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Transcript
ELECTRON TRANSPORT CHAIN Energy-rich molecules, such as glucose, are metabolized by a series of oxidation reactions ultimately yielding CO2 and water (Figure 6.6). The metabolic intermediates of these reactions donate electrons to specific coenzymes—nicotinamide adenine dinucleotide (NAD+) and flavin adenine dinucleotide (FAD)—to form the energy-rich reduced coenzymes, NADH and FADH2. These reduced coenzymes can, in turn, each donate a pair of electrons to a specialized set of electron carriers, collectively called the electron transport chain. • As electrons are passed down the electron transport chain, they lose much of their free energy. Part of this energy can be captured and stored by the production of ATP from ADP and inorganic phosphate (Pi). This process is called oxidative phosphorylation A. Mitochondrion The electron transport chain is present in the inner mitochondrial membrane and is the final common pathway by which electrons derived from different fuels of the body flow to oxygen. Electron transport and ATP synthesis by oxidative phosphorylation proceed continuously in all tissues that contain mitochondria. 1. Structure of the mitochondrion: The components of the electron transport chain are located in the inner membrane. Although the outer membrane contains special pores, making it freely permeable to most ions and small molecules, the inner mitochondrial membrane is a specialized structure that is impermeable to most small ions, including H+, Na+, and K+, small molecules such as ATP, ADP, pyruvate, and other metabolites important to mitochondrial function (Figure 6.7). Specialized carriers or transport systems are required to move ions or molecules across this membrane. The inner mitochondrial membrane is unusually rich in protein, half of which is directly involved in electron transport and oxidative phosphorylation. The inner mitochondrial membrane is highly convoluted. The convolutions, called cristae, serve to greatly increase the surface area of the membrane. • Aerobic organisms are able to capture a far greater proportion of the available free energy of respiratory substrates than anaerobic organisms. Most of this takes place inside mitochondria, which have been termed the "powerhouses" of the cell. Respiration is coupled to the generation of the high-energy intermediate, ATP, by oxidative phosphorylation. 2. ATP synthase complexes: These complexes of proteins are referred to as inner membrane particles and are attached to the inner surface of the inner mitochondrial membrane. They appear as spheres that protrude into the mitochondrial matrix. 3. Matrix of the mitochondrion: This gel-like solution in the interior of mitochondria is fifty percent protein. These molecules include the enzymes responsible for the oxidation of pyruvate, amino acids, fatty acids (by β-oxidation), and those of the tricarboxylic acid (TCA) cycle. The synthesis of urea and heme occur partially in the matrix of mitochondria. In addition, the matrix contains NAD+ and FAD (the oxidized forms of the two coenzymes that are required as hydrogen acceptors)and ADP and Pi, which are used to produce ATP. [Note: The matrix also contains mitochondrial RNA and DNA (mtRNA and mtDNA) and mitochondrial ribosomes.] B. Organization of the chain The inner mitochondrial membrane can be disrupted into five separate enzyme complexes, called complexes I,II,III, IV, and V. Complexes I to IV each contain part of the electron transport chain (Figure 6.8), whereas complex V catalyzes ATP synthesis . Each complex accepts or donates electrons to relatively mobile electron carriers, such as coenzyme Q and cytochrome c Each carrier in the electron transport chain can receive electrons from an electron donor, and can subsequently donate electrons to the next carrier in the chain. The electrons ultimately combine with oxygen and protons to form water. This requirement for oxygen makes the electron transport process the respiratory chain, which accounts for the greatest portion of the body's use of oxygen. C. Reactions of the electron transport chain With the exception of coenzyme Q, all members of this chain are proteins. These may function as enzymes as is the case with the dehydrogenases, they may contain iron as part of an iron-sulfur center, they may be coordinated with a porphyrin ring as in the cytochromes, or they may contain copper, as does the cytochrome a + a3 complex. 1. Formation of NADH: NAD+ is reduced to NADH by dehydrogenases that remove two hydrogen atoms from their substrate. (For examples of these reactions, see the discussion of the dehydrogenases found in the TCA cycle. Both electrons but only one proton (that is a hydride ion, :H ) are transferred to the NAD+, forming NADH plus a free proton, H+. 