Unit 5: Metabolism II

Metabolism II covers the oxidative core of aerobic energy production and the pathways that feed and drain it. Once glycolysis and pyruvate oxidation deliver acetyl-CoA, the citric acid cycle harvests high-energy electrons, the electron transport chain converts them into a proton gradient, and oxidative phosphorylation cashes that gradient into ATP. Lipid pathways both supply acetyl-CoA (β-oxidation) and consume it (biosynthesis, ketogenesis).

I. Orientation: The Central Oxidative Framework

Aerobic catabolism (localized largely in the mitochondrion) converts fuel carbon to CO₂ while trapping released energy in reduced coenzymes and ATP.

• Acetyl-CoA as hub: the two-carbon acetyl unit is the common entry point from carbohydrate, fat, and protein catabolism.
• Electron carriers: NAD⁺ → NADH and FAD → FADH₂ store electrons; each NADH yields ~2.5 ATP and each FADH₂ ~1.5 ATP.
• Compartmentation: citric acid cycle and ETC sit in the mitochondrial matrix and inner membrane; fatty acid synthesis is cytosolic.
• Chemiosmotic coupling: electron flow pumps protons, creating a proton-motive force (Δp) that drives ATP synthase.
• Regulation logic: high ATP/NADH ratios inhibit; high ADP/NAD⁺ ratios stimulate. Energy charge governs flux.

II. The Citric Acid Cycle

Oxidation of acetyl-CoA to CO₂

The cycle (Krebs, 1937) oxidizes one acetyl group per turn, regenerating oxaloacetate so the cycle is catalytic in its intermediates.

A. Reaction Sequence and Enzymes

Eight steps convert oxaloacetate (4C) + acetyl-CoA (2C) back to oxaloacetate.

• Citrate synthase: condenses acetyl-CoA + oxaloacetate → citrate (6C); irreversible, committed step.
• Aconitase: citrate ⇌ isocitrate via cis-aconitate (isomerization).
• Isocitrate dehydrogenase: isocitrate → α-ketoglutarate + CO₂ + NADH; rate-limiting, first oxidative decarboxylation.
• α-Ketoglutarate dehydrogenase: → succinyl-CoA + CO₂ + NADH; second decarboxylation.
• Succinyl-CoA synthetase: substrate-level phosphorylation, → succinate + GTP.
• Succinate dehydrogenase: succinate → fumarate + FADH₂; membrane-bound (Complex II).
• Fumarase: fumarate + H₂O → malate.
• Malate dehydrogenase: malate → oxaloacetate + NADH.

B. Energy Yield

Each turn tallies fixed products.

• Per acetyl-CoA: 3 NADH, 1 FADH₂, 1 GTP, 2 CO₂.
• ATP equivalent: ~10 ATP per acetyl group after ETC processing.

Acetyl-CoA + 3NAD⁺ + FAD + GDP + Pi + 2H₂O
  → 2CO₂ + 3NADH + FADH₂ + GTP + 2H⁺ + CoA

C. Regulation and Anaplerosis

Flux matches energy demand and intermediate supply.

• Allosteric control: ATP, NADH inhibit citrate synthase, isocitrate dehydrogenase, α-ketoglutarate dehydrogenase; ADP, Ca²⁺ activate.
• Amphibolic role: intermediates feed biosynthesis (α-ketoglutarate → glutamate; oxaloacetate → aspartate; succinyl-CoA → heme).
• Anaplerotic refill: pyruvate carboxylase (pyruvate + CO₂ → oxaloacetate) replenishes drained oxaloacetate.

III. The Glyoxylate Cycle

Net synthesis of carbohydrate from acetyl-CoA

In plants, fungi, and bacteria, this variant bypasses the two decarboxylation steps, conserving carbon so two acetyl units become one four-carbon unit for gluconeogenesis.

A. Bypass Reactions

Two glyoxysomal enzymes replace the CO₂-losing steps.

• Isocitrate lyase: isocitrate → succinate + glyoxylate (splits 6C into 4C + 2C).
• Malate synthase: glyoxylate + acetyl-CoA → malate (adds a second acetyl unit).
• Shared steps: citrate synthase, aconitase, malate dehydrogenase run as in the standard cycle.

B. Significance and Comparison

The pathway enables growth on two-carbon substrates (acetate, fatty acids).

1. Citric acid cycle: loses 2 CO₂, releases NADH/FADH₂, cannot net-synthesize sugar from acetyl-CoA.
2. Glyoxylate cycle: conserves both carbons of acetyl-CoA, yields succinate exported for gluconeogenesis; absent in mammals, so they cannot convert fat to glucose net.

IV. The Electron Transport Chain

Sequential redox carriers on the inner membrane

Four complexes pass electrons from NADH/FADH₂ down a redox gradient to O₂, releasing free energy used to pump protons into the intermembrane space.

A. Complexes and Mobile Carriers

Electrons flow toward increasing reduction potential.

• Complex I (NADH-Q oxidoreductase): NADH → ubiquinone; pumps 4 H⁺; contains FMN and Fe-S clusters.
• Complex II (succinate dehydrogenase): FADH₂ → ubiquinone; no proton pumping.
• Ubiquinone (Q): lipid-soluble mobile carrier ferrying electrons to Complex III.
• Complex III (cytochrome bc₁): Q → cytochrome c; Q-cycle pumps 4 H⁺.
• Cytochrome c: peripheral mobile carrier on the outer face of the inner membrane.
• Complex IV (cytochrome c oxidase): cytochrome c → O₂ → H₂O; pumps 2 H⁺; contains Cu centers.

