1. Harvesting Electrons from the Citric Acid Cycle
CHAPTER 19
Harvesting Electrons from the Citric Acid Cycle

2. Harvesting electrons from the citric acid cycle
Enzymes of the cycle are in the mitochondria of eukaryotes
Energy of the oxidation reactions is largely conserved as reducing power
(stored electrons)
Coenzymes reduced:
NAD NADH
FAD FADH2
The net effect of the cyclic pathway is the complete oxidation of an acetyl
group to 2 CO2 and the transfer of electrons to the above coenzymes.

3. Figure 19.1: An overview of the citric acid cycle
8 electrons are stored
2 carbon units are converted to CO2 and 1 ATP is made in the cycle

4. Figure 19.2: Harvested electrons will be used to drive a Proton Pump

5. 1. Citrate Synthase
Citrate is formed from the acetyl CoA and oxaloacetate
Methyl carbon from acetyl group reacts with
carbonyl of oxaloacetate
- This is the only cycle reaction with C-C bond formation

6. 1. Citrate Synthase The induced fit model revisited:
Side reaction could be the hydrolysis of acetyl CoA
A homodimeric protein
Oxaloacetate caused a major rearrangement that creates a binding
site for acetyl CoA reduces the possibility of side reactions

7. 2. Aconitase Aconitase catalyzes a near equilibrium reaction
Citrate is not a chiral molecule
Elimination of H2O from citrate to form C=C bond
of cis-aconitate
A stereospecific addition of H2O to cis-aconitate forms
2R,3S-isocitrate

8. 3. Isocitrate Dehydrogenase
Oxidative decarboxylation of isocitrate to a-ketoglutarate
(a metabolically irreversible reaction)
One of four oxidation-reduction reactions of the cycle
Hydride ion from the C-2 of isocitrate is transferred to NAD+ to
form NADH.
(unstable)

9. 4. The a-Ketoglutarate Dehydrogenase Complex
Complex is very large and is similar to the pyruvate
dehydrogenase complex
- E1 – a-ketoglutarate dehydrogenase (with TPP)
- E2 – succinyltransferase (with a flexible lipoamide prosthetic group)
- E3 – dihydrolipoamide dehydrogenase (with FAD)
Thiamine
2nd reducing
equivalence

10. Succinyl-CoA Synthetase The beginning of regenerating oxaloacetate
The high free energy stored in the thioester bond of succinyl CoA is conserved
as GTP (ATP equivalence):
Figure 13.8
Proposed mechanism of succinyl–CoA synthetase. Phosphate displaces CoA from a bound succinyl CoA molecule, forming the mixed acid anhydride succinyl phosphate as an intermediate. The phosphoryl group is then transferred from succinyl phosphate to a histidine residue of the enzyme to form a relatively stable covalent phosphoenzyme intermediate. Succinate is released, and the phosphoenzyme intermediate transfers its phosphoryl group to GDP (or ADP, depending on the organism), forming the nucleoside triphosphate product.

11. Succinyl-CoA Synthetase The beginning of regenerating oxaloacetate
The high free energy stored in the thioester bond of succinyl CoA is conserved
as GTP (or ATP)
The reaction takes place in four steps:
Figure 19.5
High energy thioester bond
Figure 13.8
Proposed mechanism of succinyl–CoA synthetase. Phosphate displaces CoA from a bound succinyl CoA molecule, forming the mixed acid anhydride succinyl phosphate as an intermediate. The phosphoryl group is then transferred from succinyl phosphate to a histidine residue of the enzyme to form a relatively stable covalent phosphoenzyme intermediate. Succinate is released, and the phosphoenzyme intermediate transfers its phosphoryl group to GDP (or ADP, depending on the organism), forming the nucleoside triphosphate product.

