Principles of Biochemistry Volume 2 - A. Lehninger 1985

Bioenergetics and Metabolism
Electron Transport, Oxidative Phosphorylation, and Regulation of ATP Synthesis
How is the redox energy of electron transport coupled to ATP synthase?

In the preceding sections of this chapter, we examined The electron transport process and explored The Structure of ATP synthase. Now, the time has come to address the core question: exactly how does the Electron Transport Chain interact with ATP synthase, and how does this drive the Oxidative Phosphorylation of ADP to form ATP? This ranks among the most fascinating yet challenging questions in biochemistry and Cell biology. Although we already know a great deal about the Utilization of ATP energy in biosynthetic reactions, the exact MOLECULAR MECHANISMS OF ATP generation during oxidative phosphorylation remain elusive. One reason for this is that the Enzymes of Electron Transport and oxidative phosphorylation are extraordinarily complex and embedded within The inner mitochondrial membrane, which greatly hinders The Study of their interaction. Three possible mechanisms for energy transfer from electron transport to ATP synthesis have been proposed.

The chemical coupling hypothesis suggests that Electron transport is coupled to ATP synthesis through a specific sequence of reactions; during these reactions, a high-energy covalent intermediate formed As a result of electron transport is cleaved, releasing its stored energy to drive The formation of ATP. This concept is analogous to the familiar role of 3-phosphoglyceroyl phosphate as a common intermediate in ATP synthesis during Glycolysis (Section 15.7.6).

The conformational coupling hypothesis proposes that the flow of electrons along the Respiratory Chain induces Conformational Changes in the protein Components of the inner mitochondrial membrane, thereby shifting them into a high-energy state. These conformational changes are transmitted to the F₀F₁-ATPase molecule, activating it. The relaxation of the activated F₀F₁-ATPase—that is, its return to its normal conformation—releases the stored energy, which is utilized for ATP synthesis and for the release of the newly synthesized ATP from the enzyme molecule.

The chemiosmotic hypothesis, formulated by the British biochemist Peter Mitchell, is based on an entirely different, novel principle. It postulates that electron transport is accompanied by the outward pumping of H⁺ ions from the matrix, across the inner mitochondrial membrane, and into the external aqueous medium. As a result, an H⁺ concentration gradient (a transmembrane gradient) is established across the Two Sides of the inner mitochondrial membrane. The synthesis of ATP, which requires an energy input, is driven precisely by the osmotic energy inherent in this gradient. It is widely accepted that the chemiosmotic theory most accurately reflects the organizing principle of oxidative phosphorylation. Let us examine several KEY FEATURES OF this process that support the chemiosmotic hypothesis.

a. No "high-energy" intermediates linking electron transport to ATP synthesis have been detected

Years of intensive research aimed at identifying such hypothetical intermediates have yielded no results: they could not be found.

b. Oxidative phosphorylation requires an intact inner mitochondrial membrane

Oxidative phosphorylation can take place only if the integrity of the inner mitochondrial membrane remains undisturbed—that is, if the membrane forms a completely closed vesicle. Any tears or breaks in the inner mitochondrial membrane abolish its capacity for oxidative phosphorylation, even though electron transport from substrate to oxygen may continue under these conditions.

c. The inner mitochondrial membrane is impermeable to H⁺, OH^-, K⁺, and Cl^- ions

This membrane property is also critical for oxidative phosphorylation. If the membrane is damaged, or if some agent causes it to become freely permeable to these or certain other ions, oxidative phosphorylation ceases. These observations indicate that a difference in ionic composition or concentration across the two sides of the inner mitochondrial membrane plays an essential role in ATP synthesis.

d. Oxidative phosphorylation can be prevented by uncoupling agents

Certain chemical substances, such as 2,4-dinitrophenol (Fig. 17-17), inhibit the phosphorylation of ADP to ATP without affecting electron transport in Mitochondria. They uncouple electron transport from ATP synthesis by breaking the necessary link between these two processes. Such compounds are therefore called uncoupling agents. In their presence, the Free energy released during electron transport is dissipated as heat rather than being conserved as ATP. Uncoupling agents dramatically increase the permeability of the inner mitochondrial membrane to H⁺ ions. These lipophilic substances have The ability to bind H⁺ ions on one side of the membrane and carry them across to the other side, where their concentration is lower.

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Fig. 17-17. Action of the typical uncoupling agent 2,4-dinitrophenol. At pH 7, this agent exists primarily as an anion lacking lipid solubility. In its protonated form, 2,4-dinitrophenol is lipid-soluble and can therefore traverse the membrane, carrying a proton with it. On the other side of the membrane, the transported proton dissociates. Acting in this manner, uncoupling agents prevent the formation of an H⁺ concentration gradient across the membrane. Such H⁺-transporting uncoupling agents are referred to as protonophores.

At one time, attempts were made to use certain uncoupling agents to treat obesity by lowering the efficiency of ATP synthesis. However, these substances proved to be extremely toxic, and consequently, this application had to be abandoned.

e. Certain ionophores are also capable of inhibiting oxidative phosphorylation

Ionophores (i.e., "ion carriers") are lipid-soluble molecules that can bind specific ions and transport them across membranes. They differ from uncoupling agents in that they transport cations other than H⁺ ions across the membrane. For example, the toxic antibiotic valinomycin (Fig. 17-18) forms a lipid-soluble complex with K⁺ ions that readily crosses the inner mitochondrial membrane, whereas in the absence of valinomycin, K⁺ ions penetrate the membrane with great difficulty. The ionophore gramicidin facilitates the transmembrane movement not only of K⁺ but also of Na⁺ and certain other monovalent cations. Thus, uncoupling agents and ionophores inhibit oxidative phosphorylation by increasing membrane permeability to H⁺, K⁺, or Na⁺ ions.

Fig. 17-18. Valinomycin is a toxic antibiotic that stimulates The transport of K⁺ ions across the membrane. This K⁺-carrying ionophore consists of L- and D-valine, lactate, and hydroxyisovalerate residues linked in a ring-like structure. Valinomycin forms a specific complex with a K⁺ ion (shown in red), which is positioned in its hydrophilic interior. Owing to the lipid-soluble outer surface of the valinomycin molecule (shown in gray), the valinomycin–K⁺ complex easily crosses the mitochondrial membrane.

f. Electron flow forces H⁺ ions to be expelled outward from respiring mitochondria

The energy released during Electron transport along the respiratory chain from substrate to oxygen can, under certain conditions, drive the translocation of H⁺ ions from the mitochondrial matrix into the surrounding medium. As a result, the pH of the mitochondrial matrix rises while the pH of the medium falls; that is, the matrix becomes more alkaline and the medium surrounding the mitochondria becomes more acidic. The inner mitochondrial membrane must therefore contain specific "pumps" for H⁺ ions that utilize the Free energy of electron flow to pump H⁺ ions outward against their concentration gradient. The outward pumping of H⁺ ions from mitochondria generates a membrane electrical potential because the exit of these positive charges into the medium makes the outer face of the membrane more electropositive and the inner face more electronegative. Thus, electron transport creates an electrochemical H⁺ gradient comprising two components, with the Membrane Potential making the major contribution to the energy of this gradient:

In this equation, Z is a conversion factor that converts pH units into millivolts—the units usually used to express ∆μ_H and ∆ψ. THE CONTRIBUTION OF the membrane potential accounts for approximately 75% of the total electrochemical H⁺ gradient generated by electron transport.