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Chapter 20 Electron Transport and Oxidative Phosphorylation

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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 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

4 20.1 - Where in the Cell Do Electron Transport and Oxidative Phosphorylation Occur?
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 mitochondria per cell; no mitochondria in human erythrocytes The mitochondria is about 0.5 ± 0.3 micron in diameter and from 0.5 to several micron long

5 Figure 20. 1 (a) A drawing of a mitochondrion
Figure 20.1 (a) A drawing of a mitochondrion. (b) Tomography of a rat liver mitochondrion. The colors represent individual cristae.

6 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 Pyruvate dehydrogenase complex TCA cycle Glutathione dehydrogenase Fatty acid oxidation Urea cycle DNA replication Transcription Translation

7 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

8 20.2 – How Is the Electron Transport Chain Organized?
Reduction potential: The tendency of an electron donor to reduce its conjugate acceptor The standard reduction potential, ℰo' (25℃, 1 M), is the tendency of a reductant to be reduced or oxidized The standard reduction potentials are determined by measuring the voltages generated in reaction half-cells

9 The hydrogen electrode (pH 0) is set at 0 volts.
The hydrogen electrode (pH 0) is set at 0 volts. Negative value of ℰo' tends to donate electrons to H electrode (oxidation) Positive value of ℰo' tends to accept electrons from H electrode

10 High ℰo' indicates a strong tendency to be reduced
Crucial equation: Go' = - n ℱ 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 negative reduction potential and are accepted by the reaction with the more positive reduction potential: ℰo ' positive, Go' negative ℰ = ℰo' + (RT/ n ℱ ) ln [ox] [red]

11 20.2 – How Is the Electron Transport Chain Organized?
NADH (reductant) + H+ + O2 (oxidant) → NAD+ + H2O Half-reaction: NAD H e- → NADH + H ℰo' = V ½ O H e- → H2O ℰo' = V Dℰo' = – (-0.32) = V DGo'= -219 kJ/mol Electros move from more negative to more positive reduction potential in the electron-transport chain

12 Figure 20.2 Eo΄ and E values for the components of the mitochondrial electron-transport chain. Values indicated are consensus values for animal mitochondria. Black bars represent Eo΄; red bars, E.

13 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 (cytochrome c) 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

14 The Electron Transport Chain
The electron-transport chain can be considered to be composed of four parts: NADH-coenzyme Q reductase succinate-coenzyme Q reductase coenzyme Q-cytochrome c reductase cytochrome c oxidase Electrons generally fall in energy through the chain - from complexes I and II to complex IV

15 Figure 20.3 An overview of the complexes and pathways in the mitochondrial electron-transport chain.


17 Complex I Oxidizes NADH and Reduces Coenzyme Q
NADH-CoQ Reductase or NADH dehydrogenase Electron transfer from NADH to CoQ More than 45 protein subunits - mass of 980 kD Path: NADH  FMN  Fe-S  CoQ Four H+ transported out per 2 e- Iron-sulfur proteins

18 coenzyme Q is a mobile electron carrier
Isoprene unit 10 (Mammal) 6 (Bacteria) Figure (a) The three oxidation states of coenzyme Q. (b) A space-filling model of coenzyme Q. coenzyme Q is a mobile electron carrier


20 Complex II Oxidizes Succinate and Reduces Coenzyme Q
Succinate-CoQ Reductase Also called succinate dehydrogenase or flavoprotein 2 (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 + CoQH Dℰo'= 0.029V

21 Figure 20.6 A probable scheme for electron flow in Complex II.

22 flavoprotein 3 (fig 20.3) Figure The fatty acyl-CoA dehydrogenase reaction, emphasizing that the reaction involves reduction of enzyme-bound FAD (indicated by brackets).

23 CoQ-Cytochrome c Reductase
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 (Fig 20.11) The principal transmembrane protein in complex III is the b cytochrome - with hemes bL(566) and bH(562) Cytochromes, like Fe in Fe-S clusters, are one- electron transfer agents The Q cycle drives 4 H+ transport CoQH2 is a lipid-soluble electron carrier cyt c is a water-soluble mobile electron carrier

24 Figure 20.8 Typical visible absorption spectra of cytochromes.
Figure The structures of iron protoporphyrin IX, heme c, and heme a.


