1. Oxidative Phosphorylation
Pratt and Cornely, Chapter 15

2. Goal: ATP Synthesis

3. Overview Redox reactions Electron transport chain Proton gradient
ATP synthesis
Shuttles

4. Standard Reduction Potential
“potential to be reduced”
Listed according to oxidized compound being reduced
High RP means that the compound is a strong oxidizer (ie O2)
Its conjugate is a strong reducing agent (ie NADH)
½ O e + 2 H+ H2O

5. Half Reactions
Reduction potential written in terms of a reduction half reaction
Aox  Ared
Example: Calculate DEo for reduction of FMN by NADH, given the Eo of FMN to be -0.30V
Then calculate DGo’
NADH + FMN  NAD+ + FMNH2

6. Redox reactions: electricity
NAD+: Eo’ = -.32
FMN: Eo’=-.30
DEo’ = +0.02V
Calculate DGo’:2 e- transfer
DGo’ = -nFDEo’
= -2(96485 J/mol V)(0.02V)
= -3.9 kJ/mol

7. Numerous Redox Substrates
Chain of increasing reduction potential (potential to accept e-)
Mobile carriers vs. within Complex
Types of redox groups
Organic cofactors
Metals (iron/sulfur clusters)
Cytochromes O2

8. Overall chain from NADH
Complex I Q
Complex III
Cytochrome c
Complex IV O2

9. Within Complexes FAD/FMN Iron-sulfur clusters Prosthetic group
Bridge from 2 e- donors to 1 e- donors
Iron-sulfur clusters

10. Coenzyme Q: Mobile Carrier
1 or 2 e- carrier, 1 e- acceptor/donor
Ubiquinone is a mobile carrier
Can diffuse through nonpolar regions easily
“Q pool” made by Complex I and Complex II (and others)

11. Cytochrome c
Mobile carrier
Protein/heme
1 electron carrier

12. Oxygen: the final electron acceptor
Water is produced—has very low reactivity, very stable
Superoxide, peroxide as toxic intermediates
Overall reaction
NADH + H+ + ½ O2  NAD+ + H2O

14. Flow Through Complexes

15. Compartmentalization

16. Protonmotive Force NADH + H+ + ½ O2  NAD+ + H2O + 10 H+ pumped
succinate + ½ O2  fumarate + H2O + 6 H+ pumped

17. Complex I NADH  Q through “Q pool” 4 protons pumped FMN
Iron-sulfur clusters
“Q pool”
4 protons pumped
Proton wire

18. Complex III QH2  cytochromes 4 protons pumped Through Q cycle
Problem 10: An iron-sulfur protein in Complex III donates an electron to cytochrome c. Use the half reactions below to calculate the standard free energy change. How can you account for the fact that this process is spontaneous in the cell?
FeS (ox) + e-  FeS (red) Eo’ = V
Cyt c (Fe3+) + e-  cyt c (Fe2+) Eo’ = V

20. Complex IV
Cytochromes  O2
Stoichiometry of half of an oxygen atom

21. Complex II (and others)
Non-NADH sources
Complex II (part of the citric acid cycle)
Fatty acid oxidation
Glycolysis NADH shuttle (Glycerol-3-phosphate)
Bypasses Complex I
Loss of 4 protons pumped

22. Two paths of NADH into Matrix
NADH of glycolysis must get “into” matrix
Not direct
Needs either
malate-aspartate shuttle (liver)
Glycerol-3-phosphate shuttle (muscle)
Costs 1 ATP worth of proton gradients, but allows for transport against NADH gradient

23. Glycerol-3-phosphate Shuttle
Glycerol phosphate shuttle (1.5 ATP/NADH)
Produces QH2
Operational in some tissues/circumstances

24. Net ATP Harvest from Glucose
Glycolysis = 2 ATP
Plus 3 or 5 ATP from NADH
What leads to difference in this case?
Pyruvate DH = 5 ATP
Citric Acid Cycle = 20 ATP
Total: ATP/glucose

25. Overall
Chemiosmosis
10 protons shuttled from matrix to intermembrane space
Makes pH gradient and ion gradient

26. Protonmotive Force and Oxidative Phosphorylation
Flow of electrons is useless if not coupled to a useful process
Battery connected to wire
Proton gradient across mitochondrial membrane

27. Proton Gradient
Gradient driven by concentration difference + charge difference
Assume DpH 0.5 and 170mV membrane potential

28. Using the Gradient Coupled to ATP synthesis
Uncouplers used to show link of oxygen uptake and ATP synthesis

29. Uncouplers “Uncouple” protonmotive force from ATP synthase
DNP pKa / solubility perfectly suitable

30. Respiratory Poisons Other respiration poisons
Cyanide—binds Complex IV in place of oxygen

31. Complex V: ATP Synthase
Molecular motor
Rotor: c, g, e
Proton channel

32. Proton Channel Protons enters channel between rotor and stator
Rotor rotates to release strain by allowing proton to enter matrix
8- 10 protons = full rotation
Species dependent

34. “Stalk” (g) moves inside the“knob”—hexameric ATP synthase
Knob held stationary by “b”

35. Hexameric Knob

36. Binding-Change Mechanism
Stalk causes ATP synthase to have three different conformations: open, loose, tight
In “tight” conformation, energy has been used to cause an energy conformation that favors ATP formation

37. Problem 39
How did these key experiments support the chemiosmotic theory of Peter Mitchell?
The pH of the intermembrane space is lower than the pH of the mitochondrial matrix.
Oxidative phosphorylation does not occur in mitochondrial preparations to which detergents have been added.
Lipid-soluble compounds inhibit oxidative phosphorylation while allowing electron transport to continue.

38. Energy Accounting ATP costs 2.7 protons
8 protons produces 3 ATP
NADH pumps 10 protons when 2 e- reduce ½ O2
4 protons in Complex I, 4 protons in Complex III, and 2 protons in Complex IV
P/O ratio--# of phosphorylation per oxygen atom
10H+/NADH (1 ATP/2.7 H+) = 3.7 ATP/NADH
6H+/QH2 (1 ATP/2.7 H+) = 2.3 ATP/QH2
In vivo, P/O ratio closer to 2.5 and 1.5 due to other proton “leaking”
i.e. importing phosphate

39. Active Transport of ATP
ATP must go out, ADP and Pi must go in
Together, use about 1 proton of protonmotive force

40. Regulation of Oxidative Phosphorylation
Electron transport is tightly coupled to ATP production
Oxygen is not used unless ATP is being made
Avoid waste of fuels
Adding ADP causes oxygen utilization
Respiratory control

42. Problem 47
A culture of yeast grown under anaerobic conditions is exposed to oxygen, resulting in dramatic decrease in glucose consumption. This is called the Pasteur effect. Explain.
The [NADH]/[NAD+] and [ATP]/[ADP] ratios also change when an anaerobic culture is exposed to oxygen. Explain how the ratios change and what effect this has on glycolysis and the citric acid cycle in yeast.