Phosphorylation of ADP to ATP

PHOSPHORYLATION OF ADP TO ATP

The transfer of electrons down the ETC is energetically favored because NADH is a strong electron donor and O₂ is an avid electron acceptor. However, the flow of electrons does not directly result in ATP synthesis.

A. Chemiosmotic hypothesis

The chemiosmotic hypothesis (also known as the Mitchell hypothesis) explains how the free energy generated by the transport of electrons by the ETC is used to produce ATP from ADP + Pi.

1. Proton pump: Electron transort is coupled to the phosphorylation of ADP by the pumping of protons across the inner mitochondrial membrane, from the matrix to the intermembrane space, at Complexes I, III, and IV. This process creates an electrical gradient (with more positive charges on the outside of the membrane than on the inside) and a pH gradient (the outside of the membrane is at a lower pH than the inside) as shown in Figure 6.13. The energy generated by this proton gradient is sufficient to drive ATP synthesis. Thus, the proton gradient serves as the common intermediate that couples oxidation to phosphorylation.

Figure 6.13 Electron transport chain shown in association with the transport of protons (H+). A total of ten H+ are pumped for each nicotinamide adenine dinucleotide (NADH) oxidized. [Note: H+ are not pumped at Complex II.]

2. ATP synthase: The multisubunit enzyme ATP synthase (Complex V; see Figure 6.14) synthesizes ATP using the energy of the proton gradient. It contains a domain (F_o) that spans the inner mitochondrial membrane, and an extramembranous domain (F₁) that appears as a sphere that protrudes into the mitochondrial matrix (see Figure 6.13). The chemiosmotic hypothesis proposes that after protons have been pumped to the cytosolic side of the inner mitochondrial membrane, they reenter the matrix by passing through a proton channel in the F_o domain, driving rotation of the c ring of F_o and, at the same time, dissipating the pH and electrical gradients. F_o rotation causes conformational changes in the β subunits of the F₁ domain that allow them to bind ADP + Pi, phosphorylate ADP to ATP, and release ATP. [Note: ATP synthase is also called F₁/Fo-ATPase because the isolated enzyme can catalyze the hydrolysis of ATP to ADP and Pi.]

Figure 6.14 ATP synthase (F1Fo-ATPase). [Note: The rotation of the ring of c subunits in the Fo domain results in conformational changes in the β subunits of the F1 domain that allow phosphorylation of adenosine diphosphate (ADP) to adenosine triphosphate (ATP). Pi = inorganic phosphate.

a. Coupling in oxidative phosphorylation: In normal mitochondria, ATP synthesis is coupled to electron transport through the proton gradient. Increasing (or decreasing) one process has the same effect on the other. For example, hydrolysis of ATP to ADP and Pi in energy-requiring reactions increases the availability of substrates for ATP synthase and, thus, increases proton flow through the enzyme. Electron transport and proton pumping by the ETC increase to maintain the proton gradient. [Note: Increased oxidation of NADH at Complex I and, consequently, an increase in NADH-producing pathways of metabolism, such as the TCA cycle, results.]

b. Oligomycin: This drug binds to the F_o (hence the letter “o”) domain of ATP synthase, closing the proton channel and preventing reentry of protons into the matrix, thereby preventing phosphorylation of ADP to ATP. Because the pH and electrical gradients cannot be dissipated in the presence of this drug, electron transport stops because of the difficulty of pumping any more protons against the steep gradients. This dependency of cellular respiration on the ability to phosphorylate ADP to ATP is known as respiratory control and is the consequence of the tight coupling of these processes.

c. Uncoupling proteins: Uncoupling proteins (UCPs) occur in the inner mitochondrial membrane of mammals, including humans. These proteins form channels that allow protons to reenter the mitochondrial matrix without energy being captured as ATP (Figure 6.15). The energy is released as heat, and the process is called nonshivering thermogenesis. UCP1, also called thermogenin, is responsible for heat production in the brown adipocytes of mammals. In brown fat, unlike the more abundant white fat, almost 90% of its respiratory energy is used for thermogenesis in response to cold in the neonate and during arousal in hibernating animals. However, humans appear to have few concentrated deposits of brown fat (except in the newborn), and UCP1 does not appear to play a major role in energy balance. [Note: Uncoupling proteins UCP2–UCP5 have been found in other tissues, but their full significance remains unclear.]

d. Synthetic uncouplers: Electron transport and phosphorylation can also be uncoupled by compounds that pick up protons in the intermembrane space and release them in the matrix, dissipating the gradient. The classic example is 2,4-dinitrophenol, a lipophilic proton carrier that readily diffuses through the mitochondrial membrane. This uncoupler causes electron transport to proceed at a rapid rate without establishing a proton gradient, much as do the UCPs (see Figure 6.15). Again, energy is released as heat rather than being used to synthesize ATP. [Note: In high doses, aspirin and other salicylates uncouple oxidative phosphorylation. This explains the fever that accompanies toxic overdoses of these drugs.]

