Module 5 Review – Cellular Respiration & Metabolic Energy

Glycolysis, Fermentation & Need for $\text{NAD}^+$

• Step 6 of glycolysis: glyceraldehyde-3-phosphate (GAP) $\rightarrow$ 1,3-bisphosphoglycerate (1,3-BPG) by GAP dehydrogenase
– Requires oxidized coenzyme $\text{NAD}^+$ .
– If $\text{NAD}^+$ is not regenerated, glycolysis halts.
• Cells regenerate $\text{NAD}^+$ when oxidative phosphorylation is unavailable or O $2$ is limiting. Two major anaerobic routes: – Ethanol fermentation (yeast & some microbes) • Pyruvate $\xrightarrow[\text{TPP}]{\text{pyruvate decarboxylase}}$ acetaldehyde $+$ CO $2$
• Acetaldehyde $+$ $\text{NADH} \rightarrow$ ethanol $+$ $\text{NAD}^+$ (alcohol dehydrogenase)
– Lactic-acid fermentation (muscle, RBCs, some bacteria)
• Pyruvate $+$ $\text{NADH} \xrightarrow{\text{lactate dehydrogenase}}$ lactate $+$ $\text{NAD}^+$

Overview of Cellular Respiration

• Major compartments
– Cytosol: glycolysis, fermentation
– Mitochondrial matrix: pyruvate oxidation, citric acid cycle (CAC)
– Inner mitochondrial membrane (IMM): electron-transport chain (ETC), ATP synthase
• ATP‐forming mechanisms
– Substrate-level phosphorylation (glycolysis, CAC step 5)
– Oxidative phosphorylation (ETC + chemiosmosis)

Pyruvate Dehydrogenase Complex (PDC) – Bridge from Glycolysis to CAC

• Overall reaction (irreversible):
$\text{Pyruvate} + \text{CoA} + \text{NAD}^+ \;\rightarrow\; \text{Acetyl-CoA} + \text{CO}_2 + \text{NADH}$
• Three sequential steps per pyruvate

Decarboxylation
Oxidation
Transfer of acetyl group to CoA
• Multienzyme machine (E. coli stoichiometry in Table 18.1)
– E1 (Pyruvate dehydrogenase, 24 chains, TPP) – oxidative decarboxylation
– E2 (Dihydrolipoyl transacetylase, 24 chains, lipoamide) – acetyl transfer
– E3 (Dihydrolipoyl dehydrogenase, 12 chains, FAD) – lipoamide re-oxidation
• Five cofactors: TPP, lipoic acid, CoA, FAD, $\text{NAD}^+$
• Regulation
– Covalent: PDH kinase (inactivates by phosphorylation) vs PDH phosphatase (reactivates). A phosphatase defect $\Rightarrow$ pyruvate cannot $\rightarrow$ acetyl-CoA.
– Allosteric: inhibited by high $\text{ATP}$ , acetyl-CoA, $\text{NADH}$ ; activated by ADP, pyruvate, Ca $^{2+}$ (muscle)

Citric Acid / TCA / Krebs Cycle – Chemistry & Energetics

• 8 steps, catalytic oxaloacetate regenerated each turn. Key net: acetate (2 C) $\rightarrow$ 2 CO $_2$ + reducing power. Two turns per glucose.
• Step-by-step (Table 19.1, favorable steps bolded)

