LEHNINGER PRINCIPLES OF BIOCHEMISTRY - VOL. 2. BIOENERGETICS AND METABOLISM - 2014

PART II. BIOENERGETICS AND METABOLISM

19. OXIDATIVE PHOSPHORYLATION AND PHOTOPHOSPHORYLATION

19.2. ATP Synthesis

It is important to examine two aspects of the mechanism by which the energy of the H⁺ gradient, generated by electron transfer, is used to drive ATP Synthesis in Cell/35.html">Mitochondria. The first is the energetic balance of Oxidative Phosphorylation in mitochondria. As shown in Figure 13-2, The formation of 1 mol of ATP requires 50 kJ (p. 31); however, The energy released during electron transfer through the Respiratory Chain and stored as a transmembrane Electrochemical Potential amounts to -200 kJ per mole of electrons transferred—and this energy can be channeled into ATP synthesis. Consequently, the bioenergetics of mitochondrial oxidative phosphorylation does not violate the Laws of Thermodynamics. The second point of interest is the molecular mechanism coupling proton transport to ATP synthesis in mitochondria, the most widely accepted explanation for which is provided by Mitchell's chemiosmotic theory.

Class="center">Peter Mitchell, 1920–1992

According to Mitchell's chemiosmotic theory (Fig. 19-19), the energy of electron transfer creates a concentration difference of H⁺ ions and a transmembrane electrical potential across The inner mitochondrial membrane. It is precisely this electrochemical gradient, known as the proton-motive force, that serves as the energy source for ATP synthesis when H⁺ ions spontaneously flow down their concentration gradient from the Cytosol back into the matrix through specialized H⁺ channels in ATP synthase. To emphasize the crucial role of the proton-motive force, the equation for ATP synthesis in mitochondria is sometimes written as follows:

ADP + Р_i + _nH⁺_«+» —> АТР + Н₂O + _nH⁺_«-» (19-10)

Figure 19-19. The chemiosmotic theory. According to the chemiosmotic theory, electrons from NADH and other oxidizable substrates pass through a chain of electron carriers asymmetrically arranged in the inner mitochondrial membrane. Electron transfer is coupled to the translocation (pumping) of protons across the membrane from the matrix to the intermembrane space, generating a chemical gradient (∆pH) and an electrical gradient (∆Ψ). The inner membrane is impermeable to protons. Protons can reenter the matrix only through specialized channels within the F₀ protein complex. The movement of H⁺ ions from a region of higher concentration to a region of lower concentration releases Free energy, which drives ATP synthesis catalyzed by the interacting F₀ and F₁ Structure/178.html">Protein Complexes.

Mitchell termed chemiosmosis those enzymatic reactions in which chemical transformations and particle transport occur simultaneously. The experimental setup for studying the coupling of Respiration and phosphorylation—that is, "chemiosmosis"—is illustrated in Figure 19-20. When a mitochondrial suspension in a buffer solution is added to a mixture of ADP, P_i, and an oxidizable substrate such as succinate, one observes (1) The oxidation of succinate to fumarate, (2) oxygen consumption, and (3) ATP synthesis. Both oxygen consumption and ATP synthesis depend on the presence of the oxidizable substrate (in this case, succinate) as well as ADP and P_i.

Because the energy released during substrate oxidation is coupled to ATP synthesis, inhibitors of electron transfer to O₂, such as cyanide ion, carbon monoxide, and antimycin A, are expected to block ATP synthesis (Fig. 19-20a). More surprisingly, in intact mitochondria, inhibiting ATP synthesis also halts electron transfer. This interdependence is observed when oxygen is added to a mixture of isolated mitochondria and oxidizable substrates in the absence of ATP (Fig. 19-20b). Under these conditions, neither ATP synthesis nor electron transfer to molecular oxygen takes place. The coupling between oxidation and phosphorylation can be demonstrated in mixtures containing oligomycin or venturicidin—toxic substances (Antibiotics) that bind to the Active Site of mitochondrial ATP synthase. Both antibiotics are potent inhibitors of both ATP synthesis and Electron transport along the carrier chain to molecular oxygen (Fig. 19-18b). Oligomycin does not interact directly with electron carriers; rather, it binds to ATP synthase. The fact that this binding blocks both oxidation and phosphorylation confirms their interdependence—neither process can proceed independently of the other.

Figure 19-20. Investigation of the coupling between electron transfer and ATP synthesis in mitochondria. The experimental Determination of the coupling sites between respiration and ATP synthesis is performed in a mitochondrial suspension in a buffer solution. Oxygen consumption (black line) is monitored with an oxygen electrode. Samples are withdrawn at regular intervals to determine ATP content (red line). (a) When only ADP and inorganic phosphate are added to the mitochondrial suspension, respiration is not stimulated, and virtually no ATP is synthesized. Upon The addition of succinate, both respiration and ATP synthesis rates increase dramatically. The addition of cyanide (CN^-), which inhibits electron transfer between cytochrome c oxidase and O₂, blocks both respiration and ATP synthesis. (b) In a mixture of mitochondria and succinate, respiration and ATP synthesis occur only upon the addition of ADP and P_i. When venturicidin or oligomycin, which inhibit ATP synthase, is added to this mixture, respiration and ATP synthesis cease. In the presence of the uncoupling agent 2,4-dinitrophenol (DNP), mitochondrial respiration continues, whereas ATP synthesis is arrested.

