Chapter 18 — Oxidative Phosphorylation

Learning Objectives

1. Describe the key components of the electron-transport chain (ETC) and how they are arranged.

2. Explain the benefits of having the electron-transport chain located in a membrane.

3. Describe how the proton-motive force is converted into ATP.

4. Identify the ultimate determinant of the rate of cellular respiration.

Learning Objective 1: Electron-Transport Chain Components and Organization

1. Overview of Cellular Respiration and Oxidative Phosphorylation

• Cellular respiration = process that transfers energy from nutrients into ATP.

• Electron-transport chain (ETC) / respiratory chain = four large protein complexes embedded in the inner mitochondrial membrane.

• Oxidative phosphorylation = process in which energy from electrons carried by NADH and FADH₂ is used to generate ATP.

• Electrons ultimately transfer to molecular oxygen (O₂), reducing it to water (H₂O).

  • Electron transfer releases energy that is used to pump protons (H⁺) from the mitochondrial matrix into the intermembrane space.

• The resulting proton gradient powers ATP synthesis.

Location of oxidative phosphorylation

• Mitochondrial matrix: Contains enzymes for most citric acid cycle reactions and fatty acid oxidation.

• Inner mitochondrial membrane: Contains the ETC complexes and ATP synthase.

• Intermembrane space: Accumulates protons pumped out of the matrix.

• Outer mitochondrial membrane: Permeable to many small molecules and ions because of porins.

• The inner membrane is impermeable to most ions and polar molecules.

• Cristae = folds of the inner mitochondrial membrane that increase surface area for oxidative phosphorylation.

2. The Four Electron-Transport Chain Complexes

Complex I — NADH-Q oxidoreductase

• Accepts electrons from NADH.

• Transfers electrons through FMN and iron–sulfur clusters to coenzyme Q (Q).

• Reduces Q to QH₂.

• Pumps 4 H⁺ from the matrix to the intermembrane space per pair of electrons.

Complex II — Succinate-Q reductase

• Contains succinate dehydrogenase from the citric acid cycle.

• Transfers electrons from FADH₂ through iron–sulfur centers to Q.

• Reduces Q to QH₂.

• Does not pump protons.

Complex III — Q-cytochrome c oxidoreductase

• Transfers electrons from QH₂ to cytochrome c.

• Uses the Q cycle to transfer electrons from a two-electron carrier to a one-electron carrier.

• Moves protons across the membrane; the lecture describes a net movement of 4 H⁺ to the intermembrane space per Q cycle.

Complex IV — Cytochrome c oxidase

• Receives electrons from cytochrome c.

• Transfers electrons to oxygen, reducing O₂ to H₂O.

• Pumps 4 H⁺ into the intermembrane space per four electrons transferred to oxygen.

• Contains heme and copper centers.

Proton-pumping summary

Important: Complexes I, III, and IV are proton pumps. Complex II is not.

3. Mobile Electron Carriers

Coenzyme Q (ubiquinone)

• A hydrophobic, lipid-soluble electron carrier that diffuses within the inner mitochondrial membrane.

• Accepts electrons from Complex I and Complex II.

• Reduced coenzyme Q is called ubiquinol (QH₂).

• Transfers electrons to Complex III.

• Can exist in oxidized, partially reduced (semiquinone), and fully reduced forms.

Cytochrome c

• A small, soluble electron-transfer protein loosely associated with the outer surface of the inner mitochondrial membrane.

• Contains a heme prosthetic group.

• Transfers electrons from Complex III to Complex IV.

• Carries one electron at a time.

Iron–sulfur clusters

• Components of iron–sulfur proteins in the ETC.

• Participate in electron-transfer reactions.

• Iron cycles between Fe²⁺ (reduced) and Fe³⁺ (oxidized).

4. Electron Flow Through the ETC

• Electrons from NADH enter through Complex I.

• Electrons from FADH₂ enter through Complex II.

• Both pathways converge at the coenzyme Q pool.

• Electrons then move from QH₂ to Complex III, to cytochrome c, and finally to Complex IV.

• Oxygen is the final electron acceptor and is reduced to water.

Pathway to memorize:

NADH → Complex I → Q → Complex III → cytochrome c → Complex IV → O₂

FADH₂ → Complex II → Q → Complex III → cytochrome c → Complex IV → O₂

Why does NADH yield more ATP than FADH₂?

• NADH donates electrons at Complex I, so its electrons contribute to proton pumping at Complexes I, III, and IV.

