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+PO4 2- • Capture gradient energy for ATP synthesis • H+ ATPase pump • ADP-ATP antiporter • Other proton co-transporters • Pyruvate, citrate • Glutamate, citruline

4. CH3 C=O COO- 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

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

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

7. NAD+ + H++2e- NADH DE0=-0.32V ½O2+2 H++ 2e- H2O DE0=0.82V NADH + H+ + ½ O2 NAD+ +H2O Electron transport • Couple NADH/FADH2 electrons to H+ export • Ideally this completes • Electron leakage

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

9. NAD+ FAD NADH FADH2 Cyclic redox reactions Oxidized CoQ/ubiquinone Cyto-C3+ O2 dihydroubiquinone Cyto-C2+ H2O Reduced NAD+  NADH E0 = -0.32V FAD FADH2 E0 = -0.22V Ubuquinone E0 = 0.10V Cytochrome C E0 = 0.22V O2  H2O E0 = 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 FADH2 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 dominates membrane potential • DY ~ -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+O2+ADPCO2+H2O+ATP ADP State 2 State 5 State 4Rest O2 CO2 Substrate (Glucose,pyruvate, NADH) H2O ATP

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

18. Extreme mitochondria redox states • Substrate+O2+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: O2 limited • <5-10 uM O2 ~ 15-30 mmHg, 2-4%

19. Control by mitochondrial redox state • Cytochrome oxidase (complex IV) • Iron (heme) redox centers (Fe2+/Fe3+) • Copper redox center (Cu+/Cu2+) • Redox centers more oxidized • More O2 • Less ADP Fe State 3 Cu State 4 DE=DE0 - RT/nF ln(Qprod/Qreac) Redox cascade backs up by accumulation of product Hoshi, et al., 1993

20. Extreme mitochondria redox states • State 1: substrate & ADP limited • Electron transport chain oxidized • High DY • State 2: substrate limited • ETC oxidized, low DY • State 3: enzyme limited • ETC reduced, low DY • State 4: ADP & Pi limited • ETC reduced, high DY • State 5: O2 limited • ETC reduced, high DY

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: O2• • 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