Bioenergetics and Oxidative Phosphorylation - Biochemistry Final Exam PDF

168397_P069-082.qxd7.0:06 Ox-phos 5-26-04 2010.4.4 1:46 PM Page 69 UNIT II: Intermediary Metabolism Bioenergetics and Ox...

168397_P069-082.qxd7.0:06 Ox-phos 5-26-04 2010.4.4 1:46 PM Page 69 UNIT II: Intermediary Metabolism Bioenergetics and Oxidative Phosphorylation 6 I. OVERVIEW ∆G: CHANGE IN FREE ENERGY Bioenergetics describes the transfer and utilization of energy in biologic systems. It makes use of a few basic ideas from the field of thermo- Energy available to do work. dynamics, particularly the concept of free energy. Changes in free Approaches zero as reaction proceeds to equilibrium. energy (∆G) provide a measure of the energetic feasibility of a chemical Predicts whether a reaction is reaction and can, therefore, allow prediction of whether a reaction or favorable. process can take place. Bioenergetics concerns only the initial and final energy states of reaction components, not the mechanism or how much time is needed for the chemical change to take place. In short, bio- energetics predicts if a process is possible, whereas kinetics measures how fast the reaction occurs (see p. 54). II. FREE ENERGY The direction and extent to which a chemical reaction proceeds is ∆S: CHANGE IN ENTROPY determined by the degree to which two factors change during the Measure of randomness. reaction. These are enthalpy (∆H, a measure of the change in heat con- Does not predict whether a tent of the reactants and products) and entropy (∆S, a measure of the reaction is favorable. change in randomness or disorder of reactants and products, Figure 6.1). Neither of these thermodynamic quantities by itself is sufficient to Figure 6.1 determine whether a chemical reaction will proceed spontaneously in Relationship between changes in the direction it is written. However, when combined mathematically (see free energy (G), enthalpy (H), and Figure 6.1), enthalpy and entropy can be used to define a third quantity, entropy (S). T is the absolute free energy (G), which predicts the direction in which a reaction will temperature in degrees Kelvin spontaneously proceed. (oK): oK = oC + 273. 69 168397_P069-082.qxd7.0:06 Ox-phos 5-26-04 2010.4.4 1:46 PM Page 73 V. Electron Transport Chain 73 energy coupling. A gear with an attached weight spontaneously turns in the direction that achieves the lowest energy state, in this case the High-energy Adenine weight seeks its lowest position (see Figure 6.4A). The reverse motion phosphate bonds (see Figure 6.4B) is energetically unfavored and does not occur sponta- NH 2 neously. Figure 6.4C shows that the energetically favored movement of N one gear can be used to turn a second gear in a direction that it would N not move spontaneously. The simplest example of energy coupling in N N biologic reactions occurs when the energy-requiring and the energy- O O O yielding reactions share a common intermediate. OP O P O PO O O O O HO HO A. Reactions are coupled through common intermediates Two chemical reactions have a common intermediate when they Ribose occur sequentially so that the product of the first reaction is a sub- strate for the second. For example, given the reactions Figure 6.5 A+B →C+D Adenosine triphosphate. D+X →Y+Z D is the common intermediate and can serve as a carrier of chemical energy between the two reactions. Many coupled reactions use ATP to generate a common intermediate. These reactions may involve the transfer of a phosphate group from ATP to another molecule. Other reactions involve the transfer of phosphate from an energy-rich inter- Metabolism mediate to adenosine diphosphate (ADP), forming ATP. B. Energy carried by ATP Carbohydrates Fatty acids ATP consists of a molecule of adenosine (adenine + ribose) to which Amino acids three phosphate groups are attached (Figure 6.5). If one phosphate is removed, ADP is produced; if two phosphates are removed, NAD+ FAD adenosine monophosphate (AMP) results. The standard free energy of hydrolysis of ATP, ∆Go, is approximately –7.3 kcal/mol for