The chemiosmotic theory successfully explains the dependence of electron transfer on ATP synthesis in mitochondria. If electron flow into the mitochondrial matrix through the proton channels of ATP synthase is blocked (e.g., by oligomycin), thereby closing the pathway for proton reentry, the continued outward "pumping" of protons driven by electron-transfer energy significantly increases the proton concentration gradient across the inner membrane. Consequently, the proton-motive force increases until the free energy required to pump protons out of the matrix against their concentration gradient equals or exceeds the free energy released during Electron transfer from NADH to O₂. At this point, electron transfer ceases, the net free energy change of the coupled electron-transfer and proton-pumping process becomes zero, and the system reaches equilibrium.

Under certain conditions or upon exposure to specific agents, the coupling between electron transfer and ATP synthesis can be disrupted (uncoupled). For example, if the integrity of intact mitochondria is disrupted by detergent Treatment or physical disruption, the resulting membrane fragments retain The ability to transfer electrons from succinate or NADH to oxygen, but lose the capacity for ATP synthesis—that is, the necessary coupling between these two processes is lost. Certain chemical compounds uncouple respiration from phosphorylation without disrupting mitochondrial structural integrity. Examples of such uncoupling agents include 2,4-dinitrophenol (DNP) and carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP) (see Table 9-4 and Fig. 19-21). These compounds are weak acids with hydrophobic properties. In their protonated form, they readily cross the inner membrane into the matrix and release a proton, thereby diminishing the proton gradient and arresting ATP synthesis. Ionophores, such as valinomycin (see Fig. 11-45), can bind inorganic ions and transport them into the matrix, bypassing the proton channels of ATP synthase; As a result, the electrical component of the electrochemical potential is dissipated, and ATP synthesis stops.

Figure 19-21. Two chemical uncouplers of oxidative phosphorylation. DNP and FCCP readily lose a proton to become ionized and possess pronounced hydrophobic properties. In their protonated form, they diffuse freely across the inner membrane into the matrix, delivering a proton there, which collapses the proton gradient and halts ATP synthesis. Similarly, DNP and FCCP uncouple Photophosphorylation (see Fig. 19-63).

According to the chemiosmotic theory, electron transfer in mitochondria plays a very straightforward role during ATP synthesis: it drives the pumping of H⁺ ions from the matrix into the intermembrane space to establish an electrical potential—the "proton-motive force"—across the inner membrane. In light of this theory, it is logical to hypothesize that an artificially generated proton gradient could substitute for the electrons transported through the respiratory chain. This hypothesis was confirmed by the following experiment (Fig. 19-22). Mitochondria treated to establish a proton concentration difference (and thus an electrical charge difference) across their inner membrane synthesized ATP even in the absence of an oxidizable substrate—solely at the expense of the artificially created proton-motive force.

Figure 19-22. Demonstration of The Role of the proton gradient in ATP synthesis. An artificially generated electrochemical gradient in mitochondria can provide the energy for ATP synthesis in the absence of an oxidizable substrate. (a) Isolated mitochondria are incubated in a buffer solution (pH 9) containing 0.1 M KCl. Through slow permeation of the buffer and KCl into the mitochondria, equilibrium is established between the interior of the matrix and the surrounding medium. Oxidizable substrates are absent. (b) The mitochondria are separated from the pH 9 buffer and transferred to a pH 7 buffer containing valinomycin instead of KCl. This buffer exchange establishes a pH gradient across the inner mitochondrial membrane. Valinomycin transports K⁺ ions across the membrane down their concentration gradient, generating an electrical charge across the inner mitochondrial membrane (making the matrix negative). As a result, the sum of the chemical potential generated by the pH gradient and the electrical potential arising from the charge Separation across the inner membrane provides a proton-motive force sufficient to sustain ATP synthesis in the absence of an oxidizable substrate.

The ATP Synthase Enzyme Consists of Two Main Components—F₁ and F₀

Mitochondrial ATP synthase (Complex V) is an F-type ATPase (see Fig. 11-39, Vol. 1). The structure and Catalytic Mechanism of mitochondrial ATP synthase are similar to those found in METABOLISM/14.html">Chloroplasts and aerobic Bacteria. This large intramembrane enzyme complex catalyzes the synthesis of ATP from ADP and phosphate (P_i) coupled to the translocation of protons across the membrane from the positively charged side to the negatively charged side (see Equation 19-10). ATP synthase consists of two principal components: F₁ and F₀. F₁ is a peripheral membrane protein, whereas F₀ is an integral protein spanning the mitochondrial membrane (the subscript "o" denotes the ability of the complex to bind the antibiotic oligomycin). The F₁ protein complex is an essential factor in oxidative phosphorylation. It was first isolated in purified form by a research group led by Efraim Racker in the early 1960s, which marked the beginning of fruitful investigations into membrane Structure and function.

