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Citric acid cycle

• What are the functions of Citric Acid Cycle?

• What are the reactions of Citric Acid Cycle?Cycle?

• How does a mitochondrion look like?

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The third step of the energy metabolism comprises the citric acid cycle and the oxidative phosphorylation. Electrons are transferred from reduced electron carriers to oxygen, and ATP is formed via a proton gradient.

The third step of the energy metabolism

The mitochondrion

The mitochondrion has two membranes of which the inner one is selective and folded. The degree of folding depends on the energy need of the cellOuter membrane: All molecules with MW<5000 can passInner membrane: Selective, contains electron transport chain and ATP-synthase Matrix: Contains the enzymes of citric acid cycle and fatty acid oxidationMatrix: Contains the enzymes of citric acid cycle and fatty acid oxidation

Outer membrane

Matrix

Inner membrane

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The pyruvate dehydrogenase complex catalyses the conversion of pyruvate to acetyl-CoA

Pyruvate + CoA + NAD+ Acetyl-CoA + CO2 + NADH +H+

Citric acid cycle

An acetyl group is oxidised in the citric acid cycle and the electrons transferred tothe electrons transferred to NAD+ or FAD

In Out

1 Acetyl group 2 CO2

3 NAD+ 3 NADH

1 FAD 1 FADH2

1 GDP 1 GTP

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The citric acid cycle can be drained of intermediaries

The second purpose of the tricarboxylic acid cycle is to provide the cell with building blocks for the biosynthesis of e.g. amino acids , heme and fatty acids. A consequence is that a lack of intermediaries can impair the oxidative function.

REFILLINGREFILLING Refilling using pyruvate carboxylase

Pyruvat + CO2 +ATP Oxaloacetate+ ADP + Pi + H2O

Summary:

• The tricarboxylic acid cycle takes place in theThe tricarboxylic acid cycle takes place in the mitochondrion

• The tricarboxylic acid cycle generates reduced electron carriers and GTP

• The tricarboxylic acid cycle has two purposes:- Oxidise acetyl-CoA- Provide the cell with building blocks for biosynthesisProvide the cell with building blocks for biosynthesis

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Oxidative phosphorylation

• How are electrons transported from NADH to oxygen?

• How is ATP formed?• How are electron transport and ATP formation

coupled?• How much ATP is formed when one moleculeHow much ATP is formed when one molecule

glucose is oxidised?

Principle of oxidative phosphorylation

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Oxidative phosphorylation (1)

Oxidative phosphorylation can be divided into three parts:1. Electron transport from reduced electron carriers to oxygen2. Creation of a membrane potential and a pH-gradient3. Synthesis of ATPThese three parts are coupled

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Oxidative phosphorylation (2) – Electron transport chainElectrons from NADH take the following path:1. Complex I (Integral membrane protein, transfers protons)2. CoQ (quinone, transfers electrons between complex I and complex III)3. Complex III (Integral membrane protein, transfers protons)4. Cytochrome c (protein, transports electrons between complex III and complex IV)5 Complex IV (Integral membrane protein transfers protons conveys electrons

C Q

cyt c

H+ H+H+ H+

5. Complex IV (Integral membrane protein, transfers protons, conveys electrons to oxygen)

6. Oxygen is reduced to waterElectrons from FADH2 are delivered to CoQ via complex II (peripheral membrane protein)

NAD+NADH + H+

2e-

CoQ

H+

IIIV

VI III

O2 + 4e- + 4H+ 2H2O

FADH2 FAD

2e-

Oxidative phosphorylation (3) - CoQ

CoQ is a quinone, which can take up two electrons. Its hydrophobic side chain makes it comfortable in the inner membrane of the mitochondrion.

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Oxidative phosphorylation (4) – Prosthetic groups

Electron transport by a number of reductions and oxidations

NADH CoQ (red) C- III (red) C- IV (red) H2OC- I (red) Cyt c (red)

Prosthetic groups in the complexes are reduced and oxidised:

Flavin mononucleotide: FMN FMNH2Heme group: Fe3+ Fe2+

Iron-Sulphur clusters: Fe3+ Fe2+

NAD CoQ(ox) C- III (ox) C- IV (ox) O2C- I (ox) Cyt c (ox)

Iron Sulphur clusters: Fe FeCopper: Cu2+ Cu+

• Prosthetic groups are organised in order to enable electron transfer • Specificity in electron transfer: E.g. NADH can't reduce cytochrome c• All components of the electron transfer diffuse in the membrane

Reactive intermediaries in the electron transport chain

Reactive oxygen species (ROS) like e.g. the superoxide radical are generated during transport of electrons from NADH and FADH2 to oxygen. They can react with other biomolecules and cause damage (and possibly diseases). We have some protection from some enzymes, which take care of ROS.

2O2- .

+ 2H+ O2 + H2O2

Superoxide dismutase

protection from some enzymes, which take care of ROS.

Catalase

2 H2O2 H2O + O2

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Uncoupler (1)

Electron transport, proton transfer and phosphorylation of ADP are usually coupled processes but they can be uncoupled under certain circumstances. Uncouplers can lead to formation of heat instead of ATP, e.g. hibernating animals and newborn children use an uncoupling protein to lead the protons b k i id th it h d iback inside the mitochondrion.

Uncoupler (2)

Also low molecular weight substances can act as uncouplers. In the example below, protonated dinitrophenol can diffuse through the membrane thereby ”destroying” the membrane potential.