each of NADH + H+ the two terminal phosphate groups. Because of this large negative FADH2 ∆Go, ATP is called a high-energy phosphate compound. CO2 + H2O V. ELECTRON TRANSPORT CHAIN Energy-rich molecules, such as glucose, are metabolized by a series of O2 oxidation reactions ultimately yielding CO2 and water (Figure 6.6). The metabolic intermediates of these reactions donate electrons to specific ADP + Pi NADH + H+ coenzymes—nicotinamide adenine dinucleotide (NAD +) and flavin FADH2 adenine dinucleotide (FAD)—to form the energy-rich reduced coen- NAD+ zymes, NADH and FADH2. These reduced coenzymes can, in turn, ATP FAD each donate a pair of electrons to a specialized set of electron carriers, collectively called the electron transport chain, described in this section. H 2O As electrons are passed down the electron transport chain, they lose much of their free energy. Part of this energy can be captured and Oxidative phosphorylation stored by the production of ATP from ADP and inorganic phosphate (Pi). This process is called oxidative phosphorylation and is described Figure 6.6 on p. 77. The remainder of the free energy not trapped as ATP is used The metabolic breakdown of energy- to drive ancillary reactions such as Ca2+ transport into mitochondria, yielding molecules. and to generate heat. 168397_P069-082.qxd7.0:06 Ox-phos 5-26-04 2010.4.4 1:46 PM Page 74 74 6. Bioenergetics and Oxidative Phosphorylation A. Mitochondrion INNER MEMBRANE Impermeable to The electron transport chain is present in the inner mitochondrial most small ions, membrane and is the final common pathway by which electrons small and large CELL molecules 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. Membranes of the mitochondrion: The components of the elec- tron transport chain are located in the inner membrane. Although the outer membrane contains special pores, making it freely per- meable to most ions and small molecules, the inner mitochondrial membrane is a specialized structure that is impermeable to most Outer small ions, including H+, Na+, and K+, and small molecules such membrane Intermembrane as ATP, ADP, pyruvate, and other metabolites important to mito- space chondrial function (Figure 6.7). Specialized carriers or transport systems are required to move ions or molecules across this mem- brane. The inner mitochondrial membrane is unusually rich in pro- tein, 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 Cristae greatly increase the surface area of the membrane. ADP ATP 2. Matrix of the mitochondrion: This gel-like solution in the interior of NAD+ mitochondria is 50% protein. These molecules include the enzymes FMN CoQ b c a a3 responsible for the oxidation of pyruvate, amino acids, fatty acids (by β-oxidation), and those of the tricarboxylic acid (TCA) cycle. The An electron synthesis of glucose, urea, and heme occur partially in the matrix of transport assembly 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: ATP synthesizing structures (ATP The matrix also contains mitochondrial RNA and DNA (mtRNA and synthase) mtDNA) and mitochondrial ribosomes.] B. Organization of the electron transport chain MATRIX The inner mitochondrial membrane can be disrupted into five sepa- rate protein complexes, called Complexes I, II, III, IV, and V. TCA cycle enzymes Complexes I–IV each contain part of the electron transport chain Fatty acid oxidation enzymes (Figure 6.8). Each complex accepts or donates electrons to relatively mtDNA, mtRNA mobile electron carriers, such as coenzyme Q and cytochrome c. Mitochondrial ribosomes Each carrier in the electron transport chain can receive electrons from an electron donor, and can subsequently donate electrons to Figure 6.7 the next carrier in the chain. The electrons ultimately combine with Structure of a mitochondrion oxygen and protons to form water. This requirement for oxygen showing schematic representation makes the electron transport process the respiratory chain, which of the electron transport chain and accounts for the greatest portion of the body’s