• FADH₂ donates electrons through Complex II, bypassing Complex I.

• Complex II does not pump protons.

• Therefore, electron transfer from FADH₂ supports less proton-motive force and produces less ATP than electron transfer from NADH.

5. The Q Cycle

• The Q cycle occurs in Complex III.

• It transfers electrons from QH₂, which carries two electrons, to cytochrome c, which carries one electron.

• One electron is passed toward cytochrome c.

• The other travels through cytochrome b to reduce Q at another binding site.

• A second QH₂ participates to complete the cycle and regenerate QH₂ at the second site.

• The process couples electron transfer to proton movement across the membrane.

6. Complex IV and Oxygen Reduction

• Complex IV transfers four electrons from four cytochrome c molecules to one O₂ molecule.

• Oxygen is reduced to form two H₂O molecules.

• Four chemical protons from the matrix are used to form water.

• Four additional protons are pumped into the intermembrane space.

Preventing reactive oxygen species

• Reactive oxygen species (ROS) form when oxygen is incompletely reduced.

• Examples include superoxide, peroxide, and hydroxyl radicals.

• These molecules can damage cellular components.

• Complex IV holds oxygen tightly between metal centers during reduction, helping prevent the premature release of reactive intermediates.

Protective enzymes

• Superoxide dismutase (SOD): Converts superoxide radicals into hydrogen peroxide and oxygen.

  • Manganese-containing form: mitochondria.

  • Copper-and-zinc-dependent form: cytoplasm.

• Catalase: Converts hydrogen peroxide into water and oxygen.

7. Respirasome

• Respirasome = a larger supramolecular assembly containing ETC complexes.

• The lecture describes the human respirasome as containing two copies of Complex I, Complex III, and Complex IV.

• Complex II can associate in a gap between Complexes I and IV.

• Organization into a larger assembly helps improve electron-transfer efficiency.

Learning Objective 2: Benefits of Locating the ETC in a Membrane

8. Why Is the Inner Mitochondrial Membrane Important?

• The inner mitochondrial membrane provides a surface for organizing the ETC complexes and ATP synthase.

• It separates the mitochondrial matrix from the intermembrane space.

• It is largely impermeable to protons and most other ions, allowing a proton gradient to develop.

• ETC complexes use energy from electron transfer to pump protons from the matrix into the intermembrane space.

• The difference in proton concentration and electrical charge across the membrane stores potential energy.

Benefits of membrane organization

1. Creates a proton gradient: Protons accumulate in the intermembrane space.

2. Stores energy: The proton gradient stores energy released by electron transfer.

3. Couples electron transport to ATP synthesis: Protons flow back into the matrix through ATP synthase, driving ATP production.

4. Organizes the machinery: ETC complexes and ATP synthase are positioned in the inner membrane for efficient energy conversion.

5. Maintains separate compartments: The matrix and intermembrane space can maintain different proton concentrations and electrical charges.

The chemiosmotic hypothesis

• Chemiosmotic hypothesis = electron transport and ATP synthesis are coupled through a proton gradient across the inner mitochondrial membrane.

• Electron transfer generates the gradient.

• ATP synthase uses the gradient to synthesize ATP.

• The gradient, rather than direct electron transfer to ADP, supplies the energy for ATP synthesis.

Key sequence:

Electron transfer → Proton pumping → Proton-motive force → ATP synthesis

9. The Proton-Motive Force

• Proton-motive force (PMF) = the energy stored in the unequal distribution of protons across a membrane.

• It has two components:

1. Chemical gradient (ΔpH): Difference in proton concentration across the membrane.

2. Electrical gradient (Δψ): Difference in electrical charge across the membrane.

• The intermembrane space becomes more acidic and more positively charged relative to the matrix.

• Protons tend to flow back into the matrix down their electrochemical gradient.

• ATP synthase harnesses this flow to drive ATP synthesis.

\[ \text{PMF} = \text{chemical gradient} + \text{electrical gradient} \]

Learning Objective 3: Conversion of Proton-Motive Force into ATP

10. ATP Synthase (Complex V)

• ATP synthase = a protein complex in the inner mitochondrial membrane that synthesizes ATP from ADP and inorganic phosphate (Pi).

• It uses energy from proton flow to power ATP synthesis and ATP release.

• It acts as a rotary molecular motor.

Two main components

F₀ component

• Embedded in the inner mitochondrial membrane.

• Contains the proton-conducting pathway.

