ATP Synthesis PQs

What are the primary roles of Complexes I, III, and IV?

Complex I: Pumps 4 H⁺, oxidizes NADH → QH₂.

Complex III: Pumps 4 H⁺ (via Q cycle), passes e⁻ to cytochrome c.

Complex IV: Pumps 2 H⁺, reduces O₂ → H₂O.

How does inhibiting Complex IV (e.g., with CO/azide) affect the ETC?

Blocks all electron flow → components stay reduced → no proton pumping or ATP synthesis.

What two components make up the proton motive force?

ΔpH (H⁺ concentration gradient).

ΔΨ (charge gradient; positive IMS, negative matrix).

How does valinomycin experimentally prove PMF requires both ΔpH and ΔΨ?

ΔpH alone (no K⁺) → no ATP synthesis. Valinomycin adds ΔΨ (K⁺ movement) → ATP synthesis resumes.

Describe the structure and function of F₀ and F₁ subunits.

F₀: Membrane-embedded c-ring; rotates clockwise with H⁺ flow.

F₁: α₃β₃ matrix unit; rotates counterclockwise to synthesize ATP.

Why is ATP synthase called a "molecular motor"?

It couples H⁺ flow (F₀ rotation) to ATP synthesis (F₁ conformational changes).

How many H⁺ are needed per ATP? Why?

4 H⁺ total: 3 for ATP synthase rotation. 1 to import Pi (phosphate symport).

Compare the malate-aspartate and glycerol-3-phosphate shuttles.

Malate-Aspartate Glycerol-3-P ATP Yield: 2.5 ATP/NADH (Complex I) 1.5 ATP/NADH (Complex II)

Complexity: Multi-step (many enzymes) Simple (2 reactions)

Energy Loss: None Loses e⁻ to FADH₂

How do uncouplers (e.g., DNP, UCP1) work?

Bind H⁺ in IMS → diffuse to matrix → dissipate ΔΨ/ΔpH as heat.

Result: ETC runs freely (↑O₂ use), but no ATP made.

Why does adding an uncoupler restore O₂ consumption in oligomycin-treated mitochondria?

Oligomycin blocks H⁺ flow → ETC stalls; uncouplers collapse gradient → ETC resumes.

How does oligomycin affect pH and O₂ consumption?

Blocks ATP synthase → H⁺ gradient builds → ETC stops (low O₂ use). pH drops (H⁺ trapped in IMS).

What happens in an O₂ pulse experiment?

+O₂: ETC activates → pH drops (H⁺ pumped).

-O₂: pH slowly recovers (H⁺ leaks back).

How does PINK1 detect damaged mitochondria?

Healthy: PINK1 imported → degraded.

Depolarized: PINK1 accumulates on OMM → recruits Parkin → ubiquitination → mitophagy.

Why does cytochrome c release trigger apoptosis?

Forms apoptosome → activates caspases → programmed cell death.

How can mitochondrial dysfunction be targeted in cancer?

Leukemia: Damage mitochondria → kill stem cells (↓relapse).

Drugs: Inhibit ETC, force pore opening, activate mitophagy.