Oxidative Phosphorylation

Oxidative Phosphorylation

Overview

• Oxidative phosphorylation is a process that uses the electron transport chain to generate a proton gradient, which is then used by ATP synthase to produce ATP.
• It takes place in the mitochondria.
  • The main goal is to generate ATP to fuel cellular activity.

Learning Objectives

• Explain how electrons flow through the electron transport chain to generate a proton gradient.
• Examine the conformational changes needed for proton transport and ATP synthesis by ATP synthase.
• Define the P/O ratio for NADH and FADH2.
• Calculate the number of ATPs and waters produced from glucose (and other molecules) under aerobic conditions.
• Examine how inhibiting one pathway can impact another, leading to symptoms and disease.

Key Components and Processes

• Electron Transport Chain (ETC):
  • NADH and FADH2 carry electrons to the ETC.
  • As electrons are passed from carrier to carrier, the change in redox potential generates free energy.
  • This energy powers conformational changes in protein complexes that pump protons from the matrix into the intermembrane space.
• Proton Gradient:
  • The proton gradient (high H+ concentration in the intermembrane space, low in the matrix) is used by ATP synthase to make ATP in the matrix.
• ATP Synthase:
  • Uses the proton gradient to synthesize ATP.

Location

The process occurs across the inner mitochondrial membrane:

• Cytosol: Outside the mitochondria.
• Outer Mitochondrial Membrane: The outer boundary of the mitochondrion.
• Intermembrane Space: The space between the outer and inner mitochondrial membranes; high proton concentration.
• Inner Mitochondrial Membrane: Where the electron transport chain complexes and ATP synthase are located.
• Matrix: The innermost compartment of the mitochondrion; low proton concentration.

Electron Flow and Complexes

• Complex I (NADH-CoQ Reductase):
  • Passes electrons from NADH to Coenzyme Q (ubiquinone).
  • It is the largest complex in the ETC, with 45 polypeptide subunits, including 14 core subunits.
  • Contains cofactors (FMN, Fe-S) and amino acid side chains that pass electrons and pump protons through 4 proton channels.
  • Conformational changes mediate the process.
• Complex II (Succinate-Q Reductase):
  • Also known as succinate dehydrogenase, links the TCA cycle to the ETC.
• Complex III:
  • Accepts electrons from Coenzyme Q.
• Complex IV:
  • Transfers electrons to oxygen, forming water.
• Electrons flow from complexes I and II to Coenzyme Q, then to Complex III, then to cytochrome c, and finally to Complex IV.
• Complexes I, III, and IV pump protons from the mitochondrial matrix to the intermembrane space, creating a proton gradient.

Electron Transfer Potential

• Standard Reduction Potential (E´_0):
  • A molecule’s tendency to be oxidized or reduced.
• The change in standard reduction potential (\Delta E´_0) is related to the change in free energy (\Delta G°') by the equation:
  • \Delta G°’ = - n F \Delta E´_0
  • Where F is Faraday's constant (96,485 J/Vmol) and n is the number of electrons transferred.

Electron Transfer Potential Calculation Example

• \frac{1}{2} O2 + 2H^+ + 2e^- \rightarrow H2O E´_0 = +0.82V
• NAD^+ + 2H^+ + 2e^- \rightarrow NADH + H^+ E´_0 = -0.32V
• \frac{1}{2} O2 + NADH + H^+ \rightarrow H2O + NAD^+
• \Delta E´_0 = +0.82V - (-0.32V) = +1.14V

Electron Transport Chain

• Electrons are passed from carrier to carrier.
• Each carrier has a standard reduction potential (E´_0).
  • Good reducing agents give up electrons easily and have negative E´_0 values.
  • Strong oxidizing agents have a greater affinity for electrons and have positive E´_0 values.
• The passage of electrons through the chain (from –ve to +ve E´_0) results in a free energy change that drives conformational changes in the complexes to set up a proton gradient for ATP synthase.

Experimental Evidence for Oxidative Phosphorylation

• Experiments monitoring oxygen consumption and ATP production in isolated mitochondria demonstrate the link between the electron transport chain and ATP synthesis.
• Adding NADH/succinate, ADP, and Pi to a mitochondrial suspension results in oxygen consumption and ATP production.
• When the supply of ADP is nearly exhausted, ATP production slows, and oxygen consumption decreases.

