BIOSCI 101 Lecture 5

Real-World Anchors & Opening Anecdotes

• Citric acid (sold as food preservative and produced by “lime ants”) flavours drinks such as field-collected gin & tonics.
• Lecture sequence: Glycolysis (yesterday) ➜ Citric Acid Cycle (today) ➜ Electron-Transport & Oxidative Phosphorylation (tomorrow) ➜ ATP-synthase focus (next Tuesday).

Alternate Names & General Significance

• Citric Acid Cycle (CAC) = Tricarboxylic Acid Cycle (TCA) = “Krebs Cycle.”
– Hans Krebs also discovered three other cycles; naming ambiguity noted.
• Central hub for catabolism (energy harvest) & anabolism (biosynthesis).
• Key outputs per turn:
– 3\;\text{NADH}
– 1\;\text{FADH}2 – 1\;\text{GTP/ATP} (substrate-level phosphorylation) – 2\;\text{CO}2 (exhaled)
– Oxaloacetate regenerated.

Mitochondrial Architecture & Location

• Matrix = site of nearly every TCA enzyme.
• Double membrane resembles Gram-negative bacterium; evidence for endosymbiosis.
• Cristae = inner-membrane folds; once thought surface-area boosters, lecturer proposes they channel proton flow.
• Succinate dehydrogenase (Complex II) is exception: anchored in inner membrane but catalyses matrix-side chemistry.
• Crowded environment (visualised with free software that imports PDB files): ribosomes, mtDNA loops, DNA-shielding proteins, megadalton enzyme assemblies (e.g., pyruvate dehydrogenase), etc.

Endosymbiotic Theory – Why Keep a Bacterium?

• Initial benefits (competing hypotheses):
– Oxygen detoxification in an ancient, O₂-rising world (mitochondria house antioxidant enzymes, scavenge superoxide).
– Surplus ATP for host archaeon ➜ larger cell volume, motility, genome expansion.
• ATP abundance permits:
– Nucleotide-intensive DNA/RNA synthesis ➜ gene duplications ➜ innovation (e.g., multiple haemoglobins).
– Energy-hungry processes like muscle contraction or intracellular transport.

Historical Background: Hans Krebs

• Teenage WWI veteran; later barred from medicine by Nazi race laws.
• Escaped to Cambridge; devised urea cycle then CAC.
• Used Warburg manometer (measures CO₂ evolution) + radiolabelled acetate ( ^{14}\text{C}) to track carbon atoms.
• Logic: stimulatory intermediates + inhibitors → identify sequence by accumulation patterns (method revisited under “Poisons”).

Connecting Glycolysis to the Cycle – Pyruvate Oxidation

Pyruvate Dehydrogenase Complex (PDC / PDH)

• Megacomplex (≈ 10 MDa) composed of three catalytic units:
– E1 = Pyruvate dehydrogenase (TPP-dependent)
– E2 = Dihydrolipoyl trans-acetylase (lipoamide “swinging arm”)
– E3 = Dihydrolipoyl dehydrogenase (FAD & NAD⁺)
• Cofactors:
– Thiamine pyrophosphate (TPP) ← dietary vitamin B₁ (Vegemite > Marmite debate).
– Lipoic acid (redox lipoamide arm).
– FAD (prosthetic), NAD⁺ (mobile).
– Coenzyme A (AMP-pantetheine thiol; same adenine motif as ATP/NAD/FAD).
• Stepwise choreography (order often mis-drawn in textbooks):

  1. E1 decarboxylates pyruvate (TPP) → hydroxy-ethyl-TPP + \text{CO}_2.

  2. Acyl transfers to oxidised lipoamide on E2 → acetyl-dihydrolipoamide.

  3. CoA-SH attacks, forming acetyl-CoA (leaves).

  4. E3 re-oxidises lipoamide via FAD → FADH₂.

  5. FADH₂ passes electrons to NAD⁺ → \text{NADH}+H^+; FAD restored.
    • Global reaction:
    \text{Pyruvate}+\text{CoA-SH}+\text{NAD}^+ \;\xrightarrow{PDH}\; \text{Acetyl-CoA}+\text{CO}_2+\text{NADH}+H^+

The Eight Reactions of the Citric Acid Cycle

(4-C = oxaloacetate unless noted)

  1. Citrate synthase – Condensation: \text{Oxaloacetate}(4C)+\text{Acetyl-CoA}(2C) \rightarrow \text{Citrate}(6C)+\text{CoA-SH}.

  2. Aconitase – Isomerisation via cis-aconitate; uses Fe-S cluster.
    – Sensitive to fluoroacetate (1080 poison); WA marsupials evolved resistance due to endemic fluoroacetate plants.

  3. Isocitrate dehydrogenase – First oxidative decarboxylation → \alpha-ketoglutarate (5C) + \text{CO}_2+\text{NADH}; key regulatory point, Ca²⁺-activated.