• Proton movement drives rotation of the membrane-associated rotor.

F₁ component

• Projects into the mitochondrial matrix.

• Contains the catalytic sites for ATP synthesis.

• Contains three β subunits that are catalytically active.

Important structural features

• The F₁ portion contains three α and three β subunits arranged in a hexameric ring, plus γ, δ, and ε subunits.

• The α subunits bind nucleotides but are not the catalytic sites.

• The β subunits contain the catalytic sites.

• The γ and ε subunits form the central stalk.

• Rotation of the central γ subunit changes the conformations of the β subunits.

• The F₀ portion includes a ring of c subunits and a membrane-associated a subunit.

11. How Proton Flow Drives ATP Synthesis

1. ETC complexes pump protons from the matrix into the intermembrane space.

2. A proton gradient develops across the inner mitochondrial membrane.

3. Protons flow back into the matrix through F₀ of ATP synthase.

4. Proton flow drives rotation of the rotor and central stalk.

5. Rotation changes the shapes of the catalytic β subunits in F₁.

6. These conformational changes allow ADP and Pi to form ATP and permit ATP to be released.

Important distinction: ATP can form while tightly bound to ATP synthase without a proton-motive force, but proton flow is needed for the efficient release of newly synthesized ATP.

12. The Binding-Change Mechanism

• Binding-change mechanism = ATP synthase produces and releases ATP through changes in the conformations of its β subunits.

• The three β subunits cycle through three conformational states.

• The rotation of the γ subunit drives these changes.

• The three β subunits occupy different states at the same time.

The three β-subunit states

Order of conformational changes

L → T → O → L

• L: ADP and Pi bind.

• T: ADP and Pi are converted into ATP, which remains tightly bound.

• O: ATP is released.

• Continued rotation allows each β subunit to cycle through all three states.

Why is rotation important?

• The γ subunit rotates in steps of approximately 120°.

• Each 120° rotation advances the β subunits to their next conformational states.

• One full 360° rotation cycles all three catalytic sites through their states.

• This rotational mechanism allows ATP synthase to function as a molecular motor.

13. Energy Coupling in Oxidative Phosphorylation

• Electron transfer from NADH to oxygen is highly exergonic and releases energy.

• ATP synthesis from ADP and Pi is endergonic and requires energy.

• Proton pumping captures energy from electron transfer as a proton-motive force.

• ATP synthase uses this stored energy to drive ATP synthesis.

Remember: Electron transport creates the proton gradient; ATP synthase uses the gradient.

14. Shuttles That Transfer Cytoplasmic NADH Electrons

• The inner mitochondrial membrane is impermeable to NADH and NAD⁺.

• Electrons from cytoplasmic NADH must be transferred into mitochondria through shuttle systems.

• The ETC regenerates NAD⁺, which is needed for glycolysis to continue.

Glycerol 3-phosphate shuttle

• Transfers electrons from cytoplasmic NADH into the ETC.

• Cytoplasmic NADH reduces dihydroxyacetone phosphate (DHAP) to glycerol 3-phosphate.

• Glycerol 3-phosphate is reoxidized by a mitochondrial, membrane-associated enzyme.

• Electrons are transferred to FAD, forming FADH₂.

• FADH₂ transfers electrons to coenzyme Q, forming QH₂.

• Electrons enter the ETC at the Q pool, bypassing Complex I.

• Produces approximately 1.5 ATP per cytoplasmic NADH according to the lecture.

• Allows rapid oxidative phosphorylation at the cost of energetic efficiency.

Malate–aspartate shuttle

• Transfers electrons from cytoplasmic NADH into mitochondria.

• Produces mitochondrial NADH.

• Prominent in the heart and liver, according to the lecture.

• Uses two membrane carriers and four enzymes.

Main steps:

1. Cytoplasmic NADH transfers electrons to oxaloacetate, forming malate.

2. Malate enters the mitochondrial matrix in exchange for α-ketoglutarate.

3. Malate is oxidized to oxaloacetate, producing mitochondrial NADH.

4. Oxaloacetate is converted to aspartate through a transamination reaction.

5. Aspartate and α-ketoglutarate participate in transport back to the cytoplasm.

6. Oxaloacetate is regenerated in the cytoplasm, allowing the cycle to continue.

Shuttle comparison

15. ATP-ADP Translocase and Phosphate Transport

• ATP-ADP translocase (ANT) = transporter that exchanges cytoplasmic ADP for mitochondrial ATP across the inner mitochondrial membrane.

