Formation of Acetyl CoA, Krebs Cycle, and Electron Transport System

Formation of Acetyl CoA

• The formation of Acetyl CoA is also known as the oxidative decarboxylation of Pyruvate.

• This process involves a complex enzyme and requires at least five essential cofactors:
    - $Mg$ ions ($Mg^{++}$)
    - Thiamine pyrophosphate (TPP)
    - $NAD^+$
    - Coenzyme A (CoA)
    - Lipoic acid

• The steps of oxidative decarboxylation are summarized as follows:
    1. $Pyruvic \, acid + TPP \rightarrow TPP \, complex + CO_{2}$
    2. $TPP \, complex + Lipoic \, acid \, (oxidised \, form) \rightarrow Acetyl \, lipoic \, acid \, complex + TPP$
    3. $Acetyl \, lipoic \, acid \, complex + CoA \rightarrow Acetyl \, CoA + Lipoic \, acid \, (reduced \, form)$
    4. $Lipoic \, acid \, (reduced \, form) + NAD^{+} \rightarrow Lipoic \, acid \, (oxidised \, form) + NADH + H^{+} \, (NADH_{2})$

• Summary Equation:
    - $Pyruvate + CoA + NAD^{+} \xrightarrow{TPP} Acetyl \, CoA + CO_{2} + NADH + H^{+}$

• The decarboxylase enzyme and transacetylase enzyme systems facilitate the movement of the acetyl group to the lipoic acid and then to Coenzyme A ($HS.CoA$).

• This reaction acts as the connecting link between the EMP pathway (glycolysis) which occurs in the cytoplasm, and the Krebs cycle which occurs in the mitochondria.

The Krebs Cycle

• Named after H.A. Kreb, the cycle is also known by several other names:
    - Tricarboxylic acid cycle (TCA cycle)
    - Organic acid cycle
    - Mitochondrial respiration
    - Oxidation of pyruvate
    - Citric acid cycle
• Location: The cycle takes place within the mitochondrial matrix.

Important Steps of the Krebs Cycle

• 1. Formation of Citric Acid:
    - Acetyl CoA (a 2-carbon compound) reacts with Oxaloacetic acid (a 4-carbon compound) and one molecule of water.
    - Enzyme: Citric acid synthetase.
    - Result: Formation of Citric acid (a 6-carbon compound) and the regeneration of Coenzyme A ($HS.CoA$).
    - $Oxaloacetic \, acid + Acetyl \, CoA \rightarrow Citric \, acid + HS.CoA$
    - CoA is released and becomes available again for the breakdown of Pyruvate. CoA has a controlling influence on the rate of respiration.
    - Anaplerotic Reaction: When TCA intermediates are diverted to other processes, Oxaloacetic acid is replenished by PEP carboxylase in the cytosol:
      - $Phosphoenol \, pyruvate + HCO_{3}^{-} \rightarrow Oxaloacetate + H_{2}PO_{4}^{-}$

• 2. Isomerization of Citric Acid:
    - Citric acid loses water to form Cis-aconitic acid.
    - Cis-aconitic acid takes back water to form Isocitric acid.
    - Enzyme: Aconitase (catalyzes both reactions).
    - $Citric \, acid \rightleftharpoons Cis\text{-}aconitic \, acid \rightleftharpoons Isocitric \, acid$

• 3. Dehydrogenation to Oxalosuccinic Acid:
    - Isocitric acid is dehydrogenated to Oxalosuccinic acid.
    - Enzyme: Isocitric acid dehydrogenase.
    - Cofactor: $Mg^{++}$.
    - Hydrogen is accepted by $NAD^{+}$ to produce $NADH + H^{+}$.
    - $Isocitric \, acid + NAD^{+} \xrightarrow{Mg^{++}} Oxalosuccinic \, acid + NADH + H^{+}$

• 4. Decarboxylation to $\alpha$-ketoglutaric Acid:
    - Oxalosuccinic acid undergoes decarboxylation.
    - Enzyme: Decarboxylase.
    - Cofactor: $Mg^{++}$.
    - Result: Production of $\alpha$-ketoglutaric acid (a 5-carbon compound) and one molecule of $CO_{2}$.
    - $Oxalosuccinic \, acid \rightarrow \alpha\text{-}ketoglutaric \, acid + CO_{2}$

• 5. Oxidation of $\alpha$-ketoglutaric Acid (Two Steps):
    - Step i: $\alpha$-ketoglutaric acid is converted into Succinyl CoA.
    - This requires cfactors: TPP, $Mg$, $NAD$, FAD, lipoic acid, and CoA.
    - This step is analogous to the oxidation of pyruvic acid to Acetyl CoA; one molecule of $NAD^{+}$ is reduced to $NADH$ and one $CO_{2}$ is liberated.
    - $\alpha\text{-}ketoglutaric \, acid + HS.CoA + NAD^{+} \rightarrow Succinyl \, CoA + NADH_{2} + CO_{2}$
    - Step ii: Succinyl CoA is hydrolyzed to Succinic acid using one molecule of $H_{2}O$.
    - Enzyme: Succinyl thiokinase (a phosphorylating enzyme).
    - This involves substrate-level phosphorylation: GDP reacts with inorganic phosphate ($P_{i}$) to form GTP.
    - $Succinyl \, CoA + GDP + P_{i} \rightarrow Succinic \, acid + GTP + HS.CoA$
    - GTP can then react with ADP to produce ATP: $GTP + ADP \rightleftharpoons GDP + ATP$.