  4. \alpha-Ketoglutarate dehydrogenase complex – Second oxidative decarboxylation → succinyl-CoA (4C) + \text{CO}_2+\text{NADH}; PDH-like, arsenite-sensitive.

  5. Succinyl-CoA synthetase ("thiokinase") – Substrate-level phosphorylation:
    \text{Succinyl-CoA}+\text{GDP(ADP)}+P_i\rightarrow \text{Succinate}+\text{GTP(ATP)}+\text{CoA-SH}.

  6. Succinate dehydrogenase (Complex II) – Oxidation: \text{Succinate}\rightarrow\text{Fumarate}; electrons go: Succinate → FADH₂ → Fe-S centres → heme → ubiquinone (Q) → \text{QH}_2; inhibited by malonate.

  7. Fumarase – Hydration: \text{Fumarate}+\text{H}_2\text{O}\rightarrow\text{Malate}.

  8. Malate dehydrogenase – Oxidation: \text{Malate}\rightarrow\text{Oxaloacetate}+\text{NADH} (endergonic but pulled forward by step 1).

Mnemonic: “Citrate Is Krebs’ Starting Substrate For Making Oxaloacetate.”

Energy & Stoichiometry Summary

Per acetyl-CoA turn:
• 3\;\text{NADH} \times (\approx 2.5\;\text{ATP each})
• 1\;\text{FADH}_2 \times (\approx 1.5\;\text{ATP})
• 1\;\text{GTP/ATP} (direct)
• Net ≈ 10\;\text{ATP} equivalents.
Full glucose (2 turns) ≈ 20\;\text{ATP} from TCA products alone (plus glycolysis & PDH yields).

Overall cycle reaction (per acetyl-CoA):
\text{Acetyl-CoA}+3\,\text{NAD}^++\text{FAD}+\text{GDP(ADP)}+Pi+2\,\text{H}2\text{O} \rightarrow 2\,\text{CO}2+3\,\text{NADH}+3\,H^++\text{FADH}2+\text{GTP(ATP)}+\text{CoA-SH}

Regulation Logic

• High-energy indicators (ATP, NADH, citrate, succinyl-CoA, acetyl-CoA) ↓ cycle.
• Low-energy indicators (ADP, AMP, NAD⁺, CoA-SH) ↑ cycle.
• Ca²⁺ (rise during muscle contraction) activates isocitrate DH & α-KG DH, boosting cardiac & skeletal ATP output.
• PDH additionally controlled by reversible phosphorylation (PDH kinase inactivates when ATP/NADH high).
• Accumulated NADH visually evident: rock-pool shrimp fluoresce blue under UV when hypoxic.

Anaplerotic & Cataplerotic Roles

• Intermediates siphoned off to build:
– Citrate → fatty acid & sterol synthesis.
– α-KG → glutamate → other amino acids & neurotransmitter pool.
– Succinyl-CoA → porphyrins & heme.
– Oxaloacetate/malate → gluconeogenesis or aspartate family AAs.
– Acetyl-CoA → acetylcholine (neurotransmitter).
• Cycle must be “refilled” (anaplerosis) by e.g., pyruvate carboxylase ((\text{Pyruvate}+\text{CO}2+ATP \rightarrow \text{Oxaloacetate}+ADP+Pi)).

Poisons & Experimental Inhibitors

• Fluoroacetate (1080) – converted to fluorocitrate; irreversibly inhibits aconitase; ecological anecdotes from NZ & Western Australia.
• Arsenite – binds lipoamide SH groups; stalls PDH & α-KG DH; “poison of princes,” cosmetic tonic historically.
• Malonate – competitive inhibitor of succinate DH.
• Krebs mapped cycle by measuring metabolite build-up after such blocks with Warburg manometer.

FAD/FADH₂ vs. NAD⁺/NADH

• FAD tightly bound (prosthetic); NAD mobile.
• FAD accepts either 1- or 2-electron transfers via alloxazine rings; essential when substrate redox potential not sufficient for NAD⁺ reduction (succinate case).
• Both nucleotides share an adenine-diphosphate “handle,” reinforcing common evolutionary origin.

Reverse / Synthetic TCA Concepts

• Many ancient microbes ran an ancestral reductive TCA: 2\,\text{CO}_2\rightarrow\text{acetyl-CoA} and biosynthetic precursors (possible pre-photosynthetic carbon fixation).
• 2022 Chinese study engineered hybrid photosynthesis: photo-energy + reversed TCA enzymes > higher CO₂-fixation efficiency than natural photosynthesis.

Upcoming Link – Electron Transport & ATP Synthase

• NADH/FADH₂ carry high-energy electrons to respiratory chain.
• Electrons travel through Complex I (NADH), II (FADH₂), III, IV ⇒ proton gradient ⇒ ATP synthase.
• Tomorrow: detailed mechanism of electron flow, proton pumping, chemiosmotic coupling.