Oxidative Phosphorylation P2

Energy Coupling: Electron Carriers and ATP Formation

• NADH oxidation is highly exergonic – it releases a large amount of energy.

• ADP rephosphorylation (forming ATP) is only moderately endergonic.

• This difference allows energy from NADH oxidation to power ATP synthesis via proton-motive force.

⚡ Proton-Motive Force (PMF)

• PMF is generated by the oxidation of NADH and FADH2.

• It is a proton gradient across the inner mitochondrial membrane = pH difference.

• PMF drives ATP synthesis by powering ATP synthase.

• Experimental evidence supports the PMF model.

• ATP synthase = Complex V, also called:

  • Mitochondrial ATPase

  • F1F0 ATPase

⚙ Structure of ATP Synthase

• Resembles a ball-and-stick model:

  • F0 ("stick"): proton channel, embedded in membrane.

  • F1 ("ball"): catalytic, extends into matrix.

• Components:

  • F1 Subunit:

    • Catalytic unit; α₃β₃γδε.

    • Three β subunits = active sites.

    • γ subunit connects to F0, breaks symmetry.

  • F0 Subunit:

    • Proton-conducting unit; a, b, c subunits.

    • C-ring (8–14 c subunits, species dependent).

    • Stabilized by dimerization → aids cristae formation.

🔁 Connecting F0 and F1: Rotary Machinery

• γε central stalk = rotor

• γ = coiled coil, rotates within α₃β₃ hexamer.

• Stator = exterior column:

  • Composed of a subunit, 2 b subunits, δ subunit.

🔄 Binding-Change Mechanism (Mechanics of ATP Formation)

• Catalysis occurs in β subunits through conformational cycling:

  • O (open): nucleotides bind/release.

  • L (loose): ADP + Pi held in place.

  • T (tight): ATP formed from ADP + Pi.

• Without proton flow, ATP cannot be released.

• Rotation of γ subunit (powered by c-ring):

  • Converts T → O → L → T by 120° steps.

  • Drives ATP release and ADP/Pi binding.

🔁 Proton Flow Powers Rotation

• F0’s a subunit has two half-channels:

  • One opens to intermembrane space, the other to matrix.

• Proton flow mechanism:

  1. Proton enters from intermembrane space → neutralizes c-subunit aspartate.

  2. C-ring rotates, releasing a different proton to the matrix.

  3. Cycle continues; each proton drives one step of rotation.

🔂 ATP Yield and Efficiency

• One full 360° rotation = 3 ATP formed.

• In yeast: 10 c-subunits → 10 H+ for 3 ATP → ~3.33 H+ per ATP.

• In humans: 8 c-subunits → ~2.7 H+ per ATP → more efficient.

• Proton pumping by ETC complexes:

  • Complex I: 4 H⁺

  • Complex III: 2 H⁺

  • Complex IV: 4 H⁺

• ~2.5 ATP per NADH (after factoring in ATP/ADP transport cost).

🔄 ATP/ADP Exchange – Translocase System

• ATP-ADP translocase exchanges cytoplasmic ADP for mitochondrial ATP.

• Accounts for ~15% of inner membrane proteins.

• ATP has more negative charge than ADP → movement aligns with membrane potential.

• Exchange costs ~1 H+ → 25% of PMF is used here.

🏗 Inner Mitochondrial Transporters

• ATP-ADP translocase: three 100-AA domains, each with 2 transmembrane segments.

• Phosphate carrier: exchanges H₂PO₄⁻ for OH⁻ → net cost = 1 H⁺ per ADP import.

📊 Regulation of Oxidative Phosphorylation

• ~30 ATP per glucose; 26 from oxidative phosphorylation, 4 from glycolysis.

• ADP availability controls ETC → “Respiratory (Acceptor) Control”.

• No ADP = No electron flow through ETC.

🔬 Chemiosmotic Theory (Mitchell’s Hypothesis)

• ATP synthesis requires:

  • Intact inner membrane

  • Impermeability to ions

  • Electrochemical proton gradient

• Artificial PMF can drive ATP synthesis even without ETC.

🔄 Uses of Proton Gradient Beyond ATP Synthesis

• PMF used in:

  • Ca²⁺ active transport

  • Sugar/amino acid import in bacteria

  • NADPH synthesis (photosynthesis)

  • Flagella motion (bacterial propulsion)

  • Heat generation (thermogenesis)

🔐 Regulation & Uncoupling

• IF1 (Inhibitory Factor 1):

  • Inhibits ATP synthase hydrolysis mode when O₂ is limited.

  • Overexpressed in some cancers.

• UCP-1 (thermogenin):

  • In brown adipose tissue (BAT), causes nonshivering thermogenesis.

  • UCP-2/3 involved in energy regulation, may affect body weight.

  • BAT is active in cold exposure and hibernation.

☠ Inhibition of Oxidative Phosphorylation

• Three major inhibition routes:

  1. ETC inhibition → no PMF

  2. ATP synthase inhibition

  3. Uncoupling (e.g. 2,4-dinitrophenol) → protons bypass synthase.

• Key inhibitors:

  • Rotenone, amytal: block Complex I → ubiquinone.

  • Antimycin A: blocks Complex III.

  • Cyanide, azide, CO: block Complex IV.

⚠ Mitochondrial Diseases

• Complex I dysfunction is most common.

• Defects → ↓ ATP + ↑ reactive oxygen species (ROS) → mitochondrial damage.

💀 Mitochondria in Apoptosis

• Mitochondria mediate apoptosis (programmed cell death).

• Upon outer membrane permeabilization, cytochrome c is released → activates caspase cascade → cell death.

• Vital in development, tissue remodeling, and removal of damaged cells.