10/21/2014 Lecture 6B – outline Mitochondrial function – general features 1) citric acid cycle as an energy source a)) py pyruvate or α-ketoglutarate g dehydrogenase y g b) lipoic acid therapy 2) the respiratory chain as an energy source 3) oxidative phosphorylation and uncouplers 4) membrane transporters and shuttles a) cytosolic NADH oxidation b) acetyl CoA (NADPH export) c) transport systems in the mitochondria d) gluconeogenesis and glucose transport 1 Compartmentalization of the major pathways of metabolism 2 1 10/21/2014 1. CITRIC ACID CYCLE AS AN ENERGY SOURCE An overview of the citric acid cycle Stryer 3 Acetyl CoA + 3 NAD+ + FAD + GDP + Pi + 2 H2O Æ 2 CO2 + 3 NADH + FADH2 + GTP + 2H+ + CoA toxic! i ! 120uM plasma citrate complexes Fe 4 2 10/21/2014 The citric acid cycle is a source of biosynthetic precursors Glucose Pyruvate ATP, CO2 Phosphoenolpyruvate Acetyl CoA ADP, Pi Amino acids Oxaloacetate Succinyl CoA Porphyrins Citrate Stryer Fig. 20-17. Biosynthetic y roles of the citric acid cycle. Intermediates drawn off for biosyntheses are replenished by the formation of oxaloacetate from pyruvate. α−ketoglutarate Amino acids 5 Control of the citric acid cycle Stryer Fig. 20-22. Control of the citric acid cycle and the oxidative decarboxylation of pyruvate: * indicates steps that require an electron acceptor (NAD+ or FAD) that is regenerated by the respiratory chain. 6 3 10/21/2014 22. THE MITOCHONRIAL RESPIRATORY CHAIN AS AN ENERGY SOURCE 7 The mitochondrial respiratory chain NADH Diagram of a mitochondrion FMNH 2 complex I NADH-Q reductase 2Fe-2S 4Fe-4S Q FADH 2 in flavoproteins succinate:Q reductase (complex II) complex III Cytochrome reductase Chemiosmotic theory of oxidative phosphorylation cyt c complex IV Cytochrome oxidase O2 Sequence of electron carriers in the respiratory chain 8 4 10/21/2014 Origin of mitochondria: the endosymbiont hypothesis The endosymbiont hypothesis suggests that mitochondria have evolved from anaerobic bacteria which were phagocytosed by eukaryote cells at the time oxygen appeared on earth, Similarities between mitochondria and bacteria include the presence of: • cardiolipin •transporters • ribosomes • circular RNA and DNA y should be inhibited by: y Therefore mitochondria pprotein synthesis • TETRACYCLINE • CHLORAMPHENICOL. E.g. The extensive use of these drugs can inhibit 1. Bone marrow mitochondrial protein synthesis leading to a decline in the production of white or red cells. 2. Intestinal epithelial cells causing them to cease dividing. 9 NADH coenzyme Q reductase: complex I FMN NADH NAD+ Q Reduced Fe-S FMNH2 Oxidised Fe-S Fe S QH2 NADH-Q reductase O O C H3 CO C C CH3 CH3 H3 CO C C (CH2C H C C O e- + H+ CH2 )10 H OH e- + H+ C H3 CO C H3 CO C C CH3 H3 CO C C R H3 CO C C OH Coenzyme Q10 (UBIQUINONE Semiquinone Intermediate (Q ) C C CH3 C R C OH Reduced Coenzyme Q10 ( UBIQUINOL) The reduction of ubiquinone to ubiquinol proceeds through a semiquinone anion intermediate. 