use of oxygen. ATP synthesizing structures on the Complex V is a protein complex that contains a domain (Fo) that inner membrane. spans the inner mitochondrial membrane, and a domain (F1) that mtDNA = mitochondrial DNA; mtRNA = mitochondrial RNA. appears as a sphere that protrudes into the mitochondrial matrix [Note: In contrast to the inner (see p. 78). Complex V catalyzes ATP synthesis and so is referred to membrane, the outer membrane as ATP synthase. is highly permeable and the milieu of the intermembrane space is like C. Reactions of the electron transport chain that of the cytosol.] With the exception of coenzyme Q, all members of this chain are proteins. These may function as enzymes as is the case with the 168397_P069-082.qxd7.0:06 Ox-phos 5-26-04 2010.4.4 1:46 PM Page 75 V. Electron Transport Chain 75 Intermembrane MITOCHONDRION Matrix space Substrate NAD+ (reduced) Inner membrane FMNH2 1 Product 2 O2 (oxidized) NADH FMN CoQ Fe2+ Fe3+ Fe2+ + H+ Complex I NADH dehydrogenase Cyto bc1 Cyto c Cyto a + a3 Fumarate FADH2 CoQH2 Fe3+ Fe2+ Fe3+ H2O Complex III Complex IV Cytochrome bc1 Cytochrome c oxidase Succinate FAD Complex II Succinate dehydrogenase Figure 6.8 Electron transport chain. [Note: NADH, produced from a variety of oxidative (catabolic) processes, is the substrate for Complex I. Succinate, an intermediate of the TCA cycle, is the substrate for Complex II.] dehydrogenases, may contain iron as part of an iron–sulfur center, may be coordinated with a porphyrin ring as in the cytochromes, or may contain copper as does the cytochrome a + a3 complex. 1. Formation of NADH: NAD+ is reduced to NADH by dehydroge- nases that remove two hydrogen atoms from their substrate. (For examples of these reactions, see the discussion of the dehydro- genases found in the TCA cycle, p. 112.) 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 car- ried by NADH are next transferred to NADH dehydrogenase, a NADH dehydrogenase protein protein complex (Complex I) embedded in the inner mitochondrial membrane. Complex I has a tightly bound molecule of flavin S Cys mononucleotide (FMN, a coenzyme structurally related to FAD, S Fe see Figure 28.15, p. 380) that accepts the two hydrogen atoms Cys S (2e– + 2H+), becoming FMNH2. NADH dehydrogenase also con- Fe S tains iron atoms paired with sulfur atoms to make iron–sulfur cen- ters (Figure 6.9). These are necessary for the transfer of the hydrogen atoms to the next member of the chain, coenzyme Q Fe S (ubiquinone). S Fe S 3. Coenzyme Q: Coenzyme Q (CoQ) is a quinone derivative with a S Cys Cys long, hydrophobic isoprenoid tail. It is also called ubiquinone because it is ubiquitous in biologic systems. CoQ is a mobile car- Fe4S4 rier and can accept hydrogen atoms both from FMNH2, produced on NADH dehydrogenase (Complex I), and from FADH2, pro- Figure 6.9 duced on succinate dehydrogenase (Complex II), glycerophos- Iron-sulfur center of Complex I. phate dehydrogenase (see p. 79), and acyl CoA dehydrogenase [Note: Complexes II and III also (see p. 192). CoQ transfers electrons to Complex III. CoQ, then, contain iron-sulfur centers. links the flavoproteins to the cytochromes. 168397_P069-082.qxd7.0:06 Ox-phos 5-26-04 2010.4.4 1:46 PM Page 76 76 6. Bioenergetics and Oxidative Phosphorylation 4. Cytochromes: The remaining members of the electron transport Blocking electron transfer by chain are cytochromes. Each contains a heme group (a porphyrin any one of these inhibitors ring plus iron). Unlike the heme groups of hemoglobin, the stops electron flow from substrate cytochrome iron is reversibly converted from its ferric (Fe3+) to its to oxygen because the reactions of the electron transport chain are ferrous (Fe2+) form as a normal part of its function as a reversible tightly coupled like meshed gears. carrier of electrons. Electrons are passed along the chain from CoQ to cytochromes bc1 (Complex III), c, and a + a3 (Complex IV, see Figure 6.8). [Note: Cytochrome c is associated with the outer Substrate face of the inner membrane and, like CoQ, is a mobile carrier of (reduced) electrons.] e– 5. Cytochrome a + a3: This cytochrome complex is the only electron carrier in which the heme iron has an available coordination site NAD+ that can react directly with O2, and so also is called cytochrome oxidase. At this site, the transported electrons, O2, and free pro- tons are brought together, and O2 is reduced to water (see Figure e– 6.8). Cytochrome oxidase contains copper atoms that are FMN required for this complex reaction to occur. 