Principles of Biochemistry, Volume 2 - A. Lehninger 1985

Bioenergetics and Metabolism
Electron Transport, Oxidative Phosphorylation, and the Regulation of ATP Synthesis
According to the chemiosmotic hypothesis, the energy of electron transport is coupled to ATP synthesis via a proton gradient.

The very properties of Cell/35.html">Mitochondria that we have just discussed formed the basis for the chemiosmotic hypothesis (Figs. 17–19). According to this hypothesis, the function of electron transport occurring in The inner mitochondrial membrane is to pump H⁺ ions out of the mitochondrial matrix into the external medium, thereby establishing a concentration gradient of H⁺ ions across the two aqueous phases separated by this membrane, with a more acidic pH on the outside. Such a gradient, in which the concentration of H⁺ ions is higher outside than inside the mitochondrion, possesses potential energy (Sec. 14.16). The chemiosmotic hypothesis further postulates that the H⁺ ions driven outward by the energy of electron transport flow back inward into the mitochondrial matrix through special channels, or "pores," for these ions within the F₀F₁-ATPase molecules. In this process, they move down their concentration gradient, and Free energy is released as they cross the ATPase molecules. It is this energy that serves as the driving force for the coupled synthesis of ATP from ADP and phosphate.

Thus, the chemiosmotic hypothesis requires no high-energy chemical intermediate that would act as a common currency to couple electron transport with ATP synthesis. Instead, the energy transducer linking these two processes is the concentration gradient of H⁺ ions across the Two Sides of the mitochondrial membrane. In light of this hypothesis, the requirement for membrane integrity—that is, its complete closure in intact mitochondria or in membrane vesicles formed from disrupted inner membranes (Fig. 17–15)—becomes readily understandable; it is clear that without this integrity, the concentration gradient of H⁺ ions across the membrane simply could not exist. It is also easy to see that upon the "leakage" of H⁺ ions through the membrane in the presence of uncoupling agents (Fig. 17–17), the H⁺ gradient must "discharge," meaning that energy coupling is weakened. Finally, it has been demonstrated that the extrusion of H⁺ ions from mitochondria during Electron Transport and the uptake of external H⁺ ions by ATP synthase molecules are comparable in rate to The process of Oxidative Phosphorylation in intact mitochondria.

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Fig. 17–19. Coupling of electron transport with ATP Synthesis in the context of the chemiosmotic hypothesis. According to this hypothesis, the Electron Transport Chain can be viewed as a pump that translocates H⁺ ions. The energy released during Electron transport is utilized to move H⁺ ions outward from the mitochondrial matrix, resulting in the generation of an electrochemical H⁺ gradient with a higher concentration of H⁺ ions in the outer aqueous phase. This same process leads to The formation of a transmembrane electrical potential, with the outer side of the membrane becoming electropositive.

H⁺ ions from the surrounding medium rush back inward—that is, into the mitochondrial matrix—this time down their electrochemical gradient through F₀F₁-ATPase molecules. This translocation of H⁺ ions from a region of higher to a region of lower concentration is accompanied by the release of free energy, which drives the synthesis of ATP.

Thus, the chemiosmotic hypothesis suggests that a continuous cycle of H⁺ ions takes place between the mitochondrion and its surrounding environment, driven by electron transport (see Box 17–1).

However, while the chemiosmotic hypothesis satisfactorily accounts for most features of oxidative phosphorylation, certain aspects of this process still remain unclear. In particular, the mechanism by which The electron transport chain "pumps" H⁺ ions out of the mitochondrial matrix remains enigmatic (Box 17–1).

Box 17–1. Much about the Mechanism of Oxidative phosphorylation remains unclear

Although the chemiosmotic hypothesis has gained wide acceptance regarding its core organizing principle—The transfer of energy from electron transport to ATP synthesis in mitochondria, bacterial Cells, and METABOLISM/14.html">Chloroplasts (Chap. 23)—it nevertheless leaves many important questions unanswered. Perhaps the most controversial issue is the mechanism by which electron transport occurring in the inner membrane causes H⁺ ions to be pumped out of the mitochondrial matrix. Mitchell proposed an ingenious solution to this problem (Fig. 1). His solution was based on the fact that reducing equivalents are carried by some carriers (e.g., ubiquinone) in the form of H atoms, and by others (e.g., iron-sulfur centers or Cytochromes) in the form of electrons. Mitchell hypothesized that hydrogen-carrying and electron-carrying Proteins alternate in the Respiratory Chain, forming three "loops" within it. In each such loop, two H atoms are translocated across the membrane to the outside and release two H⁺ ions into the surrounding medium; the corresponding pair of electrons is then transferred back from the outer surface of the membrane to the inner surface (Fig. 1). Each pair of reducing equivalents passing through such a loop transfers two H⁺ ions from the matrix to the surrounding medium. It is assumed that each loop supplies the osmotic energy required for the formation of one ATP molecule.

This hypothetical mechanism appears quite attractive, yet it is not supported by all experimental data. First, it postulates a specific sequence and alternation of hydrogen- and electron-carrying centers, which does not entirely agree with available evidence. Second, under such a mechanism, only two H⁺ ions could be transferred per pair of electrons in a single loop, since each outwardly moving electron is accompanied by only one proton. Meanwhile, recent studies have shown that at least three, and possibly four, H⁺ ions are transferred per pair of electrons in a loop, and that three or four H⁺ ions return to the matrix for every synthesized ATP molecule.

Other questions also arise. Does the normal process of oxidative phosphorylation truly involve the efflux of H⁺ ions from mitochondria and their return to the matrix? It appears that at least a portion of this H⁺ translocation may occur within the membrane or on its surface, rather than between the two aqueous phases separated by the membrane. Furthermore, the question remains as to precisely how the flux of H⁺ ions through the complex ATP synthase system creates the new covalent bond by which the terminal phosphate group of ATP is attached.

Fig. 1. Mechanism of H⁺ ion transport postulated by the chemiosmotic hypothesis. Successive carriers of the respiratory chain (A–F) are assumed to form three H⁺-transporting "loops." Each such loop translocates two H⁺ ions outward from the mitochondrial matrix via a carrier (red arrows) that transports reducing equivalents in the form of hydrogen atoms.

The two electrons remaining after the extrusion of two H⁺ ions into the medium are returned—that is, transferred to the opposite side of the membrane—by a carrier (gray arrows) that transports reducing equivalents in the form of electrons. For every pair of electrons flowing from the substrate RH₂ to oxygen, these three loops transfer six hydrogen ions (3∙2 = 6H⁺) from the mitochondrial matrix into the medium. All Components of the respiratory chain are assumed to be fixed to the membrane, which ensures their required spatial arrangement relative to one another.

Thus, we still have a great deal to learn about the molecular components and properties of energy-transducing membranes in mitochondria, bacterial cells, and chloroplasts. Someday, following numerous experiments and the testing of new ideas, we will find Answers to these questions, but for now they remain unresolved, largely due to the complex Structure OF THE inner membrane. Such is the path of scientific inquiry: researchers build hypotheses based on experimental observations and test them again and again to ensure that no discovered fact is left unexplained. In a certain sense, biological research never truly ends. Frequently, what once seemed firmly established turns out to be merely an approximation of the truth—a step along the way to a deeper understanding revealed by new facts and concepts. Exploring the molecular logic of living cells is truly a boundless field.