Oxidative Phosphorylation and ATP Synthesis: Mechanism and Regulation

Define Oxidative Phosphorylation and quantify the scale of ATP recycling it performs daily in humans.

Definition: ATP formed from electron transfer (NADH/FADH2 → O2) via inner mitochondrial membrane complexes. Scale: Humans recycle ~83 kg ATP daily with only 250 g stored; each ATP recycled ~300 times/day.

What is the sequence of energy conversion that drives ATP synthesis?

Electron motive force (NADH/FADH2) → Proton motive force (H+ gradient) → ATP synthesis via ATP synthase.

Where are the ETC and Krebs cycle enzymes located within mitochondria?

ETC and ATP synthase: inner membrane. Pyruvate dehydrogenase, Krebs cycle, β-oxidation: matrix.

Describe ubiquinone (CoQ) and its role.

Small, hydrophobic, diffuses in inner membrane. Accepts 1e- (semiquinone, QH*) or 2e- (ubiquinol, QH2). Transfers electrons between complexes.

Identify Complex I and its cofactors. What coupled processes does it perform?

Complex I = NADH-ubiquinone oxidoreductase. Cofactors: FMN, Fe-S centers. Couples NADH e- transfer to Q with pumping of 4 H+.

Name three inhibitors of Complex I.

Amital (barbiturate), Rotenone (insecticide), Piericidin A (antibiotic).

Identify Complex II and its role.

Succinate dehydrogenase. Unique Krebs cycle enzyme bound to membrane. Contains cytochrome b, Fe-S, FAD. No proton pumping.

What are the electron acceptors and proton pumping roles of Complex III and IV?

Complex III: transfers e- QH2 → cytochrome c, pumps protons. Complex IV: transfers e- cyt c → O2, pumps protons.

According to Mitchell’s chemiosmotic model, what drives ATP synthesis? Functions of Fo and F1?

Proton gradient drives ATP synthesis. Fo: membrane channel for H+. F1: matrix-facing catalytic subunits.

Describe γ subunit’s role in ATP synthase.

Acts as central rotor axis. Proton flow rotates Fo → γ rotates (120° steps), inducing conformational changes in β subunits.

What are the three conformations of β subunits and which releases ATP?

β-ADP: binds ADP+Pi. β-ATP: tight, ATP formed. β-empty: low affinity, releases ATP.

What is the ATP yield per NADH and FADH2?

NADH pumps 10 H+ → 2.5 ATP. FADH2 pumps 6 H+ → 1.5 ATP. (4 H+ per ATP, incl. transport).

Why are shuttle systems needed? Which tissues use malate-aspartate shuttle?

Inner membrane impermeable to NADH. Malate-Aspartate Shuttle (liver, kidney, heart) allows cytosolic NADH use.

Contrast Malate-Aspartate vs Glycerol 3-Phosphate Shuttle yields.

Malate-Asp: NADH equivalents enter as malate → mitochondrial NADH, yield 2.5 ATP. G3P Shuttle: transfers e- to Q (bypasses Complex I), yield 1.5 ATP.

What regulates oxidative phosphorylation and how is coupling shown?

Regulated by ADP availability. Coupling: blocking ATP synthase (e.g., oligomycin) halts electron transport (O2 consumption stops).

How does DNP uncouple mitochondria?

Hydrophobic weak acid. Carries protons across inner membrane, dissipating gradient → ATP synth blocked.

How is wasteful ATP hydrolysis by ATP synthase prevented in hypoxia?

Low pH activates IF1 inhibitor, which blocks ATP hydrolysis by ATP synthase.

What happens when electrons escape the ETC? Defense steps?

Escaped e- partially reduce O2 → superoxide (O2*-). Defense: SOD converts to H2O2; glutathione peroxidase detoxifies H2O2 using NADPH+GSH.

What is HIF1’s role during hypoxia?

Transcription factor activating protective responses: PDH inhibition, altered Complex IV isoform, reduces ROS production.