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

Reaction 1: Formation of Citrate

• Oxaloacetate combines with the two-carbon acetyl group to form citrate

citrateoxaloacetate

+ HS-CoA + H+

COO-

CH2

CHO COO-

CH2

COO-

+ CH3 C

O

SCoA

COO-

C

CH2

COO-

O

acetyl CoA

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

Citrate

COO-

CH2

CHO COO-

CH2

COO-

COO-

CH2

C COO-

CH

COO-

COO-

CH2

C COO-

C

COO-

H

HO H

H2O H2O

Aconitase Aconitase

Aconitate Isocitrate

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

COO-

CH2

C COO-

C

COO-

H

HO H

Isocitrate

+ NAD+

COO-

CH2

C H

C

COO-

H

O + CO2 + NADH

Isocitratedehydrogenase

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

+ NAD+

COO-

CH2

CH2

C

COO-

O

+ CO2 + NADH

COO-

CH2

CH2

C

S

O

CoA

Succinyl CoA

+ CoASH

-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

+ GTP + CoA-SH+ GDP + Pi

Succinyl CoA

CH2

CH2

COO-

COO-

Succinyl CoA synthetase

CH2

CH2

C

COO-

O

S CoA

Succinate

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

+ FAD

Succinate Fumarate

+ FADH2

C

C

COO-

COO-

H

HCH2

CH2

COO-

COO-

Succinatedehydrogenase

Reaction 7: Hydration of Fumarate

• Water is added to the double bond in fumarate to form malate

Fumarase

C

C

COO-

COO-

H

H

Fumarate Malate

COO-

CHO H

C

COO-

HHH2O+

Reaction 8: Dehydration of Malate

• Another oxidation forms a C=O double bond

• The hydrogens from the oxidation form NADH + H+

+ H++ NAD+COO-

CHO H

C

COO-

HH

Malate Oxaloacetate

NADH+

Malatedehydrogenase

C

CH2

O

COO-

COO-

Summary of Products from Citric Acid CycleIn 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

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 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

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

electron carrier AH2(reduced)

electron carrier BH2(reduced)

electron carrier B(oxidized)

electron carrier A(oxidized)

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

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+

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, 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

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

Electron Transport Chain

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

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

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+)

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

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

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

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

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

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

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

ATP Regulation

• ATP levels are maintained through control of glucose metabolism