Citric acid cycle. What are the functions of Citric Acid Cycle? What are the reactions of Citric Acid. How does a mitochondrion look like?

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1 Citric acid cycle What are the functions of Citric Acid Cycle? What are the reactions of Citric Acid Cycle? How does a mitochondrion look like? 1

2 The third step of the energy metabolism 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 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 cell uter membrane: All molecules with MW<5000 can pass Inner membrane: Selective, contains electron transport chain and ATP-synthase Matrix: Contains the enzymes of citric acid cycle and fatty acid oxidation uter membrane Matrix Inner membrane 2

3 The pyruvate dehydrogenase complex catalyses the conversion of pyruvate to acetyl-coa Pyruvate + CoA + NAD + Acetyl-CoA + C 2 + NADH +H + Citric acid cycle An acetyl group is oxidised in the citric acid cycle and the electrons transferred to NAD + or FAD In ut 1 Acetyl group 2 C 2 3 NAD + 3 NADH 1 FAD 1 FADH 2 1 GDP 1 GTP 3

4 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. REFILLING Refilling using pyruvate carboxylase Pyruvat + C 2 +ATP xaloacetate+ ADP + P i + H 2 Summary: The 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: - xidise acetyl-coa - Provide the cell with building blocks for biosynthesis 4

5 xidative 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 molecule glucose is oxidised? Principle of oxidative phosphorylation 5

6 xidative phosphorylation (1) xidative phosphorylation can be divided into three parts: 1. Electron transport from reduced electron carriers to oxygen 2. Creation of a membrane potential and a ph-gradient 3. Synthesis of ATP These three parts are coupled 6

7 xidative phosphorylation (2) Electron transport chain Electrons 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 to oxygen) 6. xygen is reduced to water Electrons from FADH 2 are delivered to CoQ via complex II (peripheral membrane protein) H + H + H + H + cyt c I 2e - CQ CoQ II 2e - III 2 + 4e - + 4H + IV 2H 2 V NADH + H + NAD + FADH 2 FAD H + xidative 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. 7

8 xidative phosphorylation (4) Prosthetic groups Electron transport by a number of reductions and oxidations NADH C- I (red) CoQ (red) C- III (red) Cyt c (red) C- IV (red) H 2 NAD C- I (ox) CoQ(ox) C- III (ox) Cyt c (ox) C- IV (ox) 2 Prosthetic groups in the complexes are reduced and oxidised: Flavin mononucleotide: FMN FMNH 2 Heme group: Fe 3+ Fe 2+ Iron-Sulphur clusters: Fe 3+ Fe 2+ Copper: Cu 2+ 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 (RS) like e.g. the superoxide radical are generated during transport of electrons from NADH and FADH 2 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 RS. Superoxide dismutase H H 2 2 Catalase 2 H 2 2 H

9 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 back inside id the mitochondrion. i 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. utside In the membrane Inside H N 2 N 2 N 2 + H + + H + N 2 N2 N 2 Electron transfer driven transport of protons 9

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12 Summary xidative phosphorylation takes place in the mitochondrial inner membrane The membrane is an integral part of the mechanism The electron transport chain brings electrons from NADH and FADH 2 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 xidation of fatty acids Ketone bodies and their use Synthesis of fatty acids Fatty acids Phospholipids in membranes osp o p ds e b a es Glycolipids in membranes Fatty acid derivatives as hormones Fuel for the cell, energy store 12

13 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). H H C C H C C H C C H Digestion, mobilization and transport of dietary triacylglycerols 13

14 Chylomicron Lipase reactions The enzyme lipase catalyses the hydrolysis of two of the fatty acids in the intestine. 14

15 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 15

16 Degradation of fatty acid has three steps: 1. Activation 2. Transport into mitochondrion 3. β-oxidation Activation of the fatty acid consists of esterification to CoA at the expense of ATP CoA + R C - ATP R C S CoA AMP + PP i 2P i Transport of fatty acid into mitochondrion The acyl group is transported into mitochondrion using carnitine. A translocase brings in acylcarnitine and expels carnitine. Acyl-CoA Ais regenerated inside id mitochondrion. Carnitine CH 3 H + - H 3 C N C C C C H2 H 2 CH 3 H Position of the fatty acid 16

17 β-oxidation 17

18 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 8 x 1 = 8 FADH 2 8 x 1 = 8 GTP β-xidation yields: 7 NADH 7 FADH 2 In total: = 31 NADH corresponds to 31 x 2,5 = 77,5 ATP = 15 FADH 2 corresponds to 15 x 1,5 = 22,5 ATP 8GTP corresponds to 8ATP Summarised: ,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 CoA Acetoacetate Acetoacetyl CoA 2 Acetyl-CoA Citric acid cycle Ketone bodies are formed in the liver from surplus of acetyl- CoA when degradation of fat dominates 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. 18

19 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. H C C S CoA ATP + HC C C C S CoA ADP P i H 3 H2 Acetyl-CoA Malonyl-CoA Reaction sequence during fatty acid synthesis C H 3 C + S ACP C C C H2 Condensation ACP + C 2 S ACP H 3 C C C C S ACP H2 Acetoacetyl-ACP 2 Reduction H NADPH NADP + H 3 C C C C S ACP H2 D-3-Hydroxybutyryl-ACP H Dehydration H H 2 H 3 C C C C S ACP Crotonyl-ACP H Reduction NADPH NADP + H 3 C C C C S ACP H2 H2 Butyryl-ACP 19

20 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, FADH 2 and acetyl-coa Ketone bodies are formed when acetyl-coa is in surplus Acetyl-CoA (from carbohydrates) is starting material for fatty acid synthesis 20