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Chapter 20

Chapter 20. Electron Transport and Oxidative Phosphorylation Biochemistry by Reginald Garrett and Charles Grisham. Essential Question. How do cells oxidize NADH and [FADH 2 ] How do cells convert their reducing potential into the chemical energy of ATP?. Outline of chapter 20.

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Chapter 20

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  1. Chapter 20 Electron Transport and Oxidative Phosphorylation Biochemistry by Reginald Garrett and Charles Grisham

  2. Essential Question • How do cells oxidize NADH and [FADH2] • How do cells convert their reducing potential into the chemical energy of ATP?

  3. Outline of chapter 20 • Where in the Cell Are Electron Transport and Oxidative Phosphorylation Carried Out? • What Are Reduction Potentials, and How Are They Used to Account for Free Energy Changes in Redox Reactions? • How Is the Electron-Transport Chain Organized? • What Are the Thermodynamic Implications of Chemiosmotic Coupling? • How Does a Proton Gradient Drive the Synthesis of ATP? • What Is the P/O Ratio for Mitochondrial Electron Transport and Oxidative Phosphorylation? • How Are the Electrons of Cytosolic NADH Fed into Electron Transport?

  4. Electron Transport: • Electrons carried by reduced coenzymes, NADH or FADH2, are passed through a chain of proteins and coenzymes, finally reaching O2, the terminal electron acceptor • and to drive the generation of a proton gradient across the inner mitochondrial membrane • Oxidative Phosphorylation: • The proton gradient runs downhill to drive the synthesis of ATP

  5. 20.1 - Where in the Cell Are Electron Transport and Oxidative Phosphorylation Carried Out? • The processes of electron transport and oxidative phosphorylation are membrane associated • in bacteria, is carried out at the plasma membrane • In eukaryotic cells, happens in or at the inner mitochondrial membrane • The mitochondria is about 0.5 ± 0.3 micron in diameter and from 0.5 to several micron long

  6. Figure 20.1(a) A drawing of a mitochondrion with components labeled. (b) Tomography of a rat liver mitochondrion. The tubular structures in red, yellow, green, purple, and aqua represent individual cristae formed from the inner mitochondrial membrane. (b,Frey, T.G., and Mannella, C.A.,2000. The internal structure of mitochondria. Trends in Biochemical Sciences25:319-324.) 

  7. Mitochondrial functions are localized in specific compartments • Outer membrane • Fatty acid elongation • Fatty acid desaturation • Phospholipid synthesis • Monoamine oxidase • Inner membrane • Electron transport • Oxidative phosphorylation • Transport system • Fatty acid transport • Intermembrane space • Creatine kinase • Adenylate kinase • Martix

  8. Mitochondrial functions are localized in specific compartments • Outer membrane • Inner membrane • Intermembrane space • Martix • Pyruvate dehydrogenase complex • TCA cycle • Glutathione dehydrogenase • Fatty acid oxidation • Urea cycle • DNA replication • Transcription • Translation

  9. Intermembrane space -- creatine kinase -- adenylate kinase Outer membrane -- fatty acid elongation -- fatty acid desaturation -- phospholipid synthesis -- monoamine oxidase Inner membrane (cristae) -- electron transport -- oxidative phosphorylation -- transport system -- fatty acid transport Matrix -- pyruvate dehydrogenase complex -- citric acid cycle -- glutathione dehydrogenase -- fatty acid oxidation -- urea cycle -- replication -- transcription -- translation Figure 15.2: Localization of respiratory processes in the mitochondrion

  10. 20.2 – What Are Reduction Potentials, and How Are They Used to Account for Free Energy Changes in Redox Reactions? Reduction potential: • The tendency of an electron donorto reduce its conjugate acceptor • The standard reduction potential, Eo (25℃, 1 M), is the tendency of a reductant to loss an electron • The higher the standard reduction potential Eo, the higher the tendency of the oxidized membrane of a redox couple to attract electrons.