Efraim Racker, 1913–1991

Small membrane vesicles capable of Electron Transport and ATP synthesis were successfully isolated from the inner mitochondrial membrane. If the F₁ complex is subsequently extracted with care from these membrane vesicles, the respiratory chains remain intact because the vesicles still contain the F₀ component of ATP synthase, which preserves their ability to transfer electrons from NADH to molecular oxygen. However, vesicles lacking the F₁ component are unable to synthesize ATP, as no proton gradient is generated across the mitochondrial compartments separated by the inner membrane. This happens because, in the absence of the F₁ component, H⁺ ions flow freely through the F₀ proton channel into the matrix at a rate equal to the rate at which they are pumped out. If isolated F₁ complexes are added back to the F₁-depleted vesicles, the F₁ molecules reassociate with the F₀ component, blocking its proton channel. In these "repaired" mitochondrial vesicles, the energy coupling between electron transfer and ATP synthesis is restored. On its own, the isolated F₁ component cannot catalyze ATP synthesis, but it can hydrolyze ATP into ADP and phosphate—which is why F₁ is referred to as F₁-ATPase.

ATP molecules are bound to The surface of the enzyme complex more tightly than ADP molecules

Studies of the catalytic mechanism of the purified F₁ enzyme using isotope exchange demonstrated that the synthesis of ATP from ADP and phosphate on its surface (ADP + P_i ⇄ ATP + H₂O) is reversible, meaning The change in free energy during this process is close to zero. The Hydrolysis of ATP catalyzed by the F₁ enzyme was carried out in vitro in the Presence of Water labeled with the ¹⁸O isotope. The label was subsequently detected in the resulting phosphate. More meticulous measurements revealed that the generated phosphate contained not one, but three or four ¹⁸O atoms (Fig. 19-23). This led to the Conclusion that the terminal pyrophosphate bond in the ATP molecule undergoes continuous turnover during the reaction—breaking and reforming repeatedly until the final product P_i dissociates from the surface of the F₁ enzyme. At any stage of hydrolysis, the phosphate located in the active site of the F₁ enzyme can randomly incorporate the ¹⁸O label into one of four possible positions. This exchange reaction occurs without any alteration in the proton gradient. The isolated F₁ enzyme also carries out ATP hydrolysis without Energy Expenditure.

Fig. 19-23. Catalytic mechanism of the F₁ enzyme. (a) Study via isotope exchange. The enzyme isolated from mitochondrial membranes is incubated with ATP in water labeled with the ¹⁸O isotope. Samples are then collected at short intervals to determine the ¹⁸O content in the resulting phosphate. Three or four ¹⁸O atoms are very rapidly detected in the phosphate, indicating repeated cycles of ATP synthesis and hydrolysis. (b) The most probable STRUCTURE OF THE F₁ enzyme in the Transition State of the reversible hydrolysis-synthesis reaction of ATP. The α-subunit is shown as a green helix, and the β-subunit as a gray helix. Positively charged residues β-Arg¹⁸² and α-Arg³⁷⁶ are coordinated with two oxygen atoms of the pentavalent phosphate. β-Lys¹⁵⁵ is linked to a third oxygen atom, while the Mg²⁺ ion (green sphere) forms a coordination complex with the enzyme. The blue sphere designates an H₂O molecule retained within the complex. Interactions within the active sites of the F₁ enzyme maintain the balance between ATP and (ADP + P_i).

Kinetic studies of ATP synthesis and hydrolysis reactions On the surface of the F₁ enzyme showed that the change in Standard Free Energy ∆G′° for the ATP synthesis reaction is close to zero. If the rate constant for surface ATP hydrolysis is r₁ = 10 s^-1 and the synthesis rate constant is r_-1 = 24 s^-1, the Equilibrium Constant for the reversible reaction involving enzyme-substrate complexes can be calculated using the following equation:

Enzyme-ATP ⇌ Enzyme-(ADP + P_i)

K′_eq = r_-1 / r₁ = 24 s^-1 / 10 s^-1 = 2.4

The apparent value of ∆G′°, calculated using this K′_eq, is close to zero. However, for the hydrolysis of free ATP in solution in the absence of the enzyme, K′_eq ≈ 10⁵, giving a ∆G′° of -30.5 kJ/mol.

The reason for this dramatic difference in rate constants and Free Energy Changes between the two reactions is that ATP molecules are far more stable on the surface of ATP synthase than (ADP + P_i). The ATP synthase enzyme complex forms tight bonds with ATP molecules, releasing enough energy in the process to drive ATP formation. Precise binding constant measurements revealed that the affinity of the F₀F₁ enzyme is significantly higher for ATP (K_d ≤ 10^-12 M) than for ADP (K_d ≤ 10^-5 M). Expressed in energetic terms, this difference in K_d values amounts to approximately 40 kJ/mol, serving as the driving force that shifts the equilibrium toward ATP formation.

