L4: Kreb cycle and Electron Transport Chain

Describe Pyruvate

• End product of glycolysis

• A simple alpha-keto acid (3C)

• Contains 2 reactive groups: Ketone group (C=O) and Carboxyl group (-COOH)

  • Each group can be further reduced or acted upon to form complex molecules and polymers.

Draw the structure of pyruvate

What happens to pyruvate after glycolysis under aerobic conditions?

Oxidative decarboxylation

• After glycolysis, pyruvate (3C) transported via transport protein into mitochondria

• In mitochondria, pyruvate dehydrogenase requires coenzyme A to convert pyruvate to Acetyl CoA (2C)

• CO2 is produced and NAD+ is reduced to NADH

• Acetyl CoA enters Krebs cycle

  Note: 2x pyruvate → 2NADH = 2x3ATP (ETC) decarboxylation because pyruvate (3C) is converted to acetyl CoA (2C) and lost 1C to form CO2

What is the full name of acetyl CoA?

Acetyl Coenzyme A

What is Pi?

Inorganic phosphate

What is GTP?

Guanosine Triphosphate

What is ATP?

Adenosine Triphosphate

What is the Krebs cycle also known as?

Tricarboxylic acid (TCA) cycle or Citric acid cycle

Describe the process of Krebs cycle. Draw out the entire Krebs cycle with the structures of each step.

1. Acetyl CoA (2C) combines with oxaloacetate (4C) to form citrate/citric acid (6C)

2. Citrate/ Citric acid (6C) loses 1 H2O and gains another, as citrate is converted into its isomer isocitrate (6C) by aconitase

3. Isocitrate (6C) is oxidized to produce alpha-ketoglutarate (5C) + 1 CO2 + 2 electrons. Electrons then reduce NAD+ to NADH

4. alpha-ketoglutarate (5C) is oxidized to produced succinyl CoA (4C) + CO2 + 2 electrons by alpha-ketoglutarate dehydrogenase

   Electrons reduces NAD+ to NADH

5. Succinyl CoA (4C) is converted to succinate (4C) by succinyl CoA synthase. A phosphate group is substituted for Coenzyme A → a high energy bond is formed. This energy is used in substrate level phosphorylation to form GTP or ATP

6. Succinate (4C) is converted to fumarate (4C) by succinate dehydrogenase (dehydration process). 2H transferred to FAD to produce FADH₂. Unlike NADH, FADH₂ remains attached to the succinate dehydrogenase, electrons of FADH₂ is transported directly to electron transport chain while attached to the enzyme. The energy contained in the electrons of these atoms is insufficient to reduce NAD+ but adequate to reduce FAD.

7. Fumarate (4C) is converted to Malate (4C) by fumarase with the addition of water.

8. Malate (4C) is oxidized to produce oxaloacetate by malate dehydrogenase. NAD+ is converted to NADH. Oxaloacetate formed can then bind with Acetyl CoA again to form citrate, cycle repeats.

What is oxidative decarboxylation? Which steps in the Krebs cycle are oxidative decarboxylation steps? Why?

Oxidative decarboxylation → Molecules are oxidized and loses a carbon

Steps that remove carbon

Step 3: Isocitrate is oxidized to form alpha-ketoglutarate

Step 4: alpha-ketoglutarate is oxidized to form succinyl CoA.

These steps release electrons that reduce NAD+ to NADH and release carboxyl groups that form CO2 molecules.

Which step in the Krebs cycle is a substrate-level phosphorylation step?

Succinyl CoA is converted to Succinate (GDP to GTP)

In summary what are we doing from Step 1 - 4, Step 5 - 9 of the Kreb cycle? (What is the purpose of the Kreb cycle)

From step 1 - Step 4, we completely oxidize Acetyl CoA (2C) to succinyl CoA (4C)

From step 5 - step 9, we convert succinyl CoA back into Oxaloacetate

• To produce energy

• Completely oxidize Acetyl CoA molecule

What intermediates for anabolism are formed during the TCA cycle? What molecules can they be used to form? (OCAS)

Each intermediate contains reactive groups

1. Oxaloacetate → Aspartate → Other amino acids, purines, pyrimidines

2. Citrate → Fatty acids (occurs in cytosol), sterols

3. Alpha-ketoglutarate → Glutamate → Other amino acids → Purines

4. Succinyl CoA → Porphyrins, Heme, Chlorophyll

Summarize the products of the Kreb cycle

• 4-carbon Anabolic intermediates

• 2 molecules of CO2

• Energy in form of GTP, NADH, FADH2

For every Acetyl CoA metabolized:

• 1 GTP = 1 ATP

• 3 NADH = 9 ATP

• 1 FADH2 = 2 ATP

Total 12 ATP produced

Complete oxidation of 1 molecule of glucose → 24 ATP (2 pyruvate → 2 acetyl CoA) from TCA cycle

What is the total energy yield under Aerobic conditions?

Oxidative decarboxylation of pyruvate: Each Pyruvate molecule is converted to Acetyl-CoA, producing 1 NADH per pyruvate
Hence 2NADH produced = 6ATP

What is the total energy yield under Anaerobic conditions?