O

NO2

OH

NO2

O

NO2+ H+ + H+

Outside In the membrane Inside

NO2 NO2 NO2

Electron transfer driven transport of protons

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Summary

• Oxidative phosphorylation takes place in the mitochondrial inner membraneinner membrane

• The membrane is an integral part of the mechanism• The electron transport chain brings electrons from

NADH and FADH2 to oxygen• A proton gradient is established when electrons passes the chain• ADP is phosphorylated to ATP when the protons return to the mitochondrion

• NADH (the electrons) from glycolysis enter the mitochondrion by a shuttle

• Uncouplers generate heat

Metabolism of Fatty Acids

• Oxidation of fatty acids• Ketone bodies and their use

S th i f f tt id• Synthesis of fatty acids

Fatty acids • Phospholipids in membranesosp o p ds e b a es• Glycolipids in membranes• Fatty acid derivatives as hormones• Fuel for the cell, energy store

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Fat (Triacylglycerol)

A triacylglycerol is a glycerol esterified with three fatty acids. The fatty acids can be saturated, mono-unsaturated or poly-unsaturated. Usually they have an even number of carbon atoms (12-20).

C

C

H

H

O

H O C

O

O

C

O

C OH C

H

O

Digestion, mobilization and transport of dietary triacylglycerols

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Chylomicron

Lipase reactions

The enzyme lipase catalyses the hydrolysis of two of the fatty acids in the intestine.

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Uptake of fat in the intestine

Fat is first hydrolysed in the intestine and then taken up through the cell wall. The triacylglycerol is then regenerated before its transport as part of a chylomicron (a lipoprotein) to fat cells where it can be stored.

Triacylglycerol in fat cells is degraded to fatty acids and glycerol, which can be used in other cells

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Degradation of fatty acid has three steps:1. Activation2. Transport into mitochondrion3. β-oxidation

Activation of the fatty acid consists of esterification to CoA at the expense of ATP

CO

S CoARCO

OCoA + R - C S CoARC O

2P i

CoA + R

ATPAMP + PPi

The acyl group is transported into mitochondrion using carnitine. A translocase brings in acyl-carnitine and expels carnitine. A l C A i t d i id

Transport of fatty acid into mitochondrion

Acyl-CoA is regenerated inside mitochondrion.

N CH2

CH

CH3

CH3

C

OH

H

CH2

COO+ -

Carnitine

CH3 OH

Position of the fatty acid

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β-oxidation

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How much ATP can be formed by oxidation of fatty acid?

It is now possible to calculate the amount of ATP formed during complete oxidation of a fatty acid, e.g. palmitic acid

8 Acetyl-CoA in the tricarboxylic acid cycle: 8 x 3 = 24 NADH y y y8 x 1 = 8 FADH28 x 1 = 8 GTP

β-Oxidation yields: 7 NADH7 FADH2

In total: 7 + 24 = 31 NADH corresponds to 31 x 2,5 = 77,5 ATP7 + 8 = 15 FADH2 corresponds to 15 x 1,5 = 22,5 ATP8 GTP d t 8 ATP8 GTP corresponds to 8 ATP

Summarised: 8 + 77,5 + 22,5 = 108 ATP

Two ATP are used during the initial activation of the fatty acid The total ATP-yield will be 106

Formation and use of ketone bodies

Succinyl CoA Succinate CoAKetone bodies are formed in the liver from surplus of acetyl-CoA when degradation of fat

Acetoacetate Acetoacetyl CoA 2 Acetyl-CoA

Citric acid cycle

gdominates and the supply of carbohydrates is limited, as during starvation and fasting. The ketone bodies are exported to other organs where they can be oxidised.

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Fatty acid synthesis

• Fat is an efficient way to store energy • It is usually surplus of carbohydrates that are converted to fat • The reactions are in principle a reversed β-oxidation

The synthesis of a fatty acid starts with the carboxylation of acetyl-CoA to malonyl-CoA. This is the committed step, malonyl CoA can only be used for synthesis of fatty acid.

CH C

O

S CoA C C

O

S CoAC

O+ ATP + HCO - + ADP + Pi + H+CH3 C S CoA C

H2

C S CoAC

OATP + HCO3

ADP Pi H

Acetyl-CoA Malonyl-CoA

Reaction sequence during fatty acid synthesis

CH3 C

O

S ACP CH2

C

O

S ACPC

O

O

CH3 C CH2

C S ACP

O O

+

ACP + CO2

Condensation

Acetoacetyl-ACP2

CH3 C CH2

C S ACP

OOH

H

NADPH

NADP+

Reduction

D-3-Hydroxybutyryl-ACP

H2ODehydration

CH3 C C C S ACP

O

H

H

CH3 CH2

CH2

C S ACP

O

Crotonyl-ACP

NADPH

NADP+

Reduction

Butyryl-ACP

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Summary:

• It is efficient to store energy as fat• The fatty acid is bound to albumin during transport to target cells• The fatty acid is activated in the cytoplasm• The fatty acid is transported into the mitochondrion using carnitine• The fatty acid is oxidised by β-oxidation yielding NADH, FADH2 and

acetyl-CoA• Ketone bodies are formed when acetyl-CoA is in surplus• Acetyl-CoA (from carbohydrates) is starting material for fatty acid

synthesissynthesis