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
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 Sciences 25: ) 

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. Figure 15.2: Localization of
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 donor to 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. Reduced donor Oxidized acceptor ne-
An electrochemical cell consists of two falf-cells, each containing an electron donor and its conjugate acceptor
Reduced donor Oxidized acceptor
ne-
Oxidized donor 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 20.2 Experimental 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 negative reduction potential and are accepted by the reaction with the more positive reduction 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

14. Standard reduction potentials of compounds

15. 20.3 – 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

16. Figure 20.3 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.

17. 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

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

20. 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

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

22. Figure 20.6 Proposed 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.

23. 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'= V

24. Figure 20. 8 A probable scheme for electron flow in Complex II
Figure 20.8 A 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.

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

26. 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
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

27. Figure 20. 12 The Q cycle in mitochondria
Figure The Q cycle in mitochondria. (a) The electron transfer pathway following oxidation of the first UQH2 at the Qp site near the cytosolic face of the membrane. (b) The pathway following oxidation of a second UQH2.

28. Figure The 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., The crystal structure of the cytochrome bc1 complex from bovine heart mitochondria. Science 277:60-66.])

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

30. M
Figure The 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.

31. 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 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+

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

33. Figure 20. 16 Molecular graphic image of cytochrome c oxidase
Figure Molecular 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. 

34. Figure 20. 17 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.

35. Figure 20. 18 (a) The CuA site of cytochrome oxidase
Figure (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 The binuclear center of cytochrome oxidase. A ligand, L (probably a cysteine S), is shown bridging the CuB and Fe of heme a3 metal sites.

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

37. 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

38. Figure A 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.

39. DG = RT ln [H +out]/[H +in] + ZFDy
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

41. 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 and Table 20.3 for details

42. Figure Molecular 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).

44. 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

45. Figure ATP 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.

46. Figure The 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.

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

48. 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

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

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

51. Uncouplers Disrupt the Coupling of Electron Transport and ATP Synthase
Uncoupling e- transport and oxidative phosphorylation
Uncouplers disrupt the tight coupling between electron transport and oxidative phosphorylation by dissipating the proton gradient
Uncouplers are hydrophobic molecules with a dissociable proton
They shuttle back and forth across the membrane, carrying protons to dissipate the gradient

52. 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.

53. ATP must be transported out of the mitochondria
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
But 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+

54. Figure Outward transport of ATP (via the ATP/ADP translocase) is favored by the membrane electrochemical potential.

55. How many ATP made per electron pair through the chain?
What Is the P/O Ratio for Mitochondrial Electron Transport and Oxidative Phosphorylation?
How many ATP made per electron pair through the chain?
e- transport chain yields 10 H+ pumped out per electron pair from NADH to oxygen
4 H+ flow back into matrix per ATP to cytosol
10/4 = 2.5 for electrons entering as NADH
For electrons entering as succinate (FADH2), about 6 H+ pumped per electron pair to oxygen
6/4 = 1.5 for electrons entering as succinate

56. 20.7 – How Are the Electrons of Cytosolic NADH Fed into Electron Transport?
Most NADH used in electron transport is cytosolic and NADH 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

57. Figure The glycerophosphate shuttle (also known as the glycerol phosphate shuttle) couples the cytosolic oxidation of NADH with mitochondrial reduction of [FAD].

58. Figure The malate (oxaloacetate)-aspartate shuttle, which operates across the inner mitochondrial membrane.

59. The Net Yield of ATP from Glucose Oxidation Depends on the Shuttle Used
30 ATP per glucose if glycerol-3-P shuttle used
32 ATP per glucose if malate-Asp shuttle used
In bacteria - no mitochondria - no extra H+ used to export ATP to cytosol, so:
10/3 = ~3ATP/NADH
6/3 = ~ 2ATP/FADH2