6. Site-specific inhibitors: Site-specific inhibitors of electron trans- Amytal e – port have been identified and are illustrated in Figure 6.10. These Rotenone compounds prevent the passage of electrons by binding to a com- CoQ ponent of the chain, blocking the oxidation/reduction reaction. Therefore, all electron carriers before the block are fully reduced, e– whereas those located after the block are oxidized. [Note: Inhibition of electron transport inhibits ATP synthesis because Cyto bc1 these processes are tightly coupled.] Incomplete reduction of oxygen to water produces Antimycin A e– reactive oxygen species (ROS), such as superoxide Cyto c (O2– ), hydrogen peroxide (H2O2) and hydroxyl radi- cals (OH ). ROS damage DNA and proteins, and cause lipid peroxidation. Enzymes such as superox- e– ide dismutase (SOD), catalase, and glutathione per- oxidase are cellular defenses against ROS. Cyto a + a3 D. Release of free energy during electron transport CN– CO Free energy is released as electrons are transferred along the elec- Sodium azide e– tron transport chain from an electron donor (reducing agent or reductant) to an electron acceptor (oxidizing agent or oxidant). The O2 electrons can be transferred as hydride ions (:H –) to NAD +, as hydrogen atoms ( H) to FMN, coenzyme Q, and FAD, or as elec- trons (e–) to cytochromes. Figure 6.10 1. Redox pairs: Oxidation (loss of electrons) of one compound is Site-specific inhibitors of electron always accompanied by reduction (gain of electrons) of a second transport shown using a mechanical model for the coupling of oxidation- substance. For example, Figure 6.11 shows the oxidation of reduction reactions. [Note: Figure NADH to NAD+ accompanied by the reduction of FMN to FMNH2. illustrates normal direction of Such oxidation-reduction reactions can be written as the sum of electron flow.] two separate half-reactions, one an oxidation reaction and the other a reduction reaction (see Figure 6.11). NAD+ and NADH form a redox pair, as do FMN and FMNH2. Redox pairs differ in their tendency to lose electrons. This tendency is a characteristic of a particular redox pair, and can be quantitatively specified by a constant, Eo (the standard reduction potential), with units in volts. 168397_P069-082.qxd7.0:06 Ox-phos 5-26-04 2010.4.4 1:46 PM Page 77 VI. Oxidative Phosphorylation 77 2. Standard reduction potential (Eo): The Eo of various redox pairs Overall oxidation-reduction reaction can be ordered from the most negative Eo to the most positive. The more negative the Eo of a redox pair, the greater the tendency of NADH FMN + H+ the reductant member of that pair to lose electrons. The more posi- tive the Eo, the greater the tendency of the oxidant member of that pair to accept electrons. Therefore, electrons flow from the pair NAD+ FMNH2 with the more negative Eo to that with the more positive Eo. The Eo Component redox reactions values for some members of the electron transport chain are shown in Figure 6.12. [Note: The components of the electron trans- NADH + H+ FMN + 2e– + 2H+ port chain are arranged in order of increasingly positive E0 values.] 