26 Heme c Figure The structure of mitochondrial cytochrome c

27 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+) H+ + O2 → 4 cyt c (Fe3+) H2O Oxygen is 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 H+ across the inner membrane

28 Figure 20. 15 The electron transfer pathway for cytochrome oxidase
Figure The 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. Figure Structures of the redox centers of bovine cytochrome c oxidase. (a) The CuA site (b) the heme a site (c) the binuclear CuB/heme a3 site

29 The Complexes of Electron Transport May Function as Supercomplexes
For many years, the complexes of the electron transport chain were thought to exist and function independently in the mitochondrial inner membrane However, growing experimental evidence supports the existence of multimeric supercomplexes of the four electron transport complexes Also known as respirasomes (Fig 20.18a) , these complexes may represent functional states Association of two or more of the individual complexes may be advantageous for the organism

30 Figure (a) An electron microscopy image of a supercomplex formed from Complex I, Complex III, and Complex IV (left), and a model of the complex (right).

31 Figure (b) A model for the electron-transport pathway in the mitochondrial inner membrane. UQ/UQH2 and cyt c are mobile carriers and transfer electrons between the complexes. The proton transport driven by Complex I, III, and IV

32 Electron Transfer Energy Stored in a Proton Gradient: The Mitchell Hypothesis
Peter Mitchell proposed a novel idea—a proton gradient across the inner membrane could be used to drive ATP synthesis The proton gradient is created by the proteins of the electron-transport pathway (Figure 20.19) This mechanism stores the energy of electron transport in an electrochemical potential As proton are driven out of the matrix, the pH rises and the matrix become negatively charged with respect to the cytosol

33 Figure The proton and electrochemical gradients existing across the inner mitochondrial membrane. Electron transport-driven proton pumping thus creates a pH gradient and an electric gradient across the inner membrane, both of which tend to attract proton back into the matrix from the cytoplasm.

34 20.3 – What Are the Thermodynamic Implications of Chemiosmotic Coupling?
How much energy can be stored in an electrochemical gradient? H+in  H +out DG = RT ln [c2]/[c1] + Z F Dy DG = RT ln [H+out]/[H+in] + Z F Dy DG = RT DpH + Z F Dy DG = 2.3 RT + F (0.18V) DG =5.9 kJ kJ = 23.3 kJ Z : the charge on a proton Dy : the potential difference across the membrane DpH = 1 Z = +1 F = kJ/V Dy = 0.18 V T= 37 ℃

35 20.4 – How Does a Proton Gradient Drive the Synthesis of ATP?
Proton diffusion through the ATP synthase drives ATP synthesis The electrochemical gradient is an energetically favorable process, drives the synthesis of ATP ATP synthase also called F1F0-ATPase Is an enzyme, a proton pump, and a rotating molecular motor Consists of two complexes: F1 and F0 F1 catalyzes ATP synthesis F0 An integral membrane protein attached to F1

36 F0 includes three hydrophobic subunits denoted a, b and c; ab2c10-15
F0 forms the transmembrane pore or channel through which protons move to drive ATP synthesis The a and b subunits comprise part of the stator and a ring of c subunits forms a rotor of the motor Protons flow through the a-c complex and cause the c-ring to rotate in the membrane

37 * Bovine mitochrodria: 10; Yeast mitochondria: 10; spinach chloroplasts: 14

38 F1 consists of five polypeptides: a, b, g, d, and e; a3b3gde
The a- and b-subunits are arranged in an alternating pattern in the hexamer which are similar but not identical The noncatalytic a-sites have similar structure, but the catalytic b-sites have three quite different conformations The g is anchored to the c-subunit rotor, then the c rotor-g complex can rotate together relative to the (ab) complex

39 Each site steps through the three conformations or states to make ATP
The three b subunits of F1 exist in three different conformations represent the three steps of ATP synthesis Each site steps through the three conformations or states to make ATP Three ATP are synthesized per turn Open (O) conformation with no affinity for ligands Loose (L) conformation with low affinity for ligands Tight (T) conformation with high affinity for ligands.

40 Proton Flow Through F0 Drives Rotation of the Motor and Synthesis of ATP

41 Figure 20. 24 (c) A view looking down into the plane of the membrane
Figure (c) A view looking down into the plane of the membrane. Transported protons flow from the inlet half-channel to Asp61 residues on the c-ring, around the ring, and then into the outlet half-channel.

42 Upon illumination, bacteriorhodopsin pumped protons into these vesicles, and the resulting proton gradient was sufficient to drive ATP synthesis by the ATP synthase. Figure The reconstituted vesicles containing ATP synthase and bacteriorhodopsin used by Stoeckenius and Racker to confirm the Mitchell chemiosmotic hypothesis.

43 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 is an ATP synthase inhibitors

44 Figure The sites of action of several inhibitors of electron transport and oxidative phosphorylation.

45 Uncouplers Disrupt the Coupling of Electron Transport and ATP Synthase
Uncouplers disrupt the tight coupling between electron transport and ATP synthase Uncouplers are hydrophobic molecules with a dissociable proton They shuttle back and forth across the inner membrane, carrying protons to dissipate the proton gradient The energy released in electron transport is dissipated as heat Hibernating Animals Generate Heat by Uncoupling Oxidative Phosphorylation

46 Figure Structures of several uncouplers, molecules that dissipate the proton gradient across the inner mitochondrial membrane and thereby destroy the tight coupling between electron transport and the ATP synthase reaction.

