1 Lecture 6B – outline Mitochondrial function (e.g. hepatocytes) 1) citric acid cycle as an energy source a) pyruvate or  -ketoglutarate dehydrogenase 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

2 Compartmentalization of the major pathways of metabolism

3 Stryer An overview of the citric acid cycle 1. CITRIC ACID CYCLE AS AN ENERGY SOURCE 1. CITRIC ACID CYCLE AS AN ENERGY SOURCE

4 120uM plasma citrate complexes Fe toxic!

5 The citric acid cycle is a source of biosynthetic precursors Stryer Fig Biosynthetic roles of the citric acid cycle. Intermediates drawn off for biosyntheses are replenished by the formation of oxaloacetate from pyruvate.

6 Control of the citric acid cycle Stryer Fig 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.

7 2. THE MITOCHONRIAL RESPIRATORY CHAIN AS AN ENERGY SOURCE

8 The mitochondrial respiratory chain Sequence of electron carriers in the respiratory chain Chemiosmotic theory of oxidative phosphorylation Diagram of a mitochondrion

9 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 Therefore mitochondria protein synthesis should be inhibited by: 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.

10 NADH coenzyme Q reductase: complex I The reduction of ubiquinone to ubiquinol proceeds through a semiquinone anion intermediate.

11 Q:Cytochrome c reductase (Complex III) Stryer Fig Model of a portion of Q: cytochrome c reductase Stryer p. 537

12 Cytochrome oxidase (Complex IV) Lodish Fig

13 Electron transport can be blocked by specific inhibitor poisons

14 Cytochrome C - catalytic site The heme in cytochromes c and c1 is covalently attached to 2 cysteine side chains by thioether linkages The iron atom of the heme group in cytochrome c is bonded to a methionine sulfur atom and a histidine nitrogen atom

15 Cytochrome C - soluble NOT membrane bound 1. 26/104 amino acids residues have been invariant for > 1.5 x 10 9 years. 2. Met 80 and His 18 - coordinate Fe residues from number 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!

16 3. OXIDATIVE PHOSPHORYLATION AND UNCOUPLERS

17 Oxidative phosphorylation

18 4. Mitochondrial MEMBRANE TRANSPORTERS A) Cytosolic NADH oxidation B) Acetyl CoA (NADPH export) C) Transport systems in the mitochondria D) Gluconeogenesis and glucose transport

19 a) Cytosolic NADH oxidation: membrane transporters glycerol phosphate shuttle (Bucher shuttle)

20 b) Acetyl CoA/NADPH export to cytosol for fatty acid synthesis/ drug metabolism

21 Isocitrate as an NADPH shuttle for drug metabolism

22 d) Gluconeogenesis and glucose export by the liver ! 3 irreversible steps Major antidiabetic drug METFORMIN Inhibits gluconeogenesis Decr Hepatic Glucose Synth.

23 Glucagon 51aa & Insulin 29aa Pancreas synthesises both peptide hormones Insulin required for cells (e.g.liver,muscle,fat) to take up glucose and synthesise glycogen. Glucagon hepatocyte receptors signals glycogenolysis (glycogen breakdown to glucose then increases gluconeogenesis pyruvate --  glucose) Drugs. Dipeptidyl peptidase-4 inhibitor (Januvia, new anti type 2 diabetes) increases incretin, a GI hormonal peptide inhibitor of glucagon which lowers plasma glucose. Metformin, antidiabetic drug inhibits gluconeogenesis but also can inhibit mitoch.complex I causing lactic acidosis.