Condensation: acetyl-CoA + oxaloacetate + H $_2$ O $\rightarrow$ citrate + CoA-SH (citrate synthase, $\Delta G'^{\circ} \approx -31\,\text{kJ}$ )
Dehydration/Re-hydration (aconitase, Fe–S) – citrate $\rightarrow$ cis-aconitate $\rightarrow$ isocitrate (re-positions OH for later oxidation)
Oxidative decarboxylation: isocitrate + $\text{NAD}^+$ $\rightarrow$ $\alpha$ -ketoglutarate + CO $_2$ + NADH (isocitrate dehydrogenase)
Oxidative decarboxylation: $\alpha$ -ketoglutarate + CoA + $\text{NAD}^+$ $\rightarrow$ succinyl-CoA + CO $_2$ + NADH (multi-enzyme complex analogous to PDC)
Substrate-level phosphorylation: succinyl-CoA + P $_i$ + ADP/GDP $\rightarrow$ succinate + ATP/GTP + CoA-SH (succinyl-CoA synthetase; liver uses GDP, muscle ADP)
Oxidation: succinate + enzyme-bound FAD $\rightarrow$ fumarate + FADH $_2$ (succinate dehydrogenase, IMM-embedded; $\Delta G'^{\circ} \approx 0$ not enough to reduce $\text{NAD}^+$ )
Hydration: fumarate + H $_2$ O $\rightarrow$ L-malate (fumarase)
Oxidation: L-malate + $\text{NAD}^+$ $\rightarrow$ oxaloacetate + NADH + H $^+$ (malate dehydrogenase; endergonic but pulled by step 1)
• Yields per turn (per acetyl-CoA):
– $3\,\text{NADH}$ , $1\,\text{FADH}2$ , $1\,\text{ATP/GTP}$ , $2\,\text{CO}2$
• Biosynthetic links (Figure 19.1)
– Citrate $\rightarrow$ FA & sterols
– $\alpha$ -KG $\rightarrow$ glutamate $\rightarrow$ other AA, purines
– Succinyl-CoA $\rightarrow$ porphyrins/heme
– Oxaloacetate $\rightarrow$ aspartate $\rightarrow$ AA, purines/pyrimidines, or $\rightarrow$ PEP (gluconeogenesis)

Regulation of CAC & Anaplerosis

• Energy status:
– Inhibitors: high $\text{ATP}$ , $\text{NADH}$ , succinyl-CoA
– Activators: ADP, Ca $^{2+}$
• Rate-limiting enzymes: citrate synthase, isocitrate DH, $\alpha$ -KG DH.
• Oxaloacetate replenishment (anaplerotic)
– Mammals cannot net-convert acetyl-CoA $\rightarrow$ OAA; instead, pyruvate carboxylase (PC) adds CO $2$ : $\text{Pyruvate} + \text{CO}2 + \text{ATP} \xrightarrow{\text{PC}} \text{Oxaloacetate} + \text{ADP} + \text{P}_i$
– Favored when: low energy charge (high ADP/AMP), high acetyl-CoA (allosteric activator of PC)

Electron-Transport Chain (ETC)

• Carriers arranged by increasing standard reduction potential $E0'$ (electron affinity). – $E0' (\tfrac12 \text{O}2/\text{H}2\text{O}) = +0.82\,\text{V}$
– $E0' (\text{NAD}^+/\text{NADH}) = -0.32\,\text{V}$ • Free-energy driving force $\Delta G'^{\circ} = -nF\Delta E0'$
For NADH $\rightarrow$ O $2$ : $n = 2,\; \Delta E0' = 1.14\,\text{V} \Rightarrow \Delta G'^{\circ} \approx -220\,\text{kJ mol}^{-1}$
• Proton pumping stoichiometry
– Complex I: 4 H $^+$ /2e $^-$
– Complex III: 4 H $^+$ /2e $^-$
– Complex IV: 2 H $^+$ /2e $^-$
⇒ ~10 H $^+$ extruded per NADH; ~6 H $^+$ per FADH $_2$ (skips Complex I)
• Stepwise electron transfer avoids explosive heat release; energy captured as proton gradient instead of lost (slide 39 comparison)

Proton Motive Force (PMF) & ATP Synthase

• Mitchell equation: $\Delta p = \Delta \psi + \Delta \text{pH}$ (units mV)
– Electrical component $\Delta \psi$ ≈ 120-160 mV (matrix negative)
– Chemical component $\Delta\text{pH}$ ≈ 0.5-1.4 pH (60-80 mV)
– Total PMF ≈ 180-240 mV
• ATP synthase (F $0$ F $1$ )
– F $0$ C-ring rotates (~100 rev/s) as protons bind/deprotonate conserved Glu/Asp. – Central γ-shaft turns inside F $1$ catalytic α $3$ β $3$ knob causing conformational changes (binding → synthesis → release).
– ~3–4 H $^+$ per ATP synthesized (depends on c-ring subunit count)