47 ATP-ADP Translocase Mediates the Movement of ATP and ADP Across the Mitochondrial Membrane
ATP must be transported out of the mitochondria ATP out, ADP in - through a "translocase" ATP movement out is favored because the cytosol is "+" relative to the "-" matrix ATP out and ADP in is net movement of a negative charge out - equivalent to a H+ going in So every ATP transported out costs one H+ One ATP synthesis costs about 3 H+. Thus, making and exporting 1 ATP = 4H+

48 Figure 20. 29 (a) The bovine ATP-ADP translocase
Figure (a) The bovine ATP-ADP translocase. (b) Outward transport of ATP (via the ATP-ADP translocase is favored by the membrane electrochemical potential.

49 20.5 - What Is the P/O Ratio for Mitochondrial Oxidative Phosphorylation?
P/O ratio is the number of molecules of ATP formed per two electrons through the chain The number of H+ pass through the ATP synthase to synthesize an ATP, depending on the number of the c-subunit in the F0 The number of c-subunit in ATP synthase ranges from 8-15, depending on the organism The ratios of H+/ATP is about 2.7(8/3) to 5(15/3) Adding one H+ for ATP-ADP translocase raises to 3.7 and 6

50 P/O ratio is the number of molecules of ATP formed per two electrons through the chain
The e- transport chain yields 10 H+ pumped out per electron pair from NADH to oxygen 10/3.7 = 2.7 for electrons entering as NADH Earlier estimates the P/O ratio for NADH at 3 For electrons entering as succinate (FADH2), about 6 H+ pumped per electron pair to oxygen 6/3.7 = 1.6 for electrons entering as succinate Earlier estimates the P/O ratio for FADH2 at 2

51 20.6 – How Are the Electrons of Cytosolic NADH Fed into Electron Transport?
Most NADH used in electron transport is produced in mitochondrial matrix Cytosolic NADH produced in glycolysis doesn't cross the inner mitochondrial membrane "Shuttle systems" effect electron movement without actually carrying NADH Glycerophosphate shuttle stores electrons in glycerol-3-P, which transfers electrons to FAD Malate-aspartate shuttle uses malate to carry electrons across the membrane

52 Figure The glycerophosphate shuttle couples cytosolic oxidation of NADH with mitochondrial reduction of [FAD].

53 Figure 20.31 The malate-aspartate shuttle.

54 The Net Yield of ATP from Glucose Oxidation Depends on the Shuttle Used
32 ATP per glucose if glycerol-3-P shuttle used 34.2 ATP per glucose if malate-Asp shuttle used In bacteria, no extra H+ used to export ATP to cytosol. Thus, bacteria have the potential to produce approximately 38 ATP per glucoose


56 20.7 – How Do Mitochondria Mediate Apoptosis?
Mitochondria play a significant role in the programmed death of cells (apoptosis) Apoptosis is a mechanism through which certain cells are eliminated from higher organisms It is central to the development and homeostasis of multicellular organisms Apoptosis is suppressed through compartmentation of the involved activators and enzymes Mitochondria do this in part, by partitioning some of the apoptotic activator molecules, e.g., cytochrome c

57 Cytochrome c resides in the intermembrane space and bound tightly to a lipid chain of cardiolipin (diphosphatidylglycerol; p240) in the membrane Oxidation of bound cardiolipins releases cytochrome c from the inner membrane Apoptotic signals, including Ca2+, ROS, a family of caspases, and certain protein kinase, can induce the opening of pores in the mitochondria Opening of pores in the outer membrane releases cytochrome c from the mitochondria

58 Trigger mitochrondrial outer membrane permeabilization (MOMP)
(chapter 31; p1100) Trigger mitochrondrial outer membrane permeabilization (MOMP) Realeasing cytochrome c Binding of cytochrome c to Apaf-1 (apoptotic protease-activating factor 1) in the cytosol leads to assembly of apoptosomes, thus triggering the events of apoptosis

59 Figure (a) Apaf-1 is a multidomain protein, consisting of an N-terminal CARD, a nucleotide-binding and oligomerization domain (NOD), and several WD40 domains. (b) Binding of cytochrome c to the WD40 domains and ATP hydrolysis unlocks Apaf-1 to form the semi-open conformation. Nucleotide exchange leads to oligomerization and apoptosome formation. (c) A model of the apoptosome, a wheel-like structure with molecules of cytochrome c bound to the WD40 domains, which extend outward like spokes.

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