Fig. 19-24. Energy profile of the catalytic action of ATP synthase compared to a typical enzyme. In most enzymatic reactions (left), the peak of the activation barrier corresponds to the transition state (≠) of the interaction between substrate and product. In the reaction catalyzed by ATP synthase (right), the maximum activation barrier corresponds to the release (dissociation, rather than synthesis!) of ATP from the enzyme surface. The change in free energy during the formation of ATP from ADP and P_i in aqueous solution is a large positive value. When this reaction is catalyzed by ATP synthase, ATP binds tightly to the enzyme surface, releasing enough energy to cleave ATP into ADP and P_i, thereby making the reaction reversible (with an equilibrium constant of 1). The release of ATP from the enzyme surface—that is, enzyme regeneration—is driven by the proton-motive force.

The proton gradient acts as the driving force for releasing ATP from the enzyme surface

The energy of the proton gradient is not required to maintain the specific concentration ratio of ATP and (ADP + P_i) on the surface of the ATP synthase enzyme. However, the release of the synthesized ATP from the enzyme surface relies entirely on the energy of the proton gradient. Figure 19-24 illustrates the energy diagrams for ATP synthesis driven by ATP synthase alongside other endergonic reactions.

During continuous ATP synthesis, the ATP synthase enzyme must undergo cyclic conformational changes, transitioning between a state that binds ATP tightly and one that promotes its release. Chemical and crystallographic studies have shown that the Structural Features of ATP synthase perfectly accommodate these alternative transitions.

Each β-subunit of ATP synthase can adopt three distinct Conformations

Mitochondrial F₁ protein complexes consist of nine subunits of five different types (3α, 3β, γ, δ, ε). Each β-subunit contains a single catalytic site where ATP synthesis takes place. Crystallographic data obtained by a research group led by John Walker have revealed the structural details of ATP synthase that explain its catalytic mechanism. The mushroom-shaped F₁ complex (“HEAD”) is an oblate sphere measuring 8 nm in height and 10 nm in diameter. Its α and β subunits are arranged in alternating pairs resembling orange segments (Fig. 19-25, a–c). The polypeptide chains of the γ subunit, which forms the central “stalk” of the F₁ crystal structure, are arranged asymmetrically. One domain of the γ subunit forms a shaft that penetrates the core of the complex head. Another domain of the γ subunit interacts primarily with one of the three β subunits, which adopts an “empty” conformation (β-empty) (Fig. 19-25, c).

John E. Walker

The conformations of the β subunits within the complex differ, even though their Amino acid sequences are identical. One reason for these conformational differences is that at any given moment, the γ subunit forms contacts with only one of the three β subunits. Crystallographic studies of the δ and ε subunits have not yet been carried out.

Due to the conformational differences among the β subunits in the F₁ complex, their ATP and ADP binding sites also exhibit structural variations, as demonstrated by the following experiment. The F₁ protein was crystallized from a mixture containing ADP and App(NH)p, a structural analog of ATP that is resistant to hydrolysis by the isolated F₁ complex. It was found that one β subunit binds App(NH)p, another binds ADP molecules, and the third β subunit binds neither Ligand—its active site remains empty. Reflecting the functional states of their active sites, these three β subunit conformations are designated as β-ATP, β-ADP, and β-empty (Fig. 19-25, c). The distinct nucleotide-binding Properties of the β subunits play a crucial role in the catalytic mechanism of the F₁ complex.

The F₀ protein complex comprises three subunits (a, b, and c) in a stoichiometric ratio of ab₂c_10–12, forming a channel dedicated to proton translocation. Subunit c is a small, highly hydrophobic polypeptide (M_r ~8,000) consisting of two transmembrane helices connected by a small loop that projects almost entirely into the mitochondrial matrix. As established in 1999 from the crystal structure of the Yeast F₀F₁ complex, the two helices of each of the 10 c subunits traverse the mitochondrial membrane almost perpendicular to its surface. The points where these helices intersect the membrane surface are arranged in two concentric circles (Fig. 19-25, d, e). The inner circle corresponds to the membrane crossings of the N-terminal helices, whereas the outer circle (diameter ~5.5 nm) corresponds to the C-terminal helices. The ε and γ subunits of the F₁ component form an L-shaped assembly that projects at one end into the mitochondrial matrix and rests at the other on a base composed of the concentrically arranged c subunits embedded in the mitochondrial membrane. The overall arrangement of the F₀F₁ complex subunits is shown in Fig. 19-25, f, based on data derived from the bovine mitochondrial F₁ enzyme and the yeast F₀F₁ complex.

