Overview of Citric Acid Cycle

1. Overview of Citric Acid Cycle • The citric acid cycle operates under aerobic conditions only • The two-carbon acetyl group in acetyl CoA is oxidized to CO2 • It produces reduced coenzymes NADH and FADH2 and one ATP directly • In the citric acid cycle: - acetyl (2C) bonds to oxaloacetate (4C) to form citrate (6C) - oxidation and decarboxylation convert citrate to oxaloacetate - oxaloacetate bonds with another acetyl to repeat the cycle

3. Reaction 1: Formation of Citrate • Oxaloacetate combines with the two-carbon acetyl group to form citrate

4. Reaction 2: Isomerization to Isocitrate • Citrate isomerizes to isocitrate • The tertiary –OH group in citrate is converted to a secondary –OH that can be oxidized

5. Reaction 3: Oxidative Decarboxylation 1 • A decarboxylation removes a carbon as CO2 from isocitrate • The –OH group is oxidized to a ketone releasing H+ and 2e- that form reduced coenzyme NADH

6. Reaction 4: Oxidative Decarboxylation 2 • In a second decarboxylation, a carbon is removed as CO2 from -ketoglutarate • The 4-carbon compound bonds to coenzyme A providing H+ and 2e- to form NADH -Ketoglutarate

7. Reaction 5: Hydrolysis of Succinyl CoA • The hydrolysis of the thioester bond releases energy to add phosphate to GDP and form GTP, a high energy compound

8. Reaction 6: Dehydrogenation of Succinate • In this oxidation, two H are removed from succinate to form a double bond in fumarate • FAD is reduced to FADH2

9. Reaction 7: Hydration of Fumarate • Water is added to the double bond in fumarate to form malate

10. Reaction 8: Dehydration of Malate • Another oxidation forms a C=O double bond • The hydrogens from the oxidation form NADH + H+

11. Summary of Products from Citric Acid Cycle In one turn of the citric acid cycle: • Two decarboxylations remove two carbons as 2CO2 • Four oxidations provide hydrogen for 3NADH and one FADH2 • A direct phosphorylation forms GTP which is used to form ATP • Overall reaction of citric acid cycle: Acetyl CoA + 3NAD+ + FAD + GDP + Pi + 2H2O 2CO2 + 3NADH + 2H+ + FADH2 + HS-CoA + GTP

12. Regulation of the Citric Acid Cycle The citric acid cycle: • Increases its reaction rate when low levels of ATP or NAD+ activate isocitrate dehydrogenase to formation of acetyl CoA for the citric acid cycle • Slows when high levels of ATP or NADH inhibit citrate synthetase (first step in cycle), decreasing the formation of acetyl CoA

13. Electron Carriers • The electron transport chain consists of electron carriers that accept H+ ions and electrons from the reduced coenzymes NADH and FADH2 • The H+ ions and electrons are passed down a chain of carriers until in the last step they combine with oxygen to form H2O • Oxidative phosphorylation is the process by which the energy from transport is used to synthesize ATP

14. Oxidation and Reduction of Electron Carriers • Electron carriers are continuously oxidized and reduced as hydrogen and/or electrons are transferred from one to the next • The energy produced from these redox reactions is used to synthesize ATP

15. FMN (Flavin Mononucleotide) • FMN coenzyme is derived from riboflavin (vitamin B2) - it contains flavin, ribitol,and a phosphate - it accepts 2H+ + 2e- to form reduced coenzyme FMNH2

16. Iron-Sulfur (Fe-S) Clusters • Fe-S clusters are groups of proteins containing iron ions and sulfide • They accept electrons to reduce Fe3+ to Fe2+, and lose electrons to re-oxidize Fe2+ to Fe3+

17. Coenzyme Q (CoQ or Q) • Coenzyme Q (Q or CoQ) is a mobile electron carrier derived from quinone • It is reduced when the keto groups accept 2H+ and 2e-

18. Cytochromes (Cyt) • Cytochromes (cyt) are proteins containing heme groups with iron ions. • In a cytochrome, Fe3+ accepts an electron to form Fe2+ (reduction), and the Fe2+ is oxidized back to Fe3+ when it passes an electron to the next carrier: Fe3+ + e- Fe2+ • They are abbreviated as cyt a, cyt a3, cyt b, cyt c, and cyt c1