10 5 10/21/2014 Q:Cytochrome c reductase (Complex III) Q cyt b (+2) QH Fe-S(+2) cyt c1(+3) cyt c(Fe+2) QH cyt b (+3) QH2 Fe-S(+3) cyt c1(+2) cyt c(Fe+3) Stryer p. 537 cytochrome c reductase Stryer Fig. 21-11 Model of a portion of Q: cytochrome c reductase 11 Cytochrome oxidase (Complex IV) Lodish Fig. 17-30 12 6 10/21/2014 Electron transport can be blocked by specific inhibitor poisons NADH NADH-Q Reductase QH2 Blocked by rotenone and amytal Cytochrome b Blocked by antimycin Sites of action of some inhibitors of electron transport Cytochrome c1 Cytochrome c Cytochrome Oxidase Blocked by CN- , N3 -, and CO O2 13 Cytochrome C - catalytic site R C CH 2 H Vinyl group of the heme + CH 3 HS C R' H2 Cysteine residue of the protein The heme in cytochromes c and c1 is covalently attached to 2 cysteine side chains by thioether linkages R C S C H H2 Thioether linkage R' The iron atom of the heme group in cytochrome c is bonded to a methionine 14 sulfur atom and a histidine nitrogen atom 7 10/21/2014 Cytochrome C - soluble NOT membrane bound 1. 26/104 amino acids residues have been invariant for > 1.5 x 109 years. 2. Met 80 and His 18 - coordinate Fe. 3. 11 residues from number 70 - 80 lining a hydrophobic crevice have remained virtually unchanged throughout all cytochrome c regardless of species or even kingdom. 4. A number of invariant arginine and lysine clusters can be found on the surface of the molecule. Cytochrome c has a dual function in the cell. Electron transport for ATP production AND the major cause of most programmed cell death (apoptosis) is initiated by the release of cytochrome c into the cytosol! 15 33. OXIDATIVE PHOSPHORYLATION AND UNCOUPLERS 16 8 10/21/2014 Oxidative phosphorylation 17 4. Mitochondrial MEMBRANE TRANSPORTERS A) Cytosolic NADH oxidation B) Acetyl CoA (NADPH export) C) Transport systems in the mitochondria D) Gluconeogenesis and glucose transport 18 9 10/21/2014 a) Cytosolic NADH oxidation: membrane transporters glycerol phosphate shuttle (Bucher shuttle) Glucose Cytosol Outer membrane Inner Membrane FAD Glycolyysis Glycerol - 3 - phosphate 2 Dihydroxyacetone phosphate H2 C OH HO CH H2 C O P Glyceraldehyde - 3 - phosphate NAD+ FADH 2 Q Glycerol - 3 - phosphate 1 QH2 1,3 - Bisphosphoglycerate RESPIRATORY CHAIN Dihydroxyacetone phosphate H2 C OH O C H2 C O P NADH, H + 1. Glycerol phosphate dehydrogenase 2. Glycerophosphate oxidase b c1 c See figure 21-30 Stryer 4th Ed. a, a 3 19 b) Acetyl CoA/NADPH export to cytosol for fatty acid synthesis/ drug metabolism Glucose Cytosol Pyruvate Acetyl CoA at high concentration Citrate Acetyl CoA Citrate Synthase Citrate +ATP +CoA fatty acid synthesis or drug metabolism (N-acetylation) ATP citrate it t lyase l Oxaloacetate Mitochondrial Matrix NADH malate dehydrogenase Oxaloacetate NAD+ Malate ADP CO2 NADP+ malic enzyme Pyruvate Pyruvate ATP NADPH CO2 fatty acid synthesis or P450 catalyzed drug metabolism Therefore malic enzyme supplies NADPH Citrate Lyase supplies acetyl CoA. Pentose Phosphate Pathway NADPH 20 10 10/21/2014 Isocitrate as an NADPH shuttle for drug metabolism Glucose Pyruvate Acetyl CoA CYTOSOL MITOCHONDRIAL MATRIX Citrate Oxaloacetate O l NADH Malate Fumarate NAD+ CITRIC ACID CYCLE Isocitrate NAD+ NADH S i t Succinate Isocitrate isocitrate dehydrogenase CO2 α-ketoglutarate NADH NADP+ isocitrate dehydrogenase NADPH α-ketoglutarate NAD+ P450 catalyzed DRUG METABOLISM Succinyl CoA CO2 21 d) Gluconeogenesis and glucose export by the liver ! 3 irreversible steps Major antidiabetic drug METFORMIN Inhibits ggluconeogenesis g Decr Hepatic Glucose Synth. 22 11
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