Fig. 19-25. Structure of mitochondrial ATP synthase. (a) Structural model of the F₁ complex based on crystallographic and biochemical studies. Three α and three β subunits (colored gray and purple, respectively) alternate like orange segments around a central stalk formed by the γ subunit (green). (b) Side view of the crystal structure of the F₁ complex isolated from bovine Heart mitochondria (PDB ID 1BMF). The central stalk consists of the γ subunit (green helix). On the β subunits, ATP-binding sites are marked by red spheres, and ADP-binding sites by yellow spheres. Two α subunits, one β subunit, as well as the δ and ε subunits, have been omitted for clarity to reveal internal structures. (c) Top view of the F₁ complex from the side facing the negatively charged surface of the mitochondrial membrane. Three α and three β subunits are arrayed around the central stalk formed by the γ subunit (green helix). Nucleotide-binding sites crucial for enzyme activity are located at the interfaces between adjacent α and β subunits. The single γ subunit interacts predominantly with only one of the three αβ subunit pairs, which accounts for the conformational differences among the three β subunits and their active sites. One β subunit, designated as the β-ADP conformation, holds an ADP molecule (yellow sphere) in its active site. Another β subunit, designated as the β-ATP conformation, binds ATP molecules (third sphere). The conformation of the third β subunit, which lacks bound NUCLEOTIDES, is designated as β-empty.

Fig. 19-25. Continued, c — structure of the F₀F₁ complex, side view. The model is based on crystallographic data for The structure of the component isolated from bovine mitochondria and the F₀ component obtained from yeast mitochondria (PDB ID 1Q01). Subunits a, b, δ, and ε are not shown in this schematic representation of the F₀F₁ complex. d — Structure of the F₀F₁ complex, cross-section, top view, looking from the positively charged surface of the mitochondrial membrane toward its negatively charged surface. The cross-section clearly shows the concentric intersections of the two helices from each of the 10 subunits that span the membrane. e — Model of the F₀F₁ complex based on biochemical and crystallographic data. Two b subunits of the F₀ component are tightly bound to the α and β subunits of the F₁ component, fixing the "head" of the F₁ component in a stationary position relative to the mitochondrial membrane. The membrane-embedded cylinder composed of c subunits is connected to the stalk formed by the γ and ε subunits, which together make up the L-shaped system within the F₁ complex. As protons pass through the membrane via the F₀ channels from the positively charged side to the negatively charged side, the cylinder and stalk rotate. During this process, the γ subunit in the F₁ component sequentially forms bonds with one of the β subunits, thereby altering the conformations of the remaining β subunits.

The Mechanism of rotational catalysis is the key to understanding changes in binding site selectivity

Based on detailed studies of kinetics and ligand-binding conditions at active sites during Reactions Catalyzed by the F₀F₁ enzyme, Paul Boyer proposed the rotational catalysis mechanism. According to this model, the catalytic cycle of ATP synthesis occurs sequentially at three active sites of the F₁ component (Fig. 19-26). In The First stage of the cycle, ADP and phosphate from the external medium bind to the active site of one of the β subunits, which is in a conformation favorable for this process (β-ADP). In the second stage, interactions within this active site induce a conformational change to the β-ATP state, which favors tight binding to the ATP molecule. Retaining the ATP molecule at the active site brings the ADP + P_i system and ATP on the enzyme surface into a state of equilibrium. In the Third Stage, events within the active site cause this same β subunit to adopt an empty β conformation characterized by low affinity for ATP, resulting in the release of the ATP molecule from the enzyme surface. The next catalytic cycle begins when this same subunit once again adopts the β-ADP conformation and binds ADP and phosphate in its active site.

Paul Boyer

Fig. 19-26. Scheme of the catalytic action of ATP synthase according to the rotational mechanism of active site selectivity changes. The F₁ component of ATP synthase has three non-equivalent adenine Nucleotide binding sites located between pairs of α and β subunits. At any given moment, one of these sites is in the β-ATP conformation (capable of binding ATP tightly), another is in the β-ADP conformation (forming weak bonds with the ligand), and the third site has an empty β conformation favorable for bond Cleavage and product release. As the central stalk rotates driven by the proton-motive force, the γ subunit sequentially contacts each pair of α and β subunits in the F₁ component, simultaneously altering the conformations of all three active sites. The site with the β-ATP conformation transitions to the empty β conformation, leading to ATP dissociation. The site with the β-ADP conformation transitions to β-ATP, resulting in ATP formation from ADP and phosphate. The site with the empty β conformation is converted into the β-ADP site, where incoming ADP and phosphate from solution are bound. According to this mechanism, which has received experimental confirmation, at least two of the three catalytic sites must undergo different processes simultaneously; specifically, an ATP molecule cannot be released from one site until ADP and phosphate enter another.

The hypothesis of cyclic Conformational Changes in the β subunits of the F₁ component forms The basis of the rotational catalysis theory for ATP Biosynthesis. ATP biosynthesis is driven by the energy released as protons flow from the intermembrane space into the matrix through the channel of the F₀ complex. With each passage of protons through the proton channel, the cylinder composed of c subunits and the attached γ-subunit stalk rotate around an axis perpendicular to the plane of the mitochondrial membrane. The γ-subunit stalk rotates inside the stationary F₁ "head" (held in place by interactions with the b₂ and δ subunits), which is composed of three pairs of α and β subunits (Fig. 19-25d). With every 120° rotation, the γ subunit comes into contact with a different β subunit, forcing that β subunit into an empty conformation favorable for ATP release.