NADH molecule comes from glycolysis (2NADH required for fermentation, 8ATP-6ATP = 2ATP)

• No ATP is produced during fermentation

• Overall 2 ATPs produced from glycolysis/substrate level phosphorylation

What happens to lactate/lactic acid after anaerobic respiration? What is this cycle known as?

Cori Cycle

1. Under oxygen-limiting conditions, pyruvate is converted to lactic acid during fermentation

2. Accumulation of lactic acid causes muscle aches

3. Lactic acid is transported out of muscle cells and carried by blood to the liver

4. In the liver, lactate is converted back to pyruvate

5. Pyruvate converted back to glucose via gluconeogenesis and is released back to the blood stream to be taken up by the muscles and other tissues

What is the mitochondria comprised of and how do they help in Electron Transport Chain (ETC) / Respiratory chain?

• Proteins (electron carriers and proton pumps) found on inner membrane of mitochondria

• ADP-ATP translocase (Transport proteins) → Transports ADP and Pi into the matrix, Transports newly synthesized ATP out of the matrix

• Specific transporters → Carry pyruvate, fatty acids and amino acids into the matrix

• Cristae (folds of inner membrane) → Increases surface area to allow for more ETC for more efficient function

What is the function of electron transport chain?

• To reoxidize NADH/FADH₂ back to NAD+ and FAD (with oxygen being the final electron acceptor)

  • O2 is reduced to H2O

• To provide proton gradient for oxidative phosphorylation (involved in producing ATP)

• Movement of electrons from one complex to another results in pumping of H+ into the intermembrane space.

Where does the Electron Transport Chain get the NADH/FADH₂ from?

• Glycolysis: 2 NADH

• Oxidative decarboxylation of pyruvate: 1NADH (X2 per pyruvate)

  • Pyruvate → Acetyl CoA

• Krebs Cycle (6NADH + 2FADH₂)

What is oxidative phosphorylation?

Oxidative: Electron transfer from NADH / FADH2 to O2 via electron carrier proteins Phosphorylation: Synthesis of ATP (ADP + P → ATP), Dependent on O2 so its Oxidative

Occurs in mitochondria and is the process of ETC + ATP synthase

What is the Electron Transport Chain composed of?

1. Proton pumps

   • Complex I

   • Complex II

   • Complex IV

2. Electron carriers

   • Ubiquinone

   • Cytochrome C

   • Complex II

What is the function of ATP synthase?

Allows for movement of H+ ions from Intermembrane space into matrix through ATP synthase which drives ATP synthesis

How is ATP synthesized during oxidative phosphorylation?

In ETC:

1. NADH donates e- to Complex I (NADH dehydrogenase) to form NAD+

   FADH2 donates 2e- to Complex II to form FAD

2. Electrons move through complexes via Ubiquinone and Cytochrome C with oxygen being the terminal electron acceptor.

3. This allows for H+ to be pumped from the mitochondria matrix to the intermembrane space.

4. H+ cannot return to matrix as inner membrane is impermeable to H+.

5. Accumulation of H+ in intermembrane space results in formation of a proton gradient.

6. H+ can only move from the intermembrane space into mitochondrial matrix via ATP synthase along the proton gradient

7. Movement of H+ ions via ATP synthase drives ATP synthesis (ADP + Pi → ATP).

What are the differences between substrate level phosphorylation and oxidative phosphorylation?

What is the proton motive force?

Movement of protons back into the mitochondrial matrix.

ETC and ATP synthase are biochemically separate systems, linked only by a proton motive force.

What is the structure of Complex 2 (succinate dehydrogenase) and what does it do ?

• 2 transmembrane subunits (SDHC & SDHD)

  • Heme b & Ubiquinone is sandwiched between SDHC & SDHB

• 2 matrix subunits (SDHA & SDHB)

  • SDHA contains a bound FAD cofactor and is the binding site for succinate

  • SDHB has 3 Fe-S centers

Function → Forms a direct link with the Kreb cycle. To transfer electrons from succinate (in Kreb cycle) through complex II to Ubiquinone and then to complex III without pumping of H+

1. When succinate is converted to Fumarate in the Krebs cycle, electron pairs move from succinate to FAD cofactor, reducing FAD to FADH2

2. Electrons is transferred from FADH2 to 3 Fe-S centers.

3. Heme b prevents reactive oxygen species formation and does not participate in electron transfer

4. Electrons is transferred from Fe-S centers to coenzyme Ubiquinone, resulting in the formation of QH2

5. QH2 then transfers e- to complex III

What is a proton pump?

Electrons transfer through complexes I, III, IV via Cytochrome C and Ubiquinone with oxygen being final electron acceptor which allows for H+ to pump into intermembrane space from mitochondrial matrix

Electron transport in ETC and ATP synthesis by ATP Synthase are coupled by a _______ across the inner mitochondrial membrane.

proton (H⁺) gradient

Oxidation and phosphorylation are coupled by a ________.

Proton gradient