Uncouplers – 2,4-Dinitrophenol (DNP) & Brown Fat

• DNP
– Lipid-soluble weak acid (pK $a$ ≈ 4.1) shuttles H $^+$ across IMM: 1. Picks up H $^+$ in acidic intermembrane space; 2. Diffuses into matrix; 3. Releases proton, dissipating PMF. – Consequences: ↓ ATP synthesis, ↑ ETC flux, ↑ O $2$ consumption, energy released as heat.
– Very narrow therapeutic window (rats): $\text{ED}{50} \approx 4\,\text{mg kg}^{-1}$ , $\text{LD}{50} \approx 50\,\text{mg kg}^{-1}$ . Human fatalities include hyperthermia (42 °C, slide 46).
• Natural uncoupling: Brown adipose tissue expresses UCP-1 (thermogenin). Fatty acids activate channel to generate heat in neonates & hibernators.
• Analogy: classroom with two doors; second door (uncoupler) diverts “proton traffic” away from ATP synthase.

Cellular Consequences of Uncoupling

• Low ATP $\Rightarrow$ feedback acceleration of glycolysis, PDC, CAC, ETC (cell attempts to restore ATP).
• Muscle contraction fails without ATP (cross-bridge cycle slide 52) → weakness, respiratory failure.
• Hyperthermia results from free-energy release as heat (first law energy fate #4).

NADH Shuttles (getting cytosolic NADH into mitochondria)

Malate–Aspartate Shuttle (heart, liver)
– Cytosolic OAA + NADH $\rightarrow$ malate + $\text{NAD}^+$
– Malate enters matrix, re-oxidized to OAA + NADH (full 2.5 ATP credit).
Glycerol-3-Phosphate Shuttle (muscle, brain)
– Cytosolic dihydroxyacetone-P + NADH $\rightarrow$ glycerol-3-P
– IMM FAD-enzyme re-oxidizes glycerol-3-P, passing electrons to Q (skip Complex I) ⇒ only ~1.5 ATP per NADH.

ATP Yield per Glucose (heart/liver example)

• Glycolysis: 2 ATP + 2 NADH (5 ATP)
• 2 Pyruvate → 2 Acetyl-CoA: 2 NADH (5 ATP)
• 2 CAC turns: 2 ATP, 6 NADH (15 ATP), 2 FADH $_2$ (3 ATP)
• Theoretical total: $2 + 5 + 5 + 2 + 15 + 3 = 32\,\text{ATP}$ (range 30-32 depending on shuttle & H $^+$ /ATP ratio)

Redox Terminology & Equations

• Half-reaction notation:
$\tfrac12 \text{O}2 + 2\,\text{H}^+ + 2\,e^- \;\leftrightarrow\; \text{H}2\text{O}$ (strong oxidant)
$\text{NAD}^+ + 2\,\text{H}^+ + 2\,e^- \;\leftrightarrow\; \text{NADH} + \text{H}^+$ (weak oxidant)
• Oxidant (electron acceptor), Reductant (donor). Oxidizing agent becomes reduced; reducing agent becomes oxidized.
• Positive $E0'$ = high e $^-$ affinity; negative $E0'$ = low affinity.

Summary – Five Main Stages of Respiration (slide 23)

Glycolysis (cytosol) – split glucose, form $\text{NADH}$ , small ATP.
Pyruvate Processing (matrix) – decarboxylate, generate $\text{NADH}$ , form acetyl-CoA.
Citric Acid Cycle – complete oxidation, generate most reducing power, small ATP.
Electron Transport Chain – pass electrons to O $_2$ , pump protons.
Oxidative Phosphorylation – PMF-driven ATP synthesis via ATP synthase.

All major biochemical energy conversions hinge on controlled electron flow, proton gradients, and tight regulation; perturbations (e.g.
uncouplers, enzyme deficiencies) have profound metabolic and physiological consequences.