The conformational changes of the β subunits occur in a strictly ordered sequence. When one β subunit transitions to the empty β conformation, one of its adjacent β subunits must adopt the β-ADP conformation, and the other the β-ATP conformation. Thus, a full 360° Rotation of the γ subunit completes a cycle of conformational changes for each β subunit—from empty to β-ATP—resulting in the synthesis and release of three ATP molecules.

In accordance with this proposed Mechanism of ATP synthase action, one would expect that in one direction of γ-subunit rotation the F₀F₁ enzyme should synthesize ATP, whereas in the opposite direction it should hydrolyze ATP. This theoretical prediction was experimentally confirmed by Masasuke Yoshida and Kazuhiko Kinoshita Jr., who attached a long-chain fluorescently labeled Actin polymer to the γ subunit and directly observed the rotation of the γ subunit within the F₁ component using a Microscope. In a similar experiment with the intact F₀F₁ enzyme, they successfully observed the coordinated rotation of both the γ subunit and the entire c-subunit cylinder (Fig. 19-27). The single γ-subunit stalk did not rotate smoothly, but rather in three discrete 120° steps in the predicted direction. The efficiency of free energy conversion into mechanical motion in this system—which Boyer termed a "magnificent molecular machine"—was calculated from The rate of F₁-catalyzed ATP hydrolysis and the drag of the long actin filament, and it turned out to be close to 100%.

Fig. 19-27. Experiment enabling the observation of F₀ component and γ-subunit rotation. The genetically modified F₁ component, containing Histidine tags, is firmly attached to a microscope slide coated with a nickel complex. Biotin is attached to the c subunit of the enzyme's F₀ component. Avidin, tightly bound to biotin, is covalently linked to a long, fluorescently labeled actin filament. The actin filaments are connected to the c subunit via a biotin-avidin linkage. During F₁-catalyzed ATP hydrolysis, continuous rotation of the fluorescent actin filament is observed, indicating rotation of the F₀ c-subunit cylinder. When the fluorescent actin filament is attached directly to the γ subunit, micrographs taken at 133 ms intervals show that the actin filament abruptly changes its spatial position every 11 frames during rotation. It is hypothesized that the c-subunit cylinder and the γ-subunit stalk rotate as a single rigid unit.

The chemiosmotic theory allows for fractional values in the ratio between consumed O₂ and generated ATP

According to the oxidative phosphorylation hypothesis prevailing prior to the chemiosmotic theory, the overall reaction for NADH oxidation in the mitochondrial respiratory chain coupled with ATP synthesis was written as follows:

x ADP + x P_i + 1/2 O₂ + H⁺ + NADH —> x ATP + H₂O + NAD⁺ (19-11)

where x (the P/O ratio or P/2e^- ratio) was expected to take integer values. The amount of ATP synthesized in a suspension of intact mitochondria and an oxidizable substrate (such as succinate or NADH) in the presence of oxygen was estimated experimentally from oxygen consumption. However, measuring the P/O ratio was difficult because intact mitochondria consume ATP in various biochemical processes, and oxygen is utilized in pathways other than oxidative phosphorylation. Consequently, most such experiments yielded fractional numbers for P/O (or ATP / 1/2 O₂)—for instance, values between 2 and 3 for NADH, and between 1 and 2 for succinate. Nevertheless, integer values of 3 and 2 for NADH and succinate, respectively, were adopted for the P/O ratio for many years.

The chemiosmotic theory, which explains the coupling of electron transfer in the mitochondrial respiratory chain with mitochondrial ATP synthesis, does not postulate integer P/O ratios, but rather approaches the stoichiometry of oxidative phosphorylation from a different perspective. According to the chemiosmotic theory, calculating the stoichiometry of oxidative phosphorylation (i.e., the P/O ratio) requires taking into account not only the number of protons pumped out of the matrix across the inner mitochondrial membrane per pair of electrons transferred from one NADH molecule to oxygen, but also the number of protons returning to the matrix via the F₀F₁ proton channel to drive ATP synthesis. Measuring the number of protons crossing the mitochondrial membrane is technically challenging. One must account for the buffering capacity of mitochondria, non-productive proton leaks across the inner membrane (independent of the F₀F₁ proton channel), and determine what fraction of the Free energy of Electron transport is used for cellular activities other than ATP synthesis, such as substrate Transport Across the mitochondrial membrane. It is generally accepted that the number of protons pumped out of the matrix during The transfer of a pair of electrons from NADH to O₂ is 10, and from succinate to O₂ is 6. Most commonly, in interpreting experimental results, the number of protons whose extrusion generates sufficient proton-motive force to synthesize one ATP molecule is assumed to be 4. The proton-motive force generated by one of these four protons is utilized to transport phosphate, ATP, and ADP across the mitochondrial membrane, as discussed in detail elsewhere. Assuming that of the 10 protons pumped out of the matrix during the oxidation of one NADH molecule, four return to the matrix down their concentration gradient, releasing free energy sufficient to synthesize one ATP molecule, then P/O = 2.5. When succinate is the electron donor, under the same assumptions, P/O = 1.5 (6/4). The authors of this book use P/O values of 2.5 and 1.5, although values of 2 and 3 are also widely found in biochemical literature. A definitive answer regarding the stoichiometry of oxidative phosphorylation reactions will likely be obtained only after a detailed elucidation of the mechanism of the F₀F₁ complex.