3. ∆Go is related to ∆Eο : The change in free energy is related directly to the magnitude of the change in Eo: NAD++ 2e– + 2H+ FMNH2 ∆Go = – n F ∆Eo Redox pair Redox pair Eo = – 0.32 volt Eo = – 0.22 volt n = number of electrons transferred (1 for a cytochrome, 2 for NADH, FADH2, and coenzyme Q) Figure 6.11 F = Faraday constant (23.1 kcal/volt. mol) Oxidation of NADH by FMN, separated into two component ∆Eo = Eo of the electron-accepting pair minus the Eo of the redox pairs. electron-donating pair ∆Go = change in the standard free energy 4. ∆Go of ATP: The standard free energy for the phosphorylation of ADP to ATP is +7.3 kcal/mol. The transport of a pair of electrons Compounds with a large negative from NADH to oxygen via the electron transport chain produces Eo (located at top of the table) 52.58 kcal. Therefore, more than sufficient energy is available to are strong reducing agents produce three ATP from three ADP and three Pi (3 x 7.3 = 21.9 (reductants)— that is, they have a strong tendency to lose electrons. kcal/mol), sometimes expressed as a P:O ratio (ATP made per O atom reduced) of 3:1. The remaining calories are used for ancil- lary reactions or released as heat. [Note: P:O for FADH2 is 2:1 Redox pair Eo because Complex I is bypassed.] + NAD /NADH -0.32 VI. OXIDATIVE PHOSPHORYLATION FMN/FMNH / 2 + + -0.22 Cytochrome c Fe3 /Fe2 +0.22 1/2 O2/H2O +0.82 The transfer of electrons down the electron transport chain is energeti- cally favored because NADH is a strong electron donor and molecular oxygen is an avid electron acceptor. However, the flow of electrons from NADH to oxygen does not directly result in ATP synthesis. Compounds at the bottom of the table are strong oxidizing agents (oxidants)—that is, they A. Chemiosmotic hypothesis accept electrons. The chemiosmotic hypothesis (also known as the Mitchell hypothesis) explains how the free energy generated by the transport of electrons Figure 6.12 by the electron transport chain is used to produce ATP from ADP + Pi. Standard reduction potentials of 1. Proton pump: Electron transport is coupled to the phosphorylation some reactions. of ADP by the transport (“pumping”) of protons (H+) across the inner mitochondrial membrane from the matrix to the intermem- brane 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, Figure 6.13). The energy generated by this proton gradient is sufficient to drive ATP 168397_P069-082.qxd7.0:06 Ox-phos 5-26-04 2010.4.4 1:46 PM Page 78 78 6. Bioenergetics and Oxidative Phosphorylation MITOCHONDRION Inner membrane Outer membrane ADP + Pi ATP Complex V (F1 domain) Intermembrane Matrix space NAD+ 1/ 2O2 H2O ATP/ADP NADH transporter MITOCHONDRIAL MATRIX e– e– e– Electron flow cytc e– Complex V (F0 domain) Complex Complex Complex I III IV INTERMEMBRANE SPACE + + + + H H H H ADP ATP Figure 6.13 Electron transport chain shown coupled to the transport of protons. [Note: Protons are not pumped at Complex II.] synthesis. Thus, the proton gradient serves as the common inter- mediate 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 Uncoupling proteins create a called F1/Fo ATPase because the isolated enzyme can catalyze “proton leak,” allowing protons the hydrolysis of ATP to ADP and Pi.] The chemiosmotic hypothe- to reenter the mitochondrial matrix without capturing any sis proposes that after protons have been pumped to the cytosolic energy as ATP. side of the inner mitochondrial membrane, they reenter the matrix Uncoupling by passing through a channel in the membrane-spanning domain + protein (Fo ) of Complex V, driving rotation of Fo and, at the same time, dis- + H H sipating the pH and electrical gradients. Fo rotation causes confor- + ATP + + H synthase mational changes in the extra-membranous F1 domain that allow it H H to bind ADP + Pi, phosphorylate ADP to ATP, and release ATP. a. Oligomycin: This drug binds to the Fo (hence the letter o) + O2 H domain of ATP synthase, closing the H+ channel, preventing reentry of protons into the mitochondrial matrix, and thus pre- H2O + ADP venting phosphorylation of ADP to ATP. Because the pH and H electrical gradients cannot be dissipated in the presence of this e– ATP drug, electron transport stops because of the difficulty of pump- ing any more protons against the steep gradients. This depen- MITOCHONDRIAL MATRIX dency 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. Electron transport and Figure 6.14 phosphorylation are, therefore, again shown to be tightly cou- Transport of H+ across the mitochondrial