12. 6. The Succinate Dehydrogenase (SDH) Complex
The active site is made of two different subunits
One subunit contains 4 Fe-S cluster
The other contains covalently bound FAD
The SDH dimer is attached to the inner mitochondrial membrane via
two membrane polypeptides
- This is in contrast to other enzymes of the CA cycle which
are dissolved in the mitochondrial matrix
The transfer of two electrons occurs from succinate  FADH2  ubiquinone (Q),
a lipid soluble mobile carrier of electrons
- The reduced ubiquinone (QH2) is released as a mobile product
Succinate-Q reductase in the OP steps Q
QH2 + FAD

13. 7. Fumarase A near equilibrium trans addition of water
to the double bond of fumarate

14. 8. Malate Dehydrogenase
A near equilibrium inter-conversion that is dependent on NAD+
Oxidation of malate to oxaloacetate, with transfer of electrons
to NAD+ to form NADH.

15. Figure 19.6
The two carbons that
enter the cycle are NOT
the same to leave as CO2
FAD + QH2 Q

16. The overall net reaction can be simplified as follows:
The OH- is ultimately donated by a phosphate group
From the 8 electrons and 7 H+:
6 electrons are transferred to 3 NAD+ along with 3 H+
2 electrons are transferred to Q along with 2 H+
2 H+ are released per cycle.
The two CO2 molecules do NOT come directly from the acetyl group added to CoA

17. Reduced Coenzymes Fuel the Production of ATP
Each acetyl CoA entering the cycle nets:
1. 3 NADH
2. 1 QH2
3. 1 GTP (or 1 ATP)
Oxidation of each NADH yields 2.5 ATP
Oxidation of each QH2 yields 1.5 ATP
Complete oxidation of 1 acetyl CoA = 10 ATP
Oxidative
phosphorylation

18. Glucose degradation via glycolysis, citric acid cycle
and oxidative phosphorylation
Figure 13.10
ATP production from the catabolism of one molecule of glucose by glycolysis, the citric acid cycle, and reoxidation of NADH and QH2 The complete oxidation of glucose leads to the formation of up to 32 molecules of ATP.

19. Regulation of the citric acid cycle
Figure 19.7
Regulation of the citric acid cycle occurs mainly in two reactions:
1. isocitrate dehydrogenase (isocitrate  a-ketoglutarate)
2. a-ketogluterate dehydrogenase complex
(a-ketoglutarate  succinyl CoA)
ADP signifies the need for more energy
Slowing isocitrate dehydrogenase allows for buildup of citrate which also
Slows PFK and citrate can be also used for biosynthesis.

20. Figure 19.8 Routes leading to and from the CA cycle

21. Figure 19.9: Pyruvate carboxylase supplies
oxaloacetate for the citric acid cycle
When energy demands are high
oxaloacetate is diverted to gluconeogenesis
When energy demands are low
oxaloacetate is shuttled into citric acid cycle

22. The Glyoxylate Cycle
Pathway for the formation of glucose from noncarbohydrate precursors
in plants, bacteria and yeast (few animals)
Glyoxylate cycle leads from 2-carbon compounds to glucose
-- Glyoxylate (a 2 C compound) combines with the acetyl group
in acetyl CoA to generate malate leads to glucose formation
In most animals, acetyl CoA is NOT a carbon source for the net formation
of glucose (2 carbons of acetyl CoA enter CA cycle, 2 carbons are
released as 2 CO2
Glyoxylate cycle provides an anabolic alternative for acetyl CoA
metabolism (Allows for the formation of glucose from acetyl CoA)
This cycle is active in oily seed plants (Stored seed oils are converted to
carbohydrates during germination)
Germination is the process in which a plant or fungus emerges from a seed or spore, respectively, and begins growth

23. The glyoxylate cycle bypasses the two decarboxylation steps
Figure 19.10
The glyoxylate cycle bypasses
the two decarboxylation steps
of the CA cycle, conserving the
two carbon atoms as glyoxylate
for synthesis of glucose
Germinating seeds use this
Pathway to synthesize sugar
(glucose) from oil (triacylglycerols)
Figure 13.14
Glyoxylate pathway. Isocitrate lyase and malate synthase are the two enzymes of the pathway. When the pathway is functioning, the acetyl carbon atoms of acetyl CoA are converted to malate rather than oxidized to CO2. Malate can be converted to oxaloacetate which is a precursor in gluconeogenesis. The succinate produced in the cleavage of isocitrate is oxidized to oxaloacetate to replace the four-carbon compound consumed in glucose synthesis.

24. Assignment
Read Chapter 19
Read Chapter 20