  11. An electrochemical cell consists of two falf-cells, each containing an electron donor and its conjugate acceptor Reduced donor Oxidized acceptor ne- Oxidizeddonor Reduced acceptor The hydrogen electrode (pH 0) is set at 0 volts. Negative value of Eo tends to donate electrons to H electrode Positive value of Eo tends to accept electrons from H electrode Figure 20.2Experimental apparatus used to measure the standard reduction potential of the indicated redox couples: (a) the acetaldehyde/ethanol couple, (b) the fumarate/succinate couple, (c) the Fe3+/Fe2+ couple.

  12. High Eo' indicates a strong tendency to be reduced • Crucial equation: Go' = -nFEo' Eo' = Eo'(acceptor) - Eo'(donor) F: Faraday’s constant (96.5 kJ/mol‧V) • Electrons are donated by the half reaction with the more negativereduction potential and are accepted by the reaction with the more positivereduction potential: Eo ' positive, Go' negative • If a given reaction is written so the reverse is true, then the Eo' will be a negative number and Go' will be positive

  13. Standard reduction potentials of compounds

  14. 20.3 – How Is the Electron Transport Chain Organized? NADH (reductant) + H+ + O2 (oxidant) → NAD+ + H2O Half-reaction: NAD+ + 2 H+ + 2 e- → NADH + H+ Eo'= -0.32 V ½ O2 + 2 H+ + 2 e- → H2O Eo'= +0.816 V DEo'= 0.816 – (-0.32) = 1.136 V DGo'= -219

  15. Figure 20.3Eo΄ and Evalues for the components of the mitochondrial electron-transport chain. Values indicated are consensus values for animal mitochondria. Black bars represent Eo΄; red bars, E.

  16. The Electron Transport Chain The electron-transport chain involves several different molecular species: • Flavoproteins: FAD and FMN • A lipid soluble coenzyme Q (UQ, CoQ) • A water soluble protein (cytochromec) • A number of iron-sulfur proteins: Fe2+ and Fe3+ • Protein-bound copper: Cu+ and Cu2+ All these intermediates except for cytochrome c are membrane associated

  17. Figure 20.4 An overview of the complexes and pathways in the mitochondrial electron-transport chain.

  18. Complex I Oxidizes NADH and Reduces Coenzyme Q NADH-CoQ Reductase or NADH dehydrogenase • Electron transfer from NADH to CoQ • More than 30 protein subunits - mass of 850 kD • Path: NADH  FMN  Fe-S  CoQ • Four H+ transported out per 2 e- Iron-sulfur proteins

  19. Isoprene unit 10 (Mammal) 6 (Bacteria) Figure 20.5 (a) The three oxidation states of coenzyme Q. (b) A space-filling model of coenzyme Q.

  20. Figure 20.6Proposed structure and electron transport pathway for Complex I. Three protein complexes have been isolated, including the flavoprotein (FP), iron-sulfur protein (IP), and hydrophobic protein (HP). FP contains three peptides (of mass 51, 24, and 10 kD) and bound FMN and has 2 Fe-S centers (a 2Fe-2S center and a 4Fe-4S center). IP contains six peptides and at least 3 Fe-S centers. HP contains at least seven peptides and one Fe-S center.

  21. Complex II Oxidizes Succinate and Reduces Coenzyme Q Succinate-CoQ Reductase • Also called succinate dehydrogenase or flavoprotein 2 (FP2) - FAD covalently bound • four subunits, including 2 Fe-S proteins • Three types of Fe-S cluster: 4Fe-4S, 3Fe-4S, 2Fe-2S • Path: Succinate  FADH2  2Fe2+  CoQH2 • Net reaction: succinate + CoQ  fumarate + CoQH2 DEo'= 0.029 V

  22. Figure 20.8A probable scheme for electron flow in Complex II. Oxidation of succinate occurs with reduction of [FAD]. Electrons are then passed to Fe-S centers and then to coenzyme Q (UQ). Proton transport does not occur in this complex.

