Oxidative Phosphorylation

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

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

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Chemiosmotic Theory

• Peter Mitchell
• Links Electron Transport to ATP Synthesis

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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

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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

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ATP Synthesis
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ATP Synthase

• Proton-pumping ATP Synthase
• F1F0ATPase

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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)

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Properties of F1ATPase

• Dissociated from F0 by urea
• Catalyzes ATP hydrolysis (ATPase) but cannot
  synthesize ATP
• Importance of Membrane!

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Structure of ATP Synthase
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Structure of ATP Synthase
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F1 Component of ATP Synthase

• Pseudo three-fold symmetry
• Composition ?3?3???
• ? subunit catalyzes ATP synthesis

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Ribbon Diagram of F1ATP Synthase from Bovine
Heart Mitochondria
? Red ? Yellow ? Blue
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Electron Density Map of F1ATP Synthase from
Bovine Heart Mitochondria
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F0 Component of ATP Synthase

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

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X-Ray Structure of Yeast Mitochondrial F1c10
Complex
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ATP is Synthesized by the Binding Change Mechanism
L loose state T tight state O open state
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F1ATPase

• Three Interacting Catalytic Protomers (??)

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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

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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

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Proton Translocation Drives Interconversion of
States
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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

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F1F0ATPase is a Rotary Engine
Stator (ab2?3?3?)
Rotor (??c12)
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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

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Rotation of F1F0ATPase
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Visualizing Rotation
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Visualizing Rotation
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P/O Ratio

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

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

• NADH 3 ATP/10 H
• FADH2 2 ATP/6 H

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Mitochondrial Electron Transport Chain
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Other Fates of Proton Gradient

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

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Actual ATP Yields

• NADH 2.5 ATP
• FADH2 1.5 ATP

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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

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

• Electron Transport and Oxidative Phosphorylation
  are Tightly Coupled

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Properties of 2,4-Dinitrophenol

• Lipophilic Weak Acid
• Proton-transporting Ionophore

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

• Generates Heat

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Distribution of Brown Adipose Tissue

• Newborn mammals lacking fur
• Hibernating mammals

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Nonshivering Thermogenesis(regulated uncoupling
of oxidative phosphorylation)

• Heat Generation

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Uncoupling Protein(UCP1 or Thermogenin)

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

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Mechanism of Hormonally-Induced Uncoupling of
Oxidative Phosphorylation in Brown Adipose Tissue
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Mechanism of Hormonally-Induced Uncoupling of
Oxidative Phosphorylation in Brown Adipose Tissue
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Mechanism of Hormonally-Induced Uncoupling of
Oxidative Phosphorylation in Brown Adipose Tissue
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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