Figure 6.15 Transport of protons across the mitochondrial membrane by an uncoupling protein. ADP = adenosine diphosphate; ATP = adenosine triphosphate.

B. Membrane transport systems

The inner mitochondrial membrane is impermeable to most charged or hydrophilic substances. However, it contains numerous transport proteins that permit passage of specific molecules from the cytosol (or more correctly, the intermembrane space) to the mitochondrial matrix.

1. ATP and ADP transport: The inner membrane requires specialized carriers to transport ADP and Pi from the cytosol (where ATP is hydrolyzed to ADP in many energy-requiring reactions) into mitochondria, where ATP can be resynthesized. An adenine nucleotide antiporter imports one ADP from the cytosol into the matrix, while exporting one ATP from the matrix into the cytosol (see Figure 6.13). A transporter moves Pi from the cytosol into mitochondria.

2. Transport of reducing equivalents: The inner mitochondrial membrane lacks an NADH transporter, and NADH produced in the cytosol (for example, in glycolysis;) cannot directly enter the mitochondrial matrix. However, two electrons (reducing equivalents) of NADH are transported from the cytosol into the matrix using substrate shuttles. In the glycerophosphate shuttle (Figure 6.16A), two electrons are transferred from NADH to dihydroxyacetone phosphate by cytosolic glycerophosphate dehydrogenase. The glycerol 3-phosphate produced is oxidized by the mitochondrial isozyme as FAD is reduced to FADH₂. CoQ of the ETC oxidizes the FADH₂. The glycerophosphate shuttle, therefore, results in the synthesis of two ATPs for each cytosolic NADH oxidized. This contrasts with the malate-aspartate shuttle (Figure 6.16B), which produces NADH (rather than FADH₂ FADH₂ FADH₂ FADH₂) in the mitochondrial matrix and, therefore, yields three ATPs for each cytosolic NADH oxidized by malate dehydrogenase as oxaloacetate is reduced to malate. A transport protein moves malate into the matrix.

Figure 6.16 Substrate shuttles for the transportof electrons across the inner mitochondrial membrane. A. Glycerophosphate shuttle. B. Malate-aspartate shuttle. DHAP = dihydroxyacetone phosphate; NAD(H) = nicotinamide adenine dinucleotide; FAD(H2) = flavin adenine dinucleotide; CoQ = coenzyme Q.

C. Inherited defects in oxidative phosphorylation

Thirteen of the approximately 90 polypeptides required for oxidative phosphorylation are coded for by mtDNA and synthesized in mitochondria, whereas the remaining proteins are coded for by nuclear DNA, synthesized in the cytosol, and transported into mitochondria posttranslationally. Defects in oxidative phosphorylation are more likely a result of alterations in mtDNA, which has a mutation rate about 10 times greater than that of nuclear DNA. Tissues with the greatest ATP requirement (for example, central nervous system, skeletal and heart muscle, and liver) are most affected by defects in oxidative phosphorylation. Mutations in mtDNA are responsible for several diseases, including some cases of mitochondrial myopathies, and Leber hereditary optic neuropathy, a disease in which bilateral loss of central vision occurs as a result of neuroretinal degeneration, including damage to the optic nerve. [Note: mtDNA is maternally inherited because mitochondria from the sperm cell do not enter the fertilized egg.]

D. Mitochondria and apoptosis

The process of apoptosis, or programmed cell death, may be initiated through the intrinsic (mitochondrial-mediated) pathway by the formation of pores in the outer mitochondrial membrane. These pores allow cytochrome c to leave the intermembrane space and enter the cytosol. There, cytochrome c, in association with proapoptotic factors, activates a family of proteolytic enzymes (the caspases), causing cleavage of key proteins and resulting in the morphologic and biochemical changes characteristic of apoptosis.