1. Mitochondrial Function Structure Citric acid cycle Electron transport Regulatory/modulatory signaling

2. Mitochondrial Structure Principal metabolic engine Symbiotic bacteria –6k-370kBP genome –Human: 13 proteins Dual membrane –ie: two bilayers –Outer membrane highly permeable –Inner membrane highly impermeable

3. Mitochondrial Matrix Highly oxidative environment Unique proton gradient –High pH (8), negative (-180 mV), ~18 kJ/mole –H + actively transported out of matrix –H + leak back as H + PO 4 2- Capture gradient energy for ATP synthesis –H + ATPase pump –ADP-ATP antiporter Other proton co-transporters –Pyruvate, citrate –Glutamate, citruline

4. Metabolic Substrates Sugars –Metabolized in cytoplasm to pyruvate –Co-transported to matrix with H+ –Bound to Coenzyme A as Acetyl-CoA Fatty acids –To intermembrane space as Acyl-CoA –To matrix as Acyl-carnitine –Metabolized to Acetyl-CoA in matrix Proteins CH 3 C=O COO -

5. Acetyl Coenzyme A Common substrate for oxidative metabolism S-linked acetate carrier

6. Oxygen Coenzyme A Carbon Isocitrate a -Ketoglutarate Succinyl CoA Succinate Fumarate = Malate Oxaloacetate CoA NADH + NADH + GTP FADH 2 NADH The Citric Acid Cycle Citrate Acetyl-Coenzyme A These carbons will be removed New carbons

7. Electron transport Couple NADH/FADH2 electrons to H+ export –Ideally this completes –Electron leakage NADH + H + + ½ O 2 NAD + +H 2 O NAD + + H + +2e - NADH  E 0 =-0.32V ½O 2 +2 H + + 2e - H 2 O  E 0 =0.82V

8. KEGG pathway KEGG http://www.genome.jp/kegg/pathway.html Enzyme Commission (EC) number Hierarchical Function-centric nomenclature Compare Gene Ontology (GO) ID Entrez RefSeq UniProt ID Metabolite

9. Cyclic redox reactions Oxidized Reduced NADH FADH 2 NAD + FAD CoQ/ubiquinone dihydroubiquinone Cyto-C 3+ Cyto-C 2+ O2O2 H2OH2O NAD+  NADH E 0 = -0.32V FAD  FADH 2 E 0 = -0.22V Ubuquinone E 0 = 0.10V Cytochrome C E 0 = 0.22V O 2  H 2 O E 0 = 0.82V

10. NADH/Complex I Nicotinamide adenine dinucleotide –H dissociates as H - Complex I (NADH reductase) Then e - to ubiquinone –46 subunit protein –Nuclear-derived proteins –mtDNA-derived proteins –Transfer e - to ubiquinone –Shuttle 2 H + /e -

11. FADH2/Complex II Flavin Adenine Dinucleotide –H disrupts C-ring –Electron transfer flavoprotein Complex II –Succinate reductase Transfers 2H from succinate to FAD No H+ transport –ETF-ubiquinone oxidoreductase Transfers 2H from FADH 2 to ubiquinone

12. Complex III and IV Ubiquinone (UQ) –2 electron carrier Complex III (cytochrome reductase) –Transfer e- from ubiquinone to cytochrome c –Coupled with H+ transport –Rieske “2Fe2S” redox center –1 Ub to 2 CyC http://bcs.whfreeman.com/stryer/pages/bcs-main.asp?s=00010&n=99000&i=99010.01&v=category&o=&ns=0&uid=0&rau=0

13. Complex IV Cytochrome oxidase –Transfer 1x e- from cytochrome C to oxygen –Coupled with H+ transport –4x cytochrome yield 2xH2O Transport complex –Supercomplex of I, III, IV –Stoichiometry of 1:2:4 –Transport 8 e- and 36 H+ per citrate –ATP?

14. Mitochondrial membrane potential Few ion channels Low H+ –High H+ flux/H+ current –Proton equilibrium potential ~+50 mV –  ~ -100 mV relative to cytoplasm H+ coupled transport –Malate, pyruvate, glutamate, Ca, Pi Charge coupled transport –ATP:ADP exchange, Ca

15. Proton ATPase/Complex V ATP driven proton pump –“Reversible” –Couples H+ gradient to ATP synthesis

16. Mitochondrial control Mitochodrial nucleotide flux –Steady state (at rest), not equilibrium –Dynamic control Membrane potential Substrate+O 2 +ADP  CO2+H2O+ATP Substrate (Glucose, pyruvate, NADH) O2O2 ADP CO 2 H2OH2O ATP State 2State 4 Rest State 5

17. Control by mitochondrial potential NADH oxidation coupled to H+ transport Greater , greater resistance, slower ox Mitochondrial Depolarization NADH content Mitochondrial uncoupling poison Leyssens et al., 1997

18. Extreme mitochondria redox states Substrate+O 2 +ADP  CO2+H2O+ATP State 1: substrate & ADP limited State 2: substrate limited State 3: enzyme limited –Maximal activity State 4: ADP & Pi limited –Rest State 5: O 2 limited –<5-10 uM O 2 ~ 15-30 mmHg, 2-4%

19. Control by mitochondrial redox state Cytochrome oxidase (complex IV) –Iron (heme) redox centers (Fe 2+ /Fe 3+ ) –Copper redox center (Cu + /Cu 2+ ) Redox centers more oxidized –More O 2 –Less ADP Hoshi, et al., 1993 State 3 State 4Cu Fe  E=  E 0 - RT/nF ln(Q prod /Q reac ) Redox cascade backs up by accumulation of product

20. Extreme mitochondria redox states State 1: substrate & ADP limited –Electron transport chain oxidized –High  State 2: substrate limited –ETC oxidized, low  State 3: enzyme limited –ETC reduced, low  State 4: ADP & Pi limited –ETC reduced, high  State 5: O 2 limited –ETC reduced, high 

21. Control by calcium Calcium activated enzymes –Pyruvate dehydrogenase –Oxoglutarate dehydrogenase –NAD + -isocitrate dehydrogenase –F0F1 Membrane potential –Na-Ca antiporter –Ca uniporter  depolarization with cytoplasmic Ca Reduce F0F1 efficiency Increase NADH oxidation

22. Electron leakage Ubiquinone rapidly releases e- –Radical formation: O 2 –Bypasses electron transport Complex I Complex III Oxidative damage –Thiol crosslinking, DNA damage, etc –Inhibition of Complex I & III –Buffered by intermembrane GSH, Mn-SOD