three, subunits is crucial for understanding the mechanism of ATP synthesis.

386

The F₀ subunit is a hydrophobic segment that spans the inner mitochondrial membrane. F₀ contains the proton channel of the complex. This channel consists of a ring comprising from 8 to 15c subunits, depending on the source of the enzyme, that are embedded in the membrane. A single a subunit binds to the outside of the ring. The F₀ and F₁ subunits are connected in two ways: by the central ge stalk and by an exterior column. The exterior column consists of one a subunit, two β subunits, and the d subunit.

ATP synthases bind to one another to form dimers, which then associate to form large oligomers of dimers. This association stabilizes the individual enzymes to the rotational forces required for catalysis and facilitates the curvature of the inner mitochondrial membrane. The formation of the cristae allows the proton pumps of the electron transport chain to localize the proton gradient in the vicinity of the synthases, which are located at the tips of the cristae, thereby enhancing the efficiency of ATP synthesis (Figure 21.4).

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

There are three active sites on the enzyme, each performing one of three different functions at any instant. These functions are (1) trapping of ADP and P_i, (2) ATP synthesis, and (3) ATP release and ADP and P_i binding. The proton-motive force causes the three active sites to sequentially change functions as protons flow through the membrane-embedded component of the enzyme. Indeed, the enzyme consists of a moving part and a stationary part: (1) the moving unit, or rotor, consists of the c ring and the γε stalk; (2) the stationary unit, or stator, is composed of the remainder of the molecule.

387

How do the three active sites of ATP synthase respond to the flow of protons? A number of experimental observations suggested a binding-change mechanism for proton-driven ATP synthesis. This proposal states that a β subunit can perform each of three sequential steps—trapping, synthesis, and release and binding—in the process of ATP synthesis by changing conformation. As already noted, interactions with the γ subunit make the three β subunits structurally distinct (Figure 21.5). At any given moment, one β subunit will be in the L, or loose, conformation. This conformation binds ADP and P_i. A second subunit will be in the T, or tight, conformation. This conformation binds ATP with great avidity, so much so that it will convert bound ADP and P_i into ATP. Both the L and the T conformations are sufficiently constrained that they cannot release bound nucleotides. The final subunit will be in the O, or open, form. This form has a more open conformation and can bind or release adenine nucleotides.

The rotation of the γ subunit drives the interconversion of these three forms (Figure 21.6). ADP and P_i bound in the subunit in the T-form combine to form ATP. Suppose that the γ subunit is rotated by 120 degrees in a counterclockwise direction (as viewed from the top). This rotation converts the T-form site into an O-form site with the nucleotide bound as ATP. Concomitantly, the L-form site is converted into a T-form site, enabling the transformation of an additional ADP and P_i into ATP. The ATP in the O-form site can now depart from the enzyme to be replaced by ADP and P_i. An additional 120-degree rotation converts this O-form site into an L-form site, trapping these substrates. Each subunit progresses from the T to the O to the L-form with no two subunits ever present in the same conformational form. This mechanism suggests that ATP can be synthesized and released by driving the rotation of the γ subunit in the appropriate direction. Note that the role of the proton gradient is not to directly participate in the formation of ATP but rather to drive the release of ATP from the enzyme.

Is it possible to observe the proposed rotation directly? Ingenious experiments have demonstrated rotation through the use of a simple experimental system consisting solely of cloned α₃β₃γ subunits (Figure 21.7). The β subunits were engineered to contain amino-terminal polyhistidine tags, which have a high affinity for nickel ions. This property of the tags allowed the α₃β₃ assembly to be immobilized on a glass surface that had been coated with nickel ions. The γ subunit was linked to a fluorescently labeled actin filament to provide a long segment that could be observed under a fluorescence microscope. Remarkably, the addition of ATP caused the actin filament to rotate unidirectionally in a counterclockwise direction. The γ subunit was rotating, driven by the hydrolysis of ATP. Thus, the catalytic activity of an individual molecule could be observed. The counterclockwise rotation is consistent with the predicted direction for hydrolysis because the molecule was viewed from below relative to the view shown in Figure 21.6.

388

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

The direct observation of the γ subunit’s rotary motion is strong evidence for the rotational mechanism for ATP synthesis. The last remaining question is: How does proton flow through F₀ drive the rotation of the γ subunit. An elegant mechanism has been postulated that provides a clear answer to this question. The mechanism depends on the structures of the a and c subunits of F₀ (Figure 21.8). The a subunit directly abuts the membrane-spanning ring formed by 8 to 15c subunits. Evidence is consistent with a structure for the a subunit that includes two hydrophilic half-channels that do not span the membrane (Figure 21.8). Thus, protons can enter into either of these channels, but they cannot move completely across the membrane. The a subunit is positioned such that each half-channel directly interacts with one c subunit.

Each polypeptide chain of the c subunit forms a pair of a helices that span the membrane. Glutamic acid (or aspartic acid) is found in the middle of one of the helices. If the glutamate is charged (unprotonated), the c subunit will not move into the hydrophobic interior of the membrane. The key to proton movement across the membrane is that, in a proton-rich environment, such as the cytoplasmic side of the inner mitochondrial membrane, a proton will enter a channel and bind to the glutamate residue, while the glutamic acid in the other half-channel will release a proton to the proton-poor environment of the matrix (Figure 21.9). The c subunit with the bound proton then moves into the hydrophobic environment of the inner membrane as the ring rotates by one c subunit. This rotation brings the newly deprotonated c subunit from the matrix half-channel to the proton-rich cytoplasmic half-channel, where it can bind a proton. The movement of protons through the half-channels from the high proton concentration of the inner membrane space to the low proton concentration of the matrix powers the rotation of the c ring. The a unit remains stationary as the c ring rotates. Each proton that enters the cytoplasmic half-channel of the a unit moves through the membrane by riding around on the rotating c ring to exit through the matrix half-channel into the proton-poor environment of the matrix (Figure 21.10). The rate of c ring rotation is remarkable, nearly 100 revolutions per second.

389

How does the rotation of the c ring lead to the synthesis of ATP? The c ring is tightly linked to the γ and e subunits. Thus, as the c ring turns, these subunits are turned inside the α₃β₃ hexamer unit of F₁. The rotation of the γ subunit in turn promotes the synthesis of ATP through the binding-change mechanism. The exterior column formed by the two b chains and the δ subunit prevents the α₃β₃ hexamer from rotating in sympathy with the c ring. Each 360-degree rotation of the γ subunit leads to the synthesis and release of 3 molecules of ATP. The number of c subunits determines the efficiency with which the proton gradient is converted into ATP synthesis. For instance, if there are 10c subunits in the ring (as is the case for yeast mitochondrial ATP synthase), each molecule of ATP generated requires the transport of 10/3 = 3.33 protons. Recent evidence shows that the c rings of all vertebrates are composed of 8 subunits, making vertebrate ATP synthase the most efficient ATP synthase known, with the transport of only 2.7 protons required for ATP synthesis. For simplicity, we will assume that 3 protons must flow into the matrix for each molecule of ATP formed. As we will see, the electrons from NADH pump enough protons to generate 2.5 molecules of ATP, whereas those from FADH₂ yield 1.5 molecules of ATP.

390

Let us return for a moment to the example with which we began this section. If a resting human being requires 85 kg of ATP per day for bodily functions, then 3.3 × 10²⁵ protons must flow through ATP synthase per day, or 3.3 × 10²¹ protons per second. Figure 21.11 summarizes the process of oxidative phosphorylation.