Oxidative Phosphorylation

1
Oxidative Phosphorylation
2
Electron Transport and ATP Synthesis
ATP Synthase (Complex V)
3
Coupling of Electron Transport and ATP Synthesis

• In intact mitochondria, electron transport
  requires simultaneous synthesis of ATP

4
Chemiosmotic Theory

• Peter Mitchell
• Links Electron Transport to ATP Synthesis

5
Evidence Supporting Chemiosmotic Theory

• Oxidative Phosphorylation requires an intact
  mitochondrial inner membrane
• Mitochondrial inner membrane is impermeable to
  ions can maintain an electrochemical gradient
• Electron transport acidifies the cytosol
• Uncouplers permeabilize the mitochondrial inner
  membrane and uncouple electron transport and
  oxidative phosphorylation

6
Electron Transport Generates a Proton
Gradient(Proton Motive Force)

• ?G 2.3RT?pH ZF??
• membrane potential
• ?pH 0.75 (inside higher)
• ?G 21.5 kJ/mol

7
ATP Synthesis
8
ATP Synthase

• Proton-pumping ATP Synthase
• F1F0ATPase

9
Properties of ATP Synthase

• Multisubunit transmembrane protein
• Molecular mass 450 kD
• Functional units
• F0 water-insoluble transmembrane protein (up to
  8 different subunits)
• F1 water-soluble peripheral membrane protein (5
  subunits)

10
Structure of ATP Synthase(Electron Micrograph of
Cristae)
11
Structure of ATP Synthase(Cryoelectron
Microscopy-Based Image)
12
F1 Component of ATP Synthase

• Dissociated from F0 by urea
• Catalyzes ATP hydrolysis (ATPase) but cannot
  synthesize ATP (F1-ATPase)
• Pseudo three-fold symmetry
• Composition ?3?3???
• ? subunit catalyzes ATP synthesis

13
Ribbon Diagram of F1ATP Synthase from Bovine
Heart Mitochondria
? Red ? Yellow ? Blue
14
Electron Density Map of F1ATP Synthase from
Bovine Heart Mitochondria
Bound AMPPNP
15
F0 Component of ATP Synthase

• Includes a transmembrane ring
• Composition (E. coli) a1b2c9-12
• Mitochondrial F0 has additional subunits
  (function unclear)

16
X-Ray Structure of Yeast Mitochondrial F1c10
Complex
17
F1ATPase

• Three Interacting Catalytic Protomers (??)

18
Properties of F1 Catalytic Protomers

• L state binds substrates and products loosely
• T state binds substrates and products tightly
• O state open state does not bind substrate or
  product

19
ATP is Synthesized by the Binding Change Mechanism
L loose state T tight state O open state
20
Functions of Catalytic Protomers in ATP Synthesis

• L state binds substrates (ADP and Pi)
• T state formation of phosphoanhydride bond (ADP
  Pi gt ATP)
• O state release of product (ATP)
• Proton translocation drives interconversion of
  states

21
Steps in ATP Synthesis

• ADP and Pi bind to L site
• Energy-dependent conformational change
• L gt T
• O gt L
• T gt O
• ATP synthesized at T site and ATP released from O
  site

22
Proton Translocation Drives Interconversion of
States
23
F1F0ATPase is a Rotary Engine
Stator (ab2?3?3?)
Rotor (??c12)
24
F1F0ATPase is a Rotary Engine

• Rotor ??-c12 ring complex
• Stator ab2-?3?3?
• Movement of protons drives rotation
• Bind to c subunit
• Exit through a subunit

25
Rotation of F1F0ATPase
Protonation/ Deprotonation
Rotation
26
Visualizing Rotation
27
Visualizing Rotation
28
P/O Ratio

• Relates the Amount of ATP Synthesized to the
  Amount of Oxygen Reduced

29
P/O Ratios Measured Using Isolated
Mitochondria(only use of proton gradient)

• NADH 3 ATP/10 H
• FADH2 2 ATP/6 H

30
Mitochondrial Electron Transport Chain
Complex II (FADH2)
31
Other Fates of Proton Gradient

• Dissipation (leakage)
• Consumption for other purposes (e.g. Pi transport)

32
Actual ATP Yields

• NADH 2.5 ATP
• FADH2 1.5 ATP

33
ATP from Glucose

• Glycolysis 2 ATP 2 NADH ( 5ATP) 7 ATP
• Pyruvate Dehydrogenase 2 NADH ( 5 ATP) 5 ATP
• Citric Acid Cycle 6 NADH ( 15 ATP) 2 FADH2 (
  3 ATP) 2 GTP ( 2 ATP) 20 ATP
• TOTAL 32 ATP/Glucose

34
Thermodynamic Yield
ATP/Glucose 32 ATP x 45 kJ/mol 1440
kJ Glucose gt CO2 2866 kJ 1440/2866 50
35
Uncouplers

• Electron Transport and Oxidative Phosphorylation
  are Tightly Coupled

36
Tight Coupling
37
Properties of 2,4-Dinitrophenol

• Lipophilic Weak Acid
• Proton-transporting Ionophore

38
Action of 2,4-Dintrophenol
39
Uncoupling in Brown Adipose Tissue

• Generates Heat

40
Distribution of Brown Adipose Tissue

• Newborn mammals lacking fur
• Hibernating mammals

41
Nonshivering Thermogenesis(regulated uncoupling
of oxidative phosphorylation)

• Heat Generation

42
Uncoupling Protein(UCP1 or Thermogenin)

• Proton channel
• Inhibited by purine nucleotides
• ADP and ATP
• GDP and GTP
• Inhibition overcome by fatty acids

43
Mechanism of Hormonally-Induced Uncoupling of
Oxidative Phosphorylation in Brown Adipose Tissue
44
Mechanism of Hormonally-Induced Uncoupling of
Oxidative Phosphorylation in Brown Adipose Tissue
45
Adult Humans UCP2 and UCP3

• May be related to fast or slow metabolism
• Possible targets for anti-obesity therapies
• Previous use of 2,4-dinitrophenol as dietary aid
  abandoned due to occasional lethality