The proton-motive force is utilized for active Transport of substances across the membrane

Although the proton-motive force generated by the proton concentration gradient across the mitochondrial membrane is used primarily for ATP synthesis, a portion of it is expended on transporting metabolites essential for oxidative phosphorylation across the membrane. The inner mitochondrial membrane is generally impermeable to charged particles, but it contains specific transport systems that enable the import of ADP and phosphate into the matrix and the export of ATP into the cytosol (Fig. 19-28).

Fig. 19-28. Adenine nucleotide translocase and phosphate translocase—transport systems of the inner mitochondrial membrane. Adenine nucleotide translocase is a specific protein functioning as an ATP/ADP antiporter. It imports an ADP^3- ion into the mitochondria in exchange for an exported ATP^4- ion from the matrix. This exchange results in the net transfer of a single negative charge across the membrane, facilitated by the positive potential on the outer face of the inner membrane. Phosphate translocase selectively transports the H₂PO₄^- ion from the cytosol into the matrix, even though at intracellular pH 7 phosphate exists as a mixture of HPO₄^2- and H₂PO₄^- species. The transport of H₂PO₄^- is coupled with the cotransport of an H⁺ ion into the matrix. Consequently, this ion movement causes no net charge transfer across the membrane, but the relatively low proton concentration in the matrix favors the inward movement of H⁺. The energy source driving ATP synthase and the two transport systems—which mediate substrate import (ADP and P_i) and export of newly synthesized ATP into the cytosol—is the proton-motive force. ATP synthase and these two transport systems can be isolated from mitochondria as a single functional complex known as the ATP synthasome.

Adenine nucleotide translocase is an integral membrane protein spanning the inner mitochondrial membrane that binds an ADP^3- molecule in the intermembrane space and transports it into the mitochondrial matrix in exchange for a simultaneously exported ATP^4- molecule. The Ionic Forms of ATP and ADP are shown in Fig. 13-11. Because the ATP/ADP antiporter introduces three negative charges into the matrix in exchange for four exported negative charges, a net single negative charge enters the matrix, helping to maintain the transmembrane electrochemical potential. The ATP/ADP exchange is driven by the proton-motive force. The activity of adenine nucleotide translocase can be specifically blocked by atractyloside, a toxic glycoside produced by a species of thistle. Atractyloside halts both ADP import and ATP export, thereby shutting down cellular ATP production.

Phosphate translocase is the second mitochondrial membrane transport system involved in oxidative phosphorylation; it mediates the symport of the H₂PO₄^- ion from the cytosol into the matrix. Phosphate transport is obligatorily coupled to the transport of a proton from the positively charged side of the inner membrane to its negatively charged side, utilizing energy released during electron transport. Both phosphate transport and ATP/ADP exchange derive their energy from the transmembrane proton gradient (Fig. 19-28).

The ATP synthase and the protein complexes of both translocases are linked together and, upon mild detergent disruption of mitochondria, can be isolated as an ATP synthasome.

Shuttle systems participate in the oxidation of extramitochondrial NADH

The NADH dehydrogenase complex of the inner mitochondrial membrane can accept electrons exclusively from NADH located in the matrix. The inner mitochondrial membrane is impermeable to cytosolic NADH molecules. How, then, can NADH produced during Glycolysis—which takes place, as is well known, outside the mitochondria—be reoxidized to NAD⁺ by molecular oxygen supplied through the respiratory chain? As it turns out, specialized shuttle systems transfer reducing equivalents from cytosolic NADH into the mitochondria via an indirect pathway.

The most active of these shuttle systems is the malate-aspartate shuttle, which operates in the mitochondria of the Liver, Kidneys, and heart. Let us turn to Fig. 19-29 to understand how this system works. First, reducing equivalents from cytosolic NADH are transferred by cytosolic malate dehydrogenase to cytosolic oxaloacetate, yielding malate. The malate, carrying the reducing equivalents derived from cytosolic NADH, crosses the inner mitochondrial membrane into the matrix via the malate-α-ketoglutarate transport system. Inside the mitochondrion, malate transfers these reducing equivalents to matrix NAD⁺ in a reaction catalyzed by matrix malate dehydrogenase. In this process, NAD⁺ is reduced to NADH, which can now pass its electrons directly into the respiratory chain of the inner mitochondrial membrane. In the malate-aspartate system, 2.5 molecules of ATP are synthesized for every pair of electrons transferred to oxygen. Cytosolic oxaloacetate is regenerated through the action of a transaminase, initiating a new turn of the shuttle cycle.