2. NADH dehydrogenase: The free proton plus the hydride ion carried by NADH are next transferred to NADH dehydrogenase, an enzyme complex (complex I)embedded in the inner mitochondrial membrane. This complex has a tightly bound molecule of flavin mononucleotide (FMN, a coenzyme structurally related to FAD, that accepts the two hydrogen atoms 2_e~ + 2H+) becoming FMNH2. • NADH dehydrogenase also contains several iron atoms paired with sulfur atoms to make iron-sulfur centers (Figure 6.9). These are necessary for the transfer of the hydrogen atoms to the next member of the chain, ubiquinone (known as coenzyme Q). 3. Coenzyme Q: Coenzyme Q is a quinone derivative with a long isoprenoid tail. It is also called ubiquinone because it is ubiquitous in biologic systems. Coenzyme Q can accept hydrogen atoms both from FMNH2, produced by NADH dehydrogenase, and from FADH2 (Complex II), which is produced by succinate dehydrogenase and acyl CoA dehydrogenase . 4. Cytochromes: The remaining members of the electron transport chain are cytochromes. Each contains a heme group made of a porphyrin ring containing an atom of iron (see p. 277). Unlike the heme groups of hemoglobin, the cytochrome iron atom is reversibly converted from its ferric (Fe+ 3) to its ferrous( Fe+2) form as a normal part of its function as a reversible carrier of electrons. Electrons are passed along the chain from coenzyme Q to cytochromes b and c (Complex III) and a +a3 (Complex IV, see Figure 6.8) 5. Cytochrome a +a3 This cytochrome complexes the only electron carrier in which the heme iron has a free ligand that can react directly with molecular oxygen. At this site, the transported electrons, molecular oxygen, and free protons are brought together to produce water (see Figure 6.8). Cytochrome a + a3(also called cytochrome oxidase) contains bound copper atoms that are required for this complex reaction to occur. • Q Accepts Electrons Via Complex I and Complex II. • The Q Cycle Couples Electron Transfer to Proton Transport in Complex III. • Molecular Oxygen Is Reduced to Water Via Complex IV. 6. Site-specific inhibitors: Site-specific inhibitors of electron transport have been identified and are illustrated in Figure (6.10) These compounds prevent the passage of electrons by binding to a component of the chain, blocking the oxidation/reduction reaction. Therefore, all electron carriers before the block are fully reduced, whereas those located after the block are oxidized. [Note: Because electron transport and oxidative phosphorylation are tightly coupled, site-specific inhibition of the electron transport chain also inhibits ATP synthesis.] Sequence of redox system in respiratory chain • The arrangement of component enzyme and coenzyme in the respiratory chain depend on the redox potential, the most negative potential is that of NAD so it ś the first member of the respiratory chain , which can receive H from substrate and become reduced. C. Release of free energy during electron transport Free energy is released as electrons are transferred along the electron transport chain from an electron donor (reducing agent or reductant) to an electron acceptor (oxidizing agent or oxidant). The (electrons can be transferred in different forms, for example, as hydride ions (:H-) to NAD+, as hydrogen atoms (-H) to FMN, coenzyme Q, and FAD, or as electrons –e-) to cytochromes. Site of ATP Production Each pair of electrons when it enter the chain from the beginning till reach with oxygen then one high energy product (ATP) is produced in 3 sites between NADH and FP, CoQ or ubiquinone and cytochrome b , cytochrome a a3 &O2 . Therefore 3molecule of ATP are produced from the cycle when started from the beginning (NAD) to the end. While when the electrons enter the respiratory chain from CoQ then 2ATP are formed as it miss or by pass the first sites of synthesis of ATP. site1 ADP+Pi NAD site3 ATP O2 ATP FP ADP+Pi Cyto aa3 site2 ADP+Pi ATP CoQ Cyto b Cyto c Cyto c1 OXIDATIVE PHOSPHORYLATION • The transfer of electrons down the electron transport chain is energetically favored because NADH is a strong electron donor and molecular oxygen is an avid electron acceptor. Mechanism of oxidative phosphorylation 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 electron transport chain is used to produce ATP from ADP +Pi. 1. Proton pump: Electron transport is coupled to the phosphorylation of ADP by the transport of protons (H+) across the inner mitochondrial membrane from the matrix to the intermembrane space. This process creates across the inner mitochondrial membrane 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; 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. 