  23. Figure 20.7The fatty acyl-CoA dehydrogenase reaction, emphasizing that the reaction involves reduction of enzyme-bound FAD (indicated by brackets).

  24. Complex III Mediates Electron Transport from Coenzyme Q to Cytochrome c CoQ-Cytochrome c Reductase • CoQ passes electrons to cyt c (and pumps H+) in a unique redox cycle known as the Q cycle • The principal transmembrane protein in complex III is the b cytochrome - with hemes bL and bH • Cytochromes, like Fe in Fe-S clusters, are one- electron transfer agents • The Q cycle • CoQH2 is a lipid-soluble electron carrier • cyt c is a water-soluble mobile electron carrier

  25. Figure 20.12The Q cycle in mitochondria. (a) The electron transfer pathway following oxidation of the first UQH2 at the Qpsite near the cytosolic face of the membrane. (b) The pathway following oxidation of a second UQH2.

  26. Figure 20.11The structure of UQ-cyt c reductase, also known as the cytochrome bc1 complex. The α-helices of cytochrome b (pale green) define the transmembrane domain of the protein. The bottom of the structure as shown extends approximately 75 Å into the mitochondrial matrix, and the top of the structure as shown extends about 38 Å into the intermembrane space. (Photograph kindly provided by Di Xia and Johann Deisenhofer [From Xia, D., Yu,C.-A., Kim, H., Xia, J.-Z., Kachurin, A. M., Zhang, L., Yu, L., and Deisenhofer, J., 1997. The crystal structure of the cytochrome bc1 complex from bovine heart mitochondria. Science 277:60-66.])

  27. Figure 20.9 Typical visible absorption spectra of cytochromes. Figure 20.10The structures of iron protoporphyrin IX, heme c, and heme a.

  28. M Figure 20.13The structure of mitochondrial cytochrome c. The heme is shown at the center of the structure, covalently linked to the protein via its two sulfur atoms (yellow). A third sulfur from a methionine residue coordinates the iron.

  29. Complex IV Transfers Electrons from Cytochrome c to Reduce Oxygen on the Matrix Side Cytochrome c Oxidase • Electrons from cyt c are used in a four-electron reduction of O2 to produce 2 H2O 4 cyt c (Fe2+) + 4 H+ + O2→ 4 cyt c (Fe3+) + 2 H2O • Oxygen is thus the terminal acceptor of electrons in the electron transport pathway • Cytochrome c oxidase utilizes 2 hemes (a and a3) and 2 copper sites (CuA and CuB) • Complex IV also transports 2 H+

  30. M Figure 20.15Molecular graphic image of subunits I,II, and III of cytochrome c oxidase. Figure 20.13The subunit structure of mitochondrial cytochrome c oxidase

  31. Figure 20.16Molecular graphic image of cytochrome c oxidase. Seven of the 10 nuclear DNA-derived subunits (IV, VIa, VIc, VIIa, VIIb, VIIc, and VIII) possess transmembrane segments. Three (Va, Vb, and VIb) do not. Subunits IV and VIc are transmembrane and dumbbell-shaped. Subunit Va is globular and bound to the matrix side of the complex, whereas VIb is a globular subunit on the cytosolic side of the membrane complex. Vb is globular and matrix-side associated as well, but it has an N-terminal extended domain. VIa has a transmembrane helix and a small globular domain. Subunit VIIa consists of a tilted transmembrane helix, with another short helical segment on the matrix side of the membrane. Subunits VIIa, VIIb, and VIII consist of transmembrane segments with short extended regions outside the membrane. 

  32. Figure 20.17The electron transfer pathway for cytochrome oxidase. Cytochrome c binds on the cytosolic side, transferring electrons through the copper and heme centers to reduce O2 on the matrix side of the membrane.