Fig. 19-29. The malate-aspartate shuttle. This system mediates the transfer of reducing equivalents from cytosolic NADH into the mitochondrial matrix and operates in liver, Kidney, and heart mitochondria. (1) Cytosolic NADH transfers two reducing equivalents to cytosolic oxaloacetate, producing malate. (2) The reducing equivalent-bearing malate is transported across the inner membrane by the malate-α-ketoglutarate transport system. (3) In the matrix, malate transfers two reducing equivalents to matrix NAD⁺. The resulting matrix NADH is oxidized by electrons from the mitochondrial respiratory chain. The product of the malate dehydrogenase reaction, oxaloacetate, cannot cross the membrane to return to the cytosol. (4) Action of a transaminase converts oxaloacetate to aspartate. (5) Aspartate is transported across the membrane by the glutamate-aspartate transport system. (6) In the cytosol, oxaloacetate is regenerated, and a new cycle of the shuttle begins.

In skeletal Muscles and the Brain, the transfer of reducing equivalents from NADH is mediated by a different type of shuttle system, known as the glycerol 3-phosphate shuttle (Fig. 19-30). It differs from the malate-aspartate system in that it transfers reducing equivalents from NADH to the respiratory chain via ubiquinone—that is, to complex III rather than complex I. Operation of the glycerol 3-phosphate shuttle yields 1.5 molecules of ATP per pair of transferred electrons during NADH oxidation.

Fig. 19-30. The glycerol 3-phosphate shuttle. This shuttle system transports reducing equivalents from cytosolic NADH into the mitochondrial matrix and Functions in Skeletal Muscle and brain tissue. Two reducing equivalents from cytosolic NADH are first transferred to dihydroxyacetone phosphate by cytosolic glycerol 3-phosphate dehydrogenase. The two reducing equivalents from cytosolic glycerol 3-phosphate are subsequently transferred to ubiquinone by an isoenzyme of glycerol 3-phosphate dehydrogenase located on the outer face of the inner membrane. The glycerol 3-phosphate transport system is not coupled to membrane transport Proteins.

Plant mitochondria possess a so-called "external" NADH dehydrogenase that channels electrons from cytosolic NADH directly into the mitochondrial respiratory chain via ubiquinone, bypassing complex I NADH dehydrogenase and its associated proton translocation. The oxidation of cytosolic NADH yields a lower ATP output than the oxidation of matrix NADH (see Box 19-1).

Summary of Section 19.2 ATP Synthesis

■ Electron flow through enzyme complexes I, III, and IV drives the outward pumping of protons from the matrix across the inner mitochondrial membrane into the intermembrane space, rendering the matrix more alkaline and the intermembrane space more acidic. An H⁺ ion concentration gradient is thus established across the inner mitochondrial membrane. This very gradient serves as the driving force for ATP synthesis from ADP and inorganic phosphate, catalyzed by the enzyme ATP synthase (the F₀F₁ complex) embedded in the inner mitochondrial membrane.

■ ATP synthase catalyzes mitochondrial ATP synthesis via a rotational mechanism. The core principle of rotational catalysis is that each translocation of protons from the cytosol to the matrix through the F₀ channel induces a cyclical conformational change in each of the three nucleotide-binding sites within the F₁ component. These sites sequentially adopt conformations favoring (ADP + P_i) binding, ATP binding, and finally an empty conformation that releases the product from the enzyme surface.

■ Only a minimal amount of Energy is required for the actual formation of ATP on the enzyme surface. In mitochondrial enzymatic ATP synthesis, the energy of the proton-motive force is utilized primarily to release the synthesized ATP from the binding site on the ATP synthase surface.

■ When electrons enter the mitochondrial respiratory chain via complex I, the P/O ratio (the number of ATP molecules synthesized per pair of electrons yielding $ ext{H}_2 ext{O}$ or oxygen atoms reduced) is approximately 2.5. When electrons enter the respiratory chain directly at coenzyme Q, the P/O ratio is 1.5.

■ The energy stored in the proton concentration gradient can be harnessed to drive the transport of solutes across the mitochondrial membrane.

■ The inner mitochondrial membrane is impermeable to cytosolic NADH and NAD⁺. Reducing equivalents from cytosolic NADH are transported into the matrix via one of two shuttle systems. The malate-aspartate system transfers reducing equivalents from cytosolic NADH to the mitochondrial respiratory chain through complex I, yielding an oxidative phosphorylation P/O ratio of 2.5. The glycerol 3-phosphate shuttle delivers reducing equivalents from cytosolic NADH to the mitochondrial respiratory chain via coenzyme Q, resulting in a P/O ratio of 1.5.