2. ATP synthase: The enzyme complex ATP synthase (complex V, see Figure )6.13(synthesizes ATP, using the energy of the proton gradient generated by the electron transport chain. [Note: It is also called ATPase, because the isolated enzyme also catalyzes the hydrolysis of ATP to ADP and inorganic phosphate.] • The chemiosmotic hypothesis proposes that after protons have been transferred to the cytosolic side of the inner mitochondrial membrane, they reenter the mitochondrial matrix by passing through a channel in the ATP synthase complex, resulting in the synthesis of ATP from ADP + Pi . A.Oligomycin: This drug binds to the stalk of ATP synthase, closing the H+ channel, and preventing reentry of protons into the mitochondrial matrix. Electron transport stops because of the difficulty of pumping any more protons against the steep gradients. Electron transport and phosphorylation are, therefore, again shown to be tightly coupled processes— inhibition of phosphorylation inhibits oxidation. b. Uncoupling proteins (UCP): UCPs occur in the inner mitochondrial membrane of mammals, including humans. These proteins create a "proton leak," that is, they allow protons to reenter the mitochondrial matrix without energy being captured as ATP (Figure 6.14). [Note: Energy is released in the form of heat.]UCP1, also called thermogenin, is responsible for the activation of fatty acid oxidation and heat production in the brown adipocytes of mammals. Brown fat, unlike the more abundant white fat, wastes a most ninety percent of its respiratory energy for thermogensis in response to cold, at birth, and during arousal in hibernating animals. However humans have little brown fat (except in the newborn), and UCP1 does not appear to play a major role in energy balance. Other uncoupling proteins (UCP2, UCP3) have been found in humans, but their significance remains controversial. c. Synthetic uncouplers: Electron transport and phosphorylation can be uncoupled by compounds that increase the permeability of the inner mitochondrial membrane to protons. The classic example is a 2,4-dinitrophenol, lipophilic proton carrier that readily diffuses through the mitochondial membrane. This uncoupler causes electron transport to proceed at a rapid rate without establishing a proton gradient, much as do the UCPs (see Figure 6.14). The energy produced by the transport of electrons is released as heat rather than being used to synthesize ATP. P/O Ratio The P/O ratio is the ratio of the number of moles of ATP generated to the number of atoms of oxygen reduced in the electron transport chain. It is used to express the efficiency of oxidative phosphorylation of the system. There are 3 sites of generation of ATP in the electron transport chain. Thus P/O ratio is 3 if reducing equivalents enter electron transport chain through NAD. On the other hand for the FAD requiring reactions P/O ratio is 2. Transport of reducing equivalents The inner mitochondrial membrane lacks an NADH transport protein, and NADH produced in the cytosol cannot directly penetrate into mitochondria. However, two electrons of NADH (also called reducing equivalents) are transported from the cytosol into the mitochondria using shuttle mechanisms. ❖glycerophosphate shuttle Through the glycerophosphate shuttle electrons are transported to the mitochondrial electron transport chain via FAD. In the first step, 3-phosphoglycerol dehydrogenase with the help of dihydroxyacetone phosphate, catalyzes the oxidation of cytosolic NADH to NAD+ which re-enters glycolysis. • In the next step, electrons from 3-phosphoglycerol (glycerophosphate ) are transferred to a flavoprotein . This oxidation of 3-phosphoglycerol results in the reduction of FAD to FADH2. Since flavoprotein dehydrogenase is situated on the outer surface of the inner mitochondrial membrane, it supplies electrons directly to the electron transport chain and results in the reoxidation of FADH2 to FAD within the mitochondria. The overall process thus results in the transport of the cytosolic reducing equivalents directly into the mitochondrial electron transport chain (Figure 13.12). ❖Malate- Aspartate Shuttle In Malate Aspartate shuttle, cytosolic oxaloacetate is converted either to aspartate through the action of aspartate aminotransferase or to malate by malate dehydrogenase. With the use of reducing equivalents (from the cytosolic NADH), oxaloacetate is reduced (by malate dehydrogenase) to malate and gets transported into the mitochondria. After its reoxidation in the matrix by the same enzyme, malate releases its reducing equivalents and reoxidized to oxaloacetate. • Oxaloacetae since is impermeable through the mitochondrial membrane, it reacts with glutamate. Glutamate oxaloacetate transaminase transminates oxaloacetate to aspartate, and glutamate to α-ketoglutarate. Aspartate and α-ketoglutarate are transported through the mitochondrial membrane to the cytosol by the α-ketoglutarate transporter and the Glutamate- Aspartate transporter, respectively. In the cytosol, Aspartate is reconverted to oxaloacetate. • As both, aspartate as well as malate are freely permeable through the inner mitochondrial membrane, reducing equivalents which are accepted by NAD+ and originated in the cytosol, are thus transported to the mitochondria for their entry into the electron transport chain(Figure 13.13).