Lecture 8 Exam (Oxidative Phosphorylation)

Mitochondrial structure (4 compartments)

Outer membrane, intermembrane space, inner membrane (folded into cristae), and matrix

Why does the inner mitochondrial membrane need special transporters?

It's impermeable to most charged/hydrophilic molecules (unlike the freely permeable outer membrane), so ADP, ATP, and other molecules require dedicated transport proteins

Where does the TCA cycle occur vs. where does oxidative phosphorylation occur?

TCA cycle: mitochondrial matrix. Oxidative phosphorylation: on and around the inner mitochondrial membrane (cristae folding maximizes surface area for this)

What does oxidative phosphorylation consist of?

The electron transport chain (ETC) + proton-motive force, working together

Why is oxidative phosphorylation so important quantitatively?

A 2000 cal/day diet needs ~83 kg of ATP, but the body holds only ~200-300g at once (<1% of daily need); oxidative phosphorylation provides the vast majority of ATP by recycling ADP (~300x/day per molecule)

ATP yield comparison: glycolysis/TCA vs. oxidative phosphorylation

Glycolysis and TCA cycle each net only 2 ATP per glucose; oxidative phosphorylation nets ~26 ATP per glucose — totaling ~30 ATP from complete glucose oxidation

Mitochondrial DNA (mtDNA) facts

Located in the matrix; believed (via endosymbiotic theory) to derive from ancient engulfed bacteria; contains 37 genes, all essential for mitochondrial function, with 13 encoding oxidative phosphorylation enzymes

The electron transport chain (ETC) — overall function

A series of membrane-bound protein complexes that accept electrons from NADH/FADH2 and use that energy to pump protons out of the matrix, ultimately reducing oxygen to water

The 4 ETC complexes

Complex I: NADH-Q reductase. Complex II: Succinate-Q reductase. Complex III: Q-cytochrome C reductase. Complex IV: Cytochrome C oxidase

What shuttles electrons between the complexes?

Ubiquinone (Q/QH2, a mobile lipid-soluble carrier) and cytochrome C (a mobile matrix-soluble protein)

Complex I — entry point and proton pumping

NADH is oxidized here; overall removes 6 protons from the matrix and pumps 4 protons into the intermembrane space per NADH

Complex II — entry point and proton pumping

FADH2 (from succinate dehydrogenase) enters here; NO protons are pumped into the intermembrane space, so less ATP is derived from FADH2 than NADH

Complex III — function

Transfers electrons from ubiquinol to cytochrome C via the Q-cycle; pumps 4 protons into the intermembrane space total and removes 2 more from the matrix into the Q-pool

Complex IV — function

Reduces oxygen to water (the reason we need to breathe); removes 8 protons from the matrix, but net effect pumps 4 protons into the intermembrane space

Total protons pumped and ATP yield per NADH vs FADH2

NADH: 8 protons pumped → ~2.5 ATP. FADH2: 4 protons pumped → ~1.5 ATP

Why does NADH yield more ATP than FADH2?

FADH2 enters at Complex II, skipping Complex I entirely, so it misses out on the 4 protons pumped there

Metabolic shuttles for cytoplasmic NADH

Since cytoplasmic NADH (from glycolysis) can't cross the inner membrane directly, the glycerol-3-phosphate shuttle and malate-aspartate shuttle transport those electrons into the matrix

Why is ADP considered the rate-limiting substrate of oxidative phosphorylation?

ATP synthesis depends on the exchange rate of ADP (in) and ATP (out) across the inner membrane via special transporters — this exchange rate limits how fast the whole process runs

Proton-motive force

The energetically favorable "downhill" flow of protons back across the inner membrane (from intermembrane space to matrix), which powers ADP phosphorylation to ATP

ATP synthase (Complex V)

A large membrane-bound protein complex that controls proton flow back into the matrix and uses that energy to synthesize ATP from ADP + Pi

ATP synthase subunit conformations

Beta subunits cycle through Open (O – releases ATP), Loose (L – binds ADP + Pi), and Tight (T – forms ATP) conformations; no two beta subunits share the same conformation at once

How does proton flow generate ATP mechanically?

Protons passing through the a-subunit rotate the c-ring, which rotates the gamma-subunit, driving conformational changes in the beta-subunit hexamer ring

ATP yield per rotation of ATP synthase

Each 120° rotation of the beta-subunit hexamer produces 1 ATP; a full 360° rotation produces 3 ATP

Protons needed per ATP synthase rotation (vertebrates)

The c-ring has 8 subunits, so 8 protons must pass through per 360° rotation to make 3 ATP (~2.6-2.7 protons per ATP, rounded to ~2.5 protons per ATP)

Complex I inhibitors

Rotenone (fish/insect poison) and amytal (barbiturate); block NADH utilization but don't impair FADH2-derived electron flow (which enters via the Q-pool, beyond Complex I)

Link between rotenone and disease

Rotenone exposure, combined with genetic predisposition, has been implicated in Parkinson disease

Complex III inhibitor

Antimycin A (an antibiotic from Streptomyces, used as a fish poison)

Complex IV inhibitors

Cyanide (CN-), azide (N3-), and carbon monoxide (CO)

Why does inhibiting any ETC complex also stop ATP synthesis?

Blocking electron flow prevents the proton-motive force from being generated, so ATP synthase has no driving force

2,4-Dinitrophenol (DNP) — mechanism

An uncoupling agent that carries protons across the inner membrane down their gradient, bypassing ATP synthase

Effect of DNP on electron transport and ATP production

Electron transport (NADH oxidation, O2 consumption) proceeds normally, but the proton gradient is continuously dissipated so ATP synthase makes no ATP — the energy is released as heat instead