19. Electron Transport System • The electron carriers in the electron transport system are attached to the inner membrane of the mitochondrion • They are organized into four protein complexes: Complex I NADH dehydrogenase Complex II Succinate dehydrogenase Complex III CoQ-Cytochrome c reductase Complex IV Cytochrome c Oxidase

20. Electron Transport Chain

21. Complex I: NADH Dehydrogenase • At Complex I, hydrogen and electrons are transferred: - from NADH to FMN: FMN + NADH + H+ FMNH2 + NAD+ - from FMNH2 to Fe-S clusters and Q, which reduces Q to QH2 and regenerates FMN Q + FMNH2 QH2 + FMN - to complex I to Complex III by Q (QH2), a mobile carrier

22. Complex II: Succinate Dehydrogenase • At Complex II, hydrogen and electrons are transferred: - from FADH2 to Complex II, which is at a lower energy level than Complex I - from FADH2 to coenzyme Q, which reduces Q and regenerates FAD Q + FADH2 QH2 + FAD - from complex II to Complex III by Q(QH2), a mobile carrier

23. Complex III: Coenzyme Q-Cytochrome c Reductase • At Complex III, electrons are transferred: - from QH2 to two Cyt b, which reduces Cyt b and regenerates Q 2Cyt b (Fe3+) + QH2  2Cyt b (Fe2+) + Q + 2H+ - from Cyt b to Fe-S clusters and to Cyt c, the second mobile carrier 2Cyt c (Fe3+) + 2Cyt b (Fe2+)  2Cyt c (Fe2+) + 2Cyt b (Fe3+)

24. Complex IV: Cytochrome c Oxidase • At Complex IV, electrons are transferred: - from Cyt c to Cyt a 2Cyt a (Fe3+) + 2Cyt c (Fe2+)  2Cyt a (Fe2+) + 2Cyt c (Fe3+) - from Cyt a to Cyt a3, which provides the electrons to combine H+ and oxygen to form water 4H+ + O2 + 4e- (from Cyt a3)  2H2O

25. Oxidative Phosphorylation and the Chemiosmotic Model • In the chemiosmotic model, complexes I, III, and IV pump protons into the intermembrane space, creating a proton gradient • Protons must pass through ATP synthase to return to the matrix • The flow of protons through ATP synthase provides the energy for ATP synthesis (oxidative phosphorylation): ADP + Pi + Energy  ATP

26. ATP Synthase • In ATP synthase protons flow back to the matrix through a channel in the F0 complex • Proton flow provides the energy that drives ATP synthesis by the F1 complex

27. ATP Synthase F1 Complex • In the F1 complex of ATP synthase, a center subunit () is surrounded by three protein subunits: loose (L), tight (T), and open (O) • Energy from the proton flow through F0 turns the center subunit (), which changes the shape (conformation) of the three subunits • As ADP and Pi enter the loose L site, the center subunit turns, changing the L site to a tight T conformation • ATP is formed in the T site where it remains strongly bound • Energy from proton flow turns the center subunit, changing the T site to an open O site, which releases the ATP

28. Electron Transport and ATP Synthesis • In electron transport, the energy level decreases for electrons: • Oxidation of NADH (Complex I) provides sufficient energy for 3ATPs NADH + 3ADP + 3Pi NAD+ + 3ATP • Oxidation of FADH2 (Complex II), which enters the chain as a lower energy, provides sufficient energy for only 2ATPs FADH2 + 2ADP + 2Pi FAD + 2ATP

29. ATP from and Regulation of Electron Transport • Low levels of ADP, Pi, oxygen, and NADH decrease electron transport activity • High levels of ADP activate electron transport • As the electrons flow through decreasing energy levels, three of the transfers provide enough energy for ATP synthesis

30. ATP from Glucose • The complete oxidation of glucose yields 6CO2, 6H2O, and 36 ATP

31. ATP Regulation • ATP levels are maintained through control of glucose metabolism