• 6. Oxidation to Fumaric Acid:
    - Succinic acid is oxidised to Fumaric acid.
    - Enzyme: Succinic acid dehydrogenase.
    - This reaction uses FAD (Flavin adenine dinucleotide) instead of NAD. FAD accepts two hydrogen ions and two electrons to become $FADH_{2}$.
    - $Succinic \, acid + FAD \rightarrow Fumaric \, acid + FADH_{2}$

• 7. Hydration to Malic Acid:
    - Fumaric acid is converted to Malic acid with the addition of one molecule of water.
    - Enzyme: Fumarase.
    - $Fumaric \, acid + H_{2}O \rightarrow Malic \, acid$

• 8. Final Oxidation to Oxaloacetic Acid:
    - Malic acid is converted back into Oxaloacetic acid, completing the cycle.
    - Enzyme: Malic acid dehydrogenase.
    - Hydrogen is accepted by $NAD^{+}$ to form $NADH_{2}$.
    - $Malic \, acid + NAD^{+} \rightarrow Oxaloacetic \, acid + NADH_{2}$

Electron Transport System (ETS Chain)

• Also referred to as the Respiratory Chain or Oxidative Phosphorylation.
• Biological oxidation of glucose: $C_{6}H_{12}O_{6} + 6O_{2} \rightarrow 6H_{2}O + 6CO_{2} + 686 \, kcal$ ($\Delta F = -686 \, kcal$).
• Types of oxidation in respiration:
    1. Gain of molecular oxygen.
    2. Removal of hydrogen (dehydrogenation) - The most common type.
    3. Change in valency.
    4. Hydration followed by dehydrogenation.
• In dehydrogenation, a pair of hydrogen dissociates: $2H \rightarrow 2H^{+} + 2e^{-}$.
• Ultimate reduction of oxygen to water: $O_{2} + 4e^{-} \rightarrow 2(O_{2}^{-})$; $2(O_{2}^{-}) + 4H^{+} \rightarrow 2H_{2}O$. This requires 4 electrons and 4 protons per oxygen molecule.

Structure and Mechanism of the ETS

• Pairs of hydrogen ($2H^{+} + 2e^{-}$) are not combined directly with oxygen but transported through a series of enzymes called the respiratory chain.
• Enzymes are found primarily in the inner membrane (cristae) of the mitochondria.
• Forked Chain Arrangement:
    - Branch 1: Channels hydrogen from substrates like 3-Phosphoglyceraldehyde, Pyruvic acid, Isocitric acid, $\alpha$-ketoglutaric acid, and Malic acid.
    - These hydrogen pairs initially reduce $NAD^{+}$: $NAD^{+} + 2H^{+} + 2e^{-} \rightarrow NADH + H^{+}$.
    - Reduced NAD is then reoxidised by FAD (Flavoprotein).
    - Branch 2: Channels hydrogen from Succinate directly into the chain using the flavoprotein Succinate dehydrogenase (which uses FAD).
    - $Succinic \, acid + FAD \rightarrow Fumaric \, acid + FADH_{2}$.
• Communal Cytochrome Chain:
    - Both branches converge into a shared chain of cytochromes leading to molecular oxygen.
    - Cytochromes involved include cyt b, cyt c, cyt a, and cyt $a_{3}$.
    - At the cytochrome stage, electrons are passed one at a time through the heme prosthetic groups.
    - Ubiquinone (Ubox/Ubred) serves as an electron reservoir in higher plants.
    - Final acceptor: Oxygen.

Differences Between Oxidative and Photophosphorylation

ATP Yield and Energetics

• Pairs of electrons are released at six specific steps across Glycolysis and Krebs Cycle:
    - (i) Oxidation of 3-Phosphoglyceraldehyde (Glycolysis) - yields $2 \, ATP$.
    - (ii) Oxidative decarboxylation of Pyruvic acid - yields $3 \, ATP$.
    - (iii) Oxidation of Isocitric acid - yields $3 \, ATP$.
    - (iv) Oxidation of $\alpha$-ketoglutaric acid - yields $3 \, ATP$.
    - (v) Oxidation of Succinic acid (uses FAD) - yields $2 \, ATP$.
    - (vi) Oxidation of Malic acid - yields $3 \, ATP$.

• Calculation Per Krebs Cycle:
    - ATP from steps using NAD (ii, iii, iv, vi): $4 \times 3 \, ATP = 12 \, ATP$.
    - ATP from step using FAD (v): $1 \times 2 \, ATP = 2 \, ATP$.
    - Total ATP from ETS per cycle: $14 \, ATP$ (Note: The text states 16 total per cycle in the mitochondria, combining ETS from glycolysis and Krebs, simplified as $12 + 4 = 16$).
    - Substrate-level phosphorylation (GTP to ATP): $1 \, ATP$.
    - Total mitochondrial ATP per cycle: $16 + 1 = 17 \, ATP$.

• Total for One Glucose Molecule:
    - One glucose produces two triose molecules, requiring two complete cycles.
    - mitochondrial yield: $17 \times 2 = 34 \, ATP$.
    - Plus net gain from glycolysis transphosphorylation: $2 \, ATP$.
    - Grand Total for Eukaryotes: $34 + 2 = 36 \, ATP$.

• Overall Respiration Equation (Eukaryotes):
    - $C_{6}H_{12}O_{6} + 6O_{2} + 36ADP + 36H_{3}PO_{4} \rightarrow 6CO_{2} + 42H_{2}O + 36ATP$