Inhibitors of Oxidative Phosphorylation

• Rotenone (insecticide) and amytal (barbiturate): Inhibit electron flow from Complex I to CoQ.
• Antimycin A: Blocks Complex III.
• Cyanide, azide, and CO: Inhibit Complex IV.
• Oligomycin: Inhibits ATP synthase (Complex V).
• Uncouplers: Disrupt the H+ gradient, affecting ATP synthesis.

Uncouplers

• Uncouplers are molecules with hydrophobic groups that can cross the membrane.
• Acidic groups can bind H+ and move them from high to low concentrations, disrupting the proton gradient and ATP synthesis.
• Examples: DNP (dinitrophenol)

Peter Mitchell’s Chemiosmotic Hypothesis

• ATP synthesis arises due to an electrochemical gradient across the mitochondrial inner membrane.
• The proton gradient is produced by electron transport using suitable electron donors (NADH, FADH2).
• Proton-motive force is the driving force behind ADP to ATP conversion.
• ATP synthase is membrane-bound, reversible, and dependent on the proton gradient.

ATP Synthase Structure and Function

• F1: The peripheral protein unit that carries out the catalytic synthesis of ATP in the matrix.
• F0: The integral membrane protein unit that anchors the enzyme complex in the inner mitochondrial membrane.
• Protons flow through the rotor, causing a rotation in the ring of c subunits of F0.
• Conformational changes in the F1 β subunits are responsible for ATP synthesis.

Boyer’s Binding Change Mechanism

• Each β subunit functions independently, and there are 3 different reactions occurring simultaneously.
• The binding of H+ in the rotor rotates the γ subunit and induces a conformational change in the β subunits.
• Each β subunit undergoes a conformational change between 3 states:
  • Open or Empty/exit (ATP leaves).
  • Loose - ADP and Pi bound.
  • Tight - ATP bound.

ATP Synthase Reversibility

• The passive, facilitated transport of H+ across the membrane by ATP synthase generates rotational energy to drive ATP synthesis in the mitochondrial matrix.
• Imaging techniques can be used to visualize this rotation in the F1 unit using recombinant fusion proteins and fluorescence microscopy.
• ATP hydrolysis can also be used to reverse the reaction mechanism.
• This is a form of active transport that can be used to drive proton transport across the membrane in the opposite direction using a similar rotation mechanism.

P/O Ratio

• The P/O ratio tells you how many ATPs (P) are made per Oxygen reduced to water (O).
• NADH = 10 H+ / (4 H+/ATP) = 2.5
• FADH2 = 6 H+ / (4 H+/ATP) = 1.5
• You can’t make ½ of an ATP, round down at the end of a calculation.

P/O Ratio Exceptions

• NADHcyt from glycolysis cannot be imported across the inner mitochondrial membrane for use in the ETC.
• The glycerophosphate shuttle passes electrons from NADHcyt to FADH2 in the mitochondria.
• P/O for NADHcytosol = 1.5 (same as FADH2)
• The P/O ratio also differs depending on the ATP synthase present (different # of c subunits and # of H+ needed for a complete rotation to make 1 ATP).

Water Formation in Oxidative Phosphorylation

• By ATP Synthase: ADP + Pi → ATP + H2O
• In electron transport (complex IV):
  • NADH + H+ + ½ O2 → NAD+ + H2O
  • FADH2 + ½ O2 → FAD + H2O
• Water ratio:
  • 3. 5 H2O for NADH.
  • 4. 5 H2O for FADH2 (or NADHcytosol).

Overall Summary Equation for the Complete Oxidation of Glucose

• Glucose + ADP + Pi + O2 → CO2 + ATP + H2O

Anaerobic Metabolism

• During periods of limited oxygen, the Electron Transport Chain shuts down, and anaerobic metabolism occurs.
• All energy production in the mitochondria shuts down, and the PDC & CAC also slow down.
• Glycolysis is the only means of generating ATP.
• Lactate dehydrogenase uses NADH made in glycolysis in the cytoplasm to replenish NAD+ for glycolysis to continue.
• Lactate can be used by liver cells in GNG.

Key Messages

• A change in redox potential in the Electron Transport Chain drives the pumping of protons across the inner mitochondrial membrane.
• The Chemiosmotic Theory describes the importance of proton-motive force set up by the electron transport chain for ATP synthesis in the matrix.
• The P/O ratio determines the number of ATPs synthesized per molecular oxygen reduced to water.
• In addition to ATP made by substrate-level phosphorylation in Glycolysis and TCA (GTP), NADH and FADH2 generate more ATP in oxidative phosphorylation.
• Inhibitors, oxygen availability, and regulation are important for cellular metabolism.