oxidative phosphorylation endergonic synthesis of atp
TRANSCRIPT
Coupling of Electron Transport and ATP Synthesis
In intact mitochondria, electron transport
requires simultaneous synthesis of ATP
Chemiosmotic Theory:The free energy from the ETC is coupled to ATP synthesis by the
generation of a pH gradient across the mitochondrial inner membrane that is
then used by ATP synthase
Peter Mitchell
Links Electron Transport
to ATP Synthesis
Evidence Supporting The 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• Acidification outside mitochondrial inner membrane stimulates ATP synthesis
• Uncouplers — permeabilize the mitochondrial inner membrane – ETC continues, but ATP synthesis is inhibited
• UNCOUPLES ETC and oxidative phosphorylation
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
ATP Synthesis
ADP + Pi ATP
² Go' = +30.5 kJ/mol(Standard Conditions)
² G = ~40-50 kJ/mol(Physiological Conditions)
2-3 protons/ATP
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)
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: 33
• β-subunit catalyzes ATP synthesis
F0 Component of ATP Synthase
• Includes a transmembrane ring
• Composition (E. coli): a1b2c9-12
• Mitochondrial F0 has additional subunits (function unclear)
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
Functions of Catalytic Protomers in ATP Synthesis
• L state: binds substrates (ADP and Pi)
• T state: formation of phosphoanhydride bond (ADP + Pi —> ATP)
• O state: release of product (ATP)
Proton translocation drives interconversion of states
Steps in ATP Synthesis
• ADP and Pi bind to L site
• Energy-dependent conformational change
– L —> T
– O —> L
– T —> O
• ATP synthesized at T site and ATP released from O site
F1F0–ATPase is a Rotary Engine:
Movement of protons drives rotationBind to c subunit Exit through a
subunit
Stator
(ab2–33)
Rotor
(–c12)
P/O Ratios Measured Using Isolated Mitochondria(only use of proton gradient)
NADH: ~3 ATP/10 H+
FADH2: ~2 ATP/6 H+
Other Fates of Proton Gradient
Dissipation (leakage)
Consumption for other purposes (e.g. Pi
transport)
1H+/Pi 4H+/ATP
ATP from Glucose
Glycolysis: 2 ATP + 2 NADH (= 5 ATP) = 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
Thermodynamic Yield
ATP/Glucose
32 ATP x ~45 kJ/mol = 1440 kJ
Glucose —> CO2 = 2866 kJ
1440/2866 = ~50%
Tight Coupling
Measuring O2 consumption of isolated Mitochondria
Time
+ ADP + Pi
+ Substrate (e.g. NADH)+ Mitochondria
O2
All ADPATP
Uncouplers
• Lipophilic Weak Acid
• Proton-transporting Ionophore
• 2,4-Dinitrophenol, FCCP, CCCP
• Valinomycin
• Protein Channels
Uncoupling in Brown Adipose Tissue
Nonshivering Thermogenesis(regulated uncoupling of oxidative
phosphorylation)
Heat Generation
Uncoupling Protein(UCP1 or Thermogenin)
• Proton channel
• Inhibited by purine nucleotides
– ADP and ATP
– GDP and GTP
• Inhibition overcome by fatty acids
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