Oxidative Phosphorylation

Endergonic Synthesis of ATP

Electron Transport and ATP Synthesis

ATP Synthase

(Complex V)

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

ATP Synthase

Proton-pumping ATP Synthase

F1F0–ATPase

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)

Structure of ATP Synthase(Cryoelectron Microscopy-Based

Image)

Matrix

InnerMembrane

IMS

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

Ribbon Diagram of F1–ATP Synthase from Bovine Heart

Mitochondria

F0 Component of ATP Synthase

• Includes a transmembrane ring

• Composition (E. coli): a1b2c9-12

• Mitochondrial F0 has additional subunits (function unclear)

Structure of E coli F1–c10-15 Complex

Composite Crystal Structure Model

F1–ATPase

Three Interacting Catalytic Protomers ()

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

ATP is Synthesized by the Binding Change Mechanism

L = loose state

T = tight state

O = open state

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

Proton Translocation Drives Interconversion of States

F1F0–ATPase is a Rotary Engine:

Movement of protons drives rotationBind to c subunit Exit through a

subunit

Stator

(ab2–33)

Rotor

(–c12)

Rotation of F1F0–ATPase

Protonation/

Deprotonation

Rotation

Visualizing Rotation

Visualizing Rotation

P/O Ratio

Relates the Amount of ATP Synthesized to the Amount

of Oxygen Reduced

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

NADH: ~3 ATP/10 H+

FADH2: ~2 ATP/6 H+

Mitochondrial Electron Transport Chain

Complex II

(FADH2)

Other Fates of Proton Gradient

Dissipation (leakage)

Consumption for other purposes (e.g. Pi

transport)

1H+/Pi 4H+/ATP

Actual ATP Yields

Based on 4H+/ATP

Demonstrated experimentally

NADH: ~2.5 ATP

FADH2: ~1.5 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%

Uncouplers

Electron Transport and Oxidative Phosphorylation

are Tightly Coupled

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

Action of 2,4-Dintrophenol(Lipophilic weak acid)

Valinomycin

Amphipathic Peptide Ring

Hydrophillic

Hydrophobic

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

Mechanism of Hormonally-Induced Uncoupling of Oxidative

Phosphorylation in Brown Adipose Tissue

Mechanism of Hormonally-Induced Uncoupling of Oxidative

Phosphorylation in Brown Adipose Tissue

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

Plants: Uncoupling Proteins

• Response to Cold Stress

• Increase Flower Temperature

(vaporization of scent to attract

pollinators)