• ADP enters the matrix so it can be used to synthesize ATP.

• ATP exits the matrix so the cell can use it.

• ATP and ADP bind to the transporter without Mg²⁺, according to the lecture.

• Inhibition of ANT inhibits cellular respiration because ATP export and ADP import are disrupted.

• A phosphate carrier also transports phosphate into the matrix for ATP synthesis.

Key concept: ATP synthase depends on coordinated function of the ETC, ATP-ADP translocase, and phosphate carrier.

Learning Objective 4: Regulation of Cellular Respiration

16. What Determines the Rate of Cellular Respiration?

• The need for ATP is the primary determinant of the rate of cellular respiration.

• The availability of ADP controls the rate of oxidative phosphorylation.

• When ATP is being used, ADP levels increase, stimulating ATP synthesis and electron transport.

• When ATP demand is low, ADP levels decrease, slowing oxidative phosphorylation.

Respiratory control (acceptor control)

• Respiratory control = regulation of the rate of oxidative phosphorylation by the availability of ADP.

• The ETC does not sustain a high rate of electron flow unless ADP is available to be converted into ATP.

When ADP levels are high

1. ATP synthesis increases.

2. Protons flow back into the matrix through ATP synthase.

3. The proton gradient is used more rapidly.

4. Electron transport and proton pumping increase.

5. NADH and FADH₂ are oxidized more rapidly.

6. NAD⁺ and FAD become available for additional metabolic reactions.

When ADP levels are low

1. ATP synthesis slows.

2. Protons accumulate in the intermembrane space.

3. The proton-motive force becomes more difficult to maintain through additional pumping.

4. Electron transport slows.

5. NADH and FADH₂ are consumed more slowly.

6. The citric acid cycle slows because less NAD⁺ and FAD are available.

High-yield concept: ATP demand → ADP availability → rate of oxidative phosphorylation → rate of cellular respiration.

17. ATP Yield from Complete Glucose Oxidation

According to the lecture:

• Approximately 30 ATP are formed when glucose is completely oxidized to CO₂.

• 26 ATP are formed through oxidative phosphorylation.

• 2 ATP are formed through the citric acid cycle, as listed in the lecture.

• 2 ATP are formed through glycolysis.

18. Uncoupling Oxidative Phosphorylation

• Uncoupling = separating electron transport from ATP synthesis by allowing protons to return to the mitochondrial matrix without passing through ATP synthase.

• Electron transport may continue, but less ATP is produced.

• The energy stored in the proton gradient is released as heat instead of being captured as ATP.

Uncoupling protein 1 (UCP-1)

• Also called thermogenin.

• Located in the inner mitochondrial membrane.

• Allows protons to flow from the intermembrane space back into the matrix without ATP synthesis.

• Helps generate heat in brown adipose tissue.

• Functions in nonshivering thermogenesis, which generates heat without muscular shivering.

• Activated in response to a drop in core body temperature.

Other uncoupling proteins

• UCP-2: Found in a wide variety of tissues.

• UCP-3: Found in skeletal muscle and brown fat.

• The lecture notes that UCP-2 and UCP-3 may contribute to energy homeostasis.

Chemical uncouplers

• 2,4-Dinitrophenol (DNP): Carries protons across the inner mitochondrial membrane, dissipating the proton-motive force.

• When the proton gradient is dissipated, ATP synthesis is reduced even if electron transport continues.

• Xanthohumol is described in the lecture as a potential mild uncoupler that also scavenges free radicals.

Key distinction: Uncoupling releases energy as heat rather than efficiently capturing it as ATP.

19. Ways Oxidative Phosphorylation Can Be Inhibited

The lecture identifies four general ways to inhibit oxidative phosphorylation:

1. Inhibition of the ETC: Prevents or reduces electron transfer and proton-motive force generation.

2. Inhibition of ATP synthase: Prevents efficient ATP synthesis through the proton gradient.

3. Uncoupling: Dissipates the proton gradient, separating electron transport from ATP synthesis.

4. Inhibition of ATP export: Prevents ATP from leaving the mitochondria and interferes with ADP import.

Effects of inhibition

• ETC inhibition reduces electron flow and proton pumping.

• ATP synthase inhibition prevents normal use of the proton gradient.

• Uncoupling reduces ATP production by dissipating the proton gradient.

• ATP-ADP translocase inhibition interferes with the exchange of mitochondrial ATP and cytoplasmic ADP.