Oxidative Phosphorylation Notes

Oxidative Phosphorylation

Learning Objectives

  • Describe the transport of electrons through the respiratory chain.
  • Explain how ATP is synthesized through chemiosmotic coupling.
  • Recognize inhibitors of the ETC and oxidative phosphorylation.
  • Describe the role of uncoupling proteins

Overview of Glucose Oxidation

  • Glucose is processed through glycolysis in the cytosol, yielding pyruvate.
  • Pyruvate can be converted to lactate or enter the mitochondrion for further oxidation.
  • Inside the mitochondrion, pyruvate is converted to acetylCoA, which enters the TCA cycle.
  • The TCA cycle produces CO2, FADH2, and NADH.

ATP Production from NADH and FADH2

  • For every turn of the TCA cycle, 1 FADH2 and 3 NADH are produced, carrying a total of 8 electrons.
  • These electrons are used in the electron transport chain to create a proton gradient.
  • The proton gradient drives ATP synthesis via ATP synthase.

Electron Transport Chain Overview

  • Electrons from NADH and FADH2 are transferred to oxygen along the electron transport chain (respiratory chain).
  • Energy released is large enough to pump protons across the inner mitochondrial membrane (IMM) to create a proton gradient.
  • Protons flow back and generate ATP (ATP synthase complex).
  • This process is tightly coupled.

Electron Transport Chain

  • Located in the inner mitochondrial membrane.
  • Comprises 4 protein complexes (Complex I - IV).
    • 3 proton pumps (Complex I, III, IV).
    • 1 link to the TCA cycle (Complex II).
  • Includes 2 small mobile components: Coenzyme Q (CoQ) and cytochrome c.

Complex I: NADH Dehydrogenase

  • Contains at least 34 polypeptides (880kDa).
  • NADH binds to Complex I.
  • Accepts electrons from NADH.
  • Transfers electrons to CoQ (via FMN and Fe-S protein).
  • Pumps 4 H+ out.

Complex II: Succinate Dehydrogenase

  • Enzyme of the TCA cycle (succinate to fumarate).
  • Accepts electrons from FADH2.
  • Transfers electrons to CoQ via Fe-S proteins.

Coenzyme Q (CoQ)

  • Also known as Ubiquinone / ubiquinol.
  • Small lipid-soluble compound (hydrophobic quinone).
  • Diffuses rapidly within the IMM, acting as a mobile carrier.
  • Accepts electrons from Fe-S proteins from Complex I and Complex II.
  • Transfers electrons to Complex III / cytochrome c (Q cycle).

Complex III: Cytochrome c Reductase

  • Contains a heme prosthetic group.
  • Accepts electrons from CoQ.
  • Transfers them to cytochrome c.
  • Pumps two protons across the IMM.

Cytochrome c

  • Peripheral membrane protein loosely bound to the IMM.
  • Binds to Complex III and transfers electrons to Complex IV.
  • Highly conserved.

Complex IV: Cytochrome c Oxidase

  • Composed of 13 protein subunits containing 2 heme groups and 3 copper ions.
  • Accepts electrons from cytochrome c.
  • Transfers them to 1/2 O2 which is reduced to form H2O.
  • Pumps 8 protons across IMM.

Electron Transfer and Redox Potential

  • Electrons are transferred because the accepting carrier has a higher affinity for electrons than the donating carrier (redox potential).
  • NADH + \rac{1}{2}O2 + H^+ \rightarrow H2O + NAD^+
  • ΔE0’=1.14V\Delta E0’ = 1.14V
  • ΔG0’=−52.6kcal⋅mol−1\Delta G0’ = -52.6 kcal \cdot mol^{-1}

ATP Synthase (Complex V)

  • Located in the IMM.
  • Composed of two subunits:
    • F1 ATPase: generates ATP.
    • F0: Coupling factor - proton channel spanning IMM.

ATP Synthase Mechanism

  • Protons pass through the channel in F0.
  • This causes a conformational change in F1, causing ATP to be synthesized from ADP + Pi.

ATP Synthase and Proton Flow

  • Protons pumped to the cytosolic side of the mitochondrial membrane re-enter the matrix by passing through the F0 proton channel.
  • As protons pass down the channel they drive the rotation of the C ring of F0 which causes conformational change in the β-subunit of F1 domain (bind ADP + Pi ). ATP formed and released.

ATP Yield from Oxidative Phosphorylation

  • NADH + \rac{1}{2}O2 + H^+ \rightarrow H2O + NAD^+
    • ΔG0’=−52.6kcal⋅mol−1\Delta G0’ = -52.6 kcal \cdot mol^{-1}
  • ADP+Pi+H+→ATP+H2OADP + Pi + H^+ \rightarrow ATP + H_2O
    • ΔG0’=+7.3kcal⋅mol−1\Delta G0’ = +7.3 kcal \cdot mol^{-1}
  • Theoretically 6-7 ATP generated.
  • Only 3 sites where \Delta G0’> 7.3 kcal \cdot mol^{-1}.

ATP Yield

  • NADH yields 3 ATP (or 2.5 ATP according to newer estimates).
  • FADH2 yields 2 ATP (or 1.5 ATP).

P/O Ratios

  • Number of ATP molecules formed per oxygen atom.
  • NADH - P/O ratio of 3 (2.5).
  • FADH2 - P/O ratio of 2 (1.5).

ATP Yield in Detail

  • NADH - 3 ATP (2.5).
  • FADH2 - 2 ATP (1.5).
  • For TCA cycle (acetyl CoA):
    • 3NADH + 1FADH2 + 1GTP = 9ATP + 2ATP + 1ATP = 12 ATP (10).
  • Overall for 1 molecule of glucose:
    • 30 ATP generated.

Agents Affecting Oxidative Phosphorylation

  • ATPase inhibitors - oligomycin
  • Site-specific inhibitors of the electron transport chain
  • Uncouplers - neutralize the proton gradient and prevent ATP synthesis

Inhibitors of Electron Transport Chain

  • Complex I: Rotenone, amytal
  • Complex III: Antimycin A
  • Complex IV: Cyanide, azide, CO

Uncouplers of Electron Transport Chain

  • Uncouple electron transport from ATP synthesis and destroy proton gradient
  • Chemicals - dinitrophenol
  • Natural uncoupling proteins

Uncoupling Protein 1 (UCP1)

  • Thermogenin
  • Mitochondria of brown adipose tissue
  • Energy from electron transport used to generate heat - nonshivering thermogenesis.
  • Important in newborns and hibernating animals

Other UCPs

  • UCP3 – 57% identical with UCP1. Found in skeletal muscle.
  • May regulate body weight - expression of UCP3 in skeletal muscle leads to mice being resistant to diet-induced obesity.