  33. Figure 20.18(a) The CuA site of cytochrome oxidase. Copper ligands include two histidine imidazole groups and two cysteine side chains from the protein. (b) The coordination of histidine imidazole ligands to the iron atom in the heme a center of cytochrome oxidase. Figure 20.19The binuclear center of cytochrome oxidase. A ligand, L (probably a cysteine S), is shown bridging the CuB and Fe of heme a3 metal sites.

  34. Figure 20.20 A model for the mechanism of O2 reduction by cytochrome oxidase.

  35. The complexes are independent Each is a multiprotein aggregate maintained by numerous strong association between peptides of the complex The four complexes are independently mobile in the membrane

  36. Figure 20.21A model for the electron transport pathway in the mitochondrial inner membrane. UQ/UQH2 and cytochrome c are mobile electron carriers and function by transferring electrons between the complexes. The proton transport driven by Complexes I, III, and IV is indicated.

  37. 20.4 – What Are the Thermodynamic Implications of Chemiosmotic Coupling? • Peter Mitchell proposed a novel idea - a proton gradient across the inner membrane could be used to drive ATP synthesis (Nobel prize in 1978) H+in H +out DG = RT ln [C2]/[C1] + ZFDy DG = RT ln [H +out]/[H +in] + ZFDy DG = RT ln DpH + ZFDy Z : the charge on a proton Dy : the potential difference across the membrane

  38. 20.5 – How Does a Proton Gradient Drive the Synthesis of ATP? Proton diffusion through the ATP synthase drives ATP synthesis • Also called F1F0-ATPase • Consists of two complexes: F1 and F0 • F1 catlyzes ATP synthesis • F0 An integral membrane protein attached to F1 • See Figure 20.25 and Table 20.3 for details

  39. Figure 20.24Molecular graphic images (a) side view and (b) top view of the F1-ATP synthase showing the individual component peptides. The g-subunit is the pink structure visible in the center of view (b).

  40. 20.5 – How Does a Proton Gradient Drive the Synthesis of ATP? • The catalytic sites are in the b-subunits • A ring of c-subunits could form a rotor that turns with respect to the a-subunit, a stator • g is anchored to the c-subunit rotor, then the c rotor-g complex can rotate together relative to the (ab) complex

  41. Figure 20.26ATP production in the presence of a proton gradient and ATP/ADP exchange in the absence of a proton gradient. Exchange leads to incorporation of 18O in phosphate as shown.

  42. Figure 20.27The binding change mechanism for ATP synthesis by ATP synthase. This model assumes that F1 has three interacting and conformationally distinct active sites. The open (O) conformation is inactive and has a low affinity for ligands; the L conformation (with “loose” affinity for ligands) is also inactive; the tight (T) conformation is active and has a high affinity for ligands. Synthesis of ATP is initiated (step 1) by binding of ADP and Pi to an L site. In the second step, an energy-driven conformational change converts the L site to a T conformation and also converts T to O and O to L. In the third step, ATP is synthesized at the T site and released from the O site. Two additional passes through this cycle produce two more ATPs and return the enzyme to its original state.

  43. Figure 20.28The reconstituted vesicles containing ATP synthase and bacteriorhodopsin used by Stoeckenius and Racker to confirm the Mitchell chemiosmotic hypothesis.

  44. Inhibitors of Oxidative Phosphorylation Reveal Insights About the Mechanism • Rotenone inhibits Complex I - and helps natives of the Amazon rain forest catch fish • Cyanide (CN-), azide (N3-) and CO inhibit Complex IV, binding tightly to the ferric form (Fe3+) of a3 • Oligomycin and DCCD are ATP synthase inhibitors

  45. Figure 20.29 The structures of several inhibitors of electron transport and oxidative phosphorylation.

  46. Figure 20.30 The sites of action of several inhibitors of electron transport and/or oxidative phosphorylation.

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