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 CO 2 It produces reduced coenzymes NADH and FADH 2 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

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

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

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

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

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

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 FADH 2

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

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

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

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

Electron Carriers The electron transport chain consists of electron carriers that accept H + ions and electrons from the reduced coenzymes NADH and FADH 2 The H + ions and electrons are passed down a chain of carriers until in the last step they combine with oxygen to form H 2 O Oxidative phosphorylation is the process by which the energy from transport is used to synthesize ATP

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

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

Iron-Sulfur (Fe-S) Clusters Fe-S clusters are groups of proteins containing iron ions and sulfide They accept electrons to reduce Fe 3+ to Fe 2+, and lose electrons to re-oxidize Fe 2+ to Fe 3+

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 -

Cytochromes (Cyt) Cytochromes (cyt) are proteins containing heme groups with iron ions. In a cytochrome, Fe 3+ accepts an electron to form Fe 2+ (reduction), and the Fe 2+ is oxidized back to Fe 3+ when it passes an electron to the next carrier: Fe 3+ + e -  Fe 2+ They are abbreviated as cyt a, cyt a 3, cyt b, cyt c, and cyt c 1

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 IISuccinate dehydrogenase Complex IIICoQ-Cytochrome c reductase Complex IVCytochrome c Oxidase

Electron Transport Chain

Complex I: NADH Dehydrogenase At Complex I, hydrogen and electrons are transferred: - from NADH to FMN: FMN + NADH + H +  FMNH 2 + NAD + - from FMNH 2 to Fe-S clusters and Q, which reduces Q to QH 2 and regenerates FMN Q + FMNH 2  QH 2 + FMN - to complex I to Complex III by Q (QH 2 ), a mobile carrier

Complex II: Succinate Dehydrogenase At Complex II, hydrogen and electrons are transferred: - from FADH 2 to Complex II, which is at a lower energy level than Complex I - from FADH 2 to coenzyme Q, which reduces Q and regenerates FAD Q + FADH 2  QH 2 + FAD - from complex II to Complex III by Q(QH 2 ), a mobile carrier

Complex III: Coenzyme Q-Cytochrome c Reductase At Complex III, electrons are transferred: - from QH 2 to two Cyt b, which reduces Cyt b and regenerates Q 2Cyt b (Fe 3+ ) + QH 2  2Cyt b (Fe 2+ ) + Q + 2H + - from Cyt b to Fe-S clusters and to Cyt c, the second mobile carrier 2Cyt c (Fe 3+ ) + 2Cyt b (Fe 2+ )  2Cyt c (Fe 2+ ) + 2Cyt b (Fe 3+ )

Complex IV: Cytochrome c Oxidase At Complex IV, electrons are transferred: - from Cyt c to Cyt a 2Cyt a (Fe 3+ ) + 2Cyt c (Fe 2+ )  2Cyt a (Fe 2+ ) + 2Cyt c (Fe 3+ ) - from Cyt a to Cyt a 3, which provides the electrons to combine H + and oxygen to form water 4H + + O 2 + 4e - (from Cyt a 3 )  2H 2 O

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 + P i + Energy  ATP

ATP Synthase In ATP synthase protons flow back to the matrix through a channel in the F 0 complex Proton flow provides the energy that drives ATP synthesis by the F 1 complex

ATP Synthase F 1 Complex In the F 1 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 F 0 turns the center subunit (  ), which changes the shape (conformation) of the three subunits As ADP and P i 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

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 + 3P i  NAD + + 3ATP Oxidation of FADH 2 (Complex II), which enters the chain as a lower energy, provides sufficient energy for only 2ATPs FADH 2 + 2ADP + 2P i  FAD + 2ATP

ATP from and Regulation of Electron Transport Low levels of ADP, P i, 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

ATP from Glucose The complete oxidation of glucose yields 6CO 2, 6H 2 O, and 36 ATP

ATP Regulation ATP levels are maintained through control of glucose metabolism