pled processes. Inhibition of one process inhibits the other. membrane by 2,4-dinitrophenol. [Note: Respiratory control also results from decreased availabil- ity of ADP or Pi.] 168397_P069-082.qxd7.0:06 Ox-phos 5-26-04 2010.4.4 1:46 PM Page 79 VI. Oxidative Phosphorylation 79 b. Uncoupling proteins (UCP): UCPs occur in the inner mito- chondrial membrane of mammals, including humans. These car- rier proteins create a “proton leak,” that is, they allow protons to A re-enter the mitochondrial matrix without energy being captured as ATP (Figure 6.14). The energy is released as heat, and the NADH + H+ NAD+ CH2OH CH2OH process is called nonshivering thermogenesis. UCP1, also C O HO C H called thermogenin, is responsible for the heat production in the Cytosolic CH2OPO3 glycerophosphate CH2OPO3 brown adipocytes of mammals. UCP1 is activated by fatty acids. dehydrogenase DHAP Glycerol Brown fat, unlike the more abundant white fat, uses almost 90% 3-phosphate of its respiratory energy for thermogenesis in response to cold in CYTOSOL the neonate, and during arousal in hibernating animals. However, humans appear to have little brown fat (except in the newborn), and UCP1 does not appear to play a major role in CoQ of the electron transport chain energy balance. [Note: Other uncoupling proteins (UCP2, UCP3) have been found in humans, but their significance FADH2 FAD CH2OH CH2OH remains unclear.] C O HO C H Mitochondrial c. Synthetic uncouplers: Electron transport and phosphoryla- CH2OPO3 glycerophosphate CH2OPO3 dehydrogenase tion can also be uncoupled by compounds that increase the DHAP Glycerol 3-phosphate permeability of the inner mitochondrial membrane to protons. The classic example is 2,4-dinitrophenol, a lipophilic proton carrier that readily diffuses through the mitochondrial mem- INNER MITOCHONDRIAL MEMBRANE brane. 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). Again, energy is released as B heat rather than being used to synthesize ATP. In high doses, Oxaloacetate Glutamate aspirin and other salicylates uncouple oxidative phos - NADH phorylation. This explains the fever that accompanies toxic + H+ Cytosolic Amino- malate overdoses of these drugs. dehydrogenase transferase NAD+ B. Membrane transport systems Malate The inner mitochondrial membrane is impermeable to most charged Aspartate α-Ketoglutarate or hydrophilic substances. However, it contains numerous transport CYTOSOL proteins that permit passage of specific molecules from the cytosol (or Aspartate α-Ketoglutarate more correctly, the intermembrane space) to the mitochondrial matrix. 1. ATP-ADP transport: The inner mitochondrial membrane requires Malate specialized carriers to transport ADP and Pi from the cytosol NAD+ Mitochondrial (where ATP is used and converted to ADP in many energy- malate Amino- dehydrogenase requiring reactions) into mitochondria, where ATP can be resyn- transferase NADH thesized. An adenine nucleotide carrier imports one molecule of + H+ ADP from the cytosol into mitochondria, while exporting one ATP Oxaloacetate Glutamate from the matrix back into the cytosol (see Figure 6.13). [Note: A Complex I of the phosphate carrier is responsible for transporting Pi from the cytosol electron transport chain into mitochondria.] MITOCHONDRIAL MATRIX 2. Transport of reducing equivalents: The inner mitochondrial mem- brane lacks an NADH transporter, and NADH produced in the Figure 6.15 cytosol cannot directly enter the mitochondrial matrix. However, Substrate shuttles for the transport of electrons across the inner mito- two electrons of NADH (also called reducing equivalents) are chondrial membrane. A. Glycerol transported from the cytosol into the matrix using substrate shut- 3-phosphate shuttle. B. Malate- tles. In the glycerophosphate shuttle (Figure 6.15A), two electrons aspartate shuttle. DHAP = are transferred from NADH to dihydroxyacetone phosphate by dihydroxyacetone phosphate. cytosolic glycerophosphate dehydrogenase. The glycerol 3-phos- phate produced is oxidized by the mitochondrial isozyme as FAD is