B. Inhibitors and Electron Flow

Blocking any point halts downstream carriers and upstream reduction.

• Rotenone/amytal: block Complex I.
• Antimycin A: blocks Complex III.
• Cyanide, CO, azide: block Complex IV, the terminal step.

NADH → I → Q → III → cyt c → IV → O₂
       FADH₂ → II ↗

V. Oxidative Phosphorylation

Converting the proton gradient into ATP

The proton-motive force generated by the ETC drives ATP synthase, coupling oxidation of fuel to phosphorylation of ADP (Mitchell's chemiosmotic theory, 1961).

A. Chemiosmotic Mechanism

The gradient has chemical and electrical components.

• Proton-motive force (Δp): Δp = ΔΨ − (2.303RT/F)ΔpH; ΔΨ is membrane potential, ΔpH the concentration gradient.
• ATP synthase (Complex V): F₀ channels protons back to the matrix; F₁ (β subunits) catalyzes ADP + Pi → ATP.
• Binding-change mechanism: proton flow rotates the γ subunit, cycling β subunits through loose, tight, and open states.

B. Coupling and Yield

Electron flow and ATP synthesis are obligatorily linked in intact mitochondria.

• P/O ratio: ~2.5 ATP per NADH, ~1.5 per FADH₂.
• Uncouplers: 2,4-dinitrophenol and thermogenin dissipate the gradient, releasing energy as heat without ATP.
• Respiratory control: ADP availability limits O₂ consumption; ATP synthesis and oxidation rise together.

VI. Fatty Acid Oxidation

Mitochondrial degradation of fatty acids to acetyl-CoA

β-Oxidation removes two carbons at a time from a fatty acyl chain, each cycle producing acetyl-CoA plus reduced coenzymes.

A. Activation and Transport

Fatty acids must be primed and shuttled.

• Activation: acyl-CoA synthetase attaches CoA, consuming 2 ATP equivalents (ATP → AMP + PPi).
• Carnitine shuttle: CPT-I converts acyl-CoA to acylcarnitine to cross the inner membrane; CPT-II regenerates acyl-CoA in the matrix. Malonyl-CoA inhibits CPT-I, blocking oxidation during synthesis.

B. The β-Oxidation Spiral

Each round has four steps yielding energy carriers.

• Oxidation 1: acyl-CoA dehydrogenase → trans-enoyl-CoA + FADH₂.
• Hydration: enoyl-CoA hydratase adds water.
• Oxidation 2: hydroxyacyl-CoA dehydrogenase → ketoacyl-CoA + NADH.
• Thiolysis: thiolase cleaves acetyl-CoA, leaving a chain shorter by two carbons.
• Palmitate (C16) example: 7 cycles → 8 acetyl-CoA, 7 FADH₂, 7 NADH; net ~106 ATP.

C. Special Cases

Odd and unsaturated chains need extra enzymes.

• Odd-chain: final propionyl-CoA → succinyl-CoA (requires B₁₂), entering the citric acid cycle.
• Unsaturated: isomerase and reductase reposition or reduce double bonds.

VII. Fatty Acid Biosynthesis

Cytosolic assembly of palmitate from acetyl-CoA

Synthesis is not the reverse of oxidation: it occurs in the cytosol, uses malonyl-CoA and NADPH, and builds chains on fatty acid synthase.

A. Initiation and the Committed Step

Acetyl-CoA is first exported and carboxylated.

• Citrate shuttle: citrate carries acetyl units to cytosol, regenerating acetyl-CoA.
• Acetyl-CoA carboxylase: acetyl-CoA + CO₂ → malonyl-CoA (biotin, ATP); rate-limiting, activated by citrate, inhibited by palmitoyl-CoA.

B. Fatty Acid Synthase Cycle

A multienzyme complex elongates the chain by two carbons per round.

• Steps: condensation, reduction (NADPH), dehydration, reduction (NADPH) on the acyl carrier protein (ACP).
• NADPH source: pentose phosphate pathway and malic enzyme.
• Product: palmitate (C16); each two-carbon addition consumes 1 malonyl-CoA and 2 NADPH.

C. Contrast With Oxidation

The pathways are reciprocally regulated.

1. Oxidation: mitochondrial, CoA carrier, FAD/NAD⁺, releases acetyl-CoA.
2. Synthesis: cytosolic, ACP carrier, NADPH, uses malonyl-CoA; malonyl-CoA simultaneously blocks CPT-I to prevent futile cycling.

VIII. Ketone Bodies

Water-soluble acetyl-CoA transport during fuel scarcity

When acetyl-CoA exceeds oxaloacetate (fasting, diabetes), the liver converts excess to ketone bodies exported for extrahepatic fuel.

A. Ketogenesis

Three products form in liver mitochondria.

• Pathway: 2 acetyl-CoA → acetoacetyl-CoA → HMG-CoA → acetoacetate; reduced to β-hydroxybutyrate, or spontaneously decarboxylated to acetone.
• Key enzyme: HMG-CoA synthase (mitochondrial, ketogenic).

B. Utilization and Significance

Peripheral tissues reconvert ketones to acetyl-CoA.

• Uptake: thiophorase (succinyl-CoA transferase) activates acetoacetate; absent in liver, so liver cannot use its own product.
• Fuel role: brain adapts to use ketones during prolonged starvation, sparing glucose.
• Ketoacidosis: overproduction lowers blood pH, a hallmark of untreated type 1 diabetes.