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MiP school 2008 Schröcken, July 2008

MiP school 2008 Schröcken, July 2008. http://www.mitophysiology.org/index.php?id=mip-textbook. Mitochondrial Respiratory Physiology. Mitochondrial respiratory control: Electron transport system, oxidative phosphorylation and leak – ETS , OXPHOS and LEAK . Erich Gnaiger

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MiP school 2008 Schröcken, July 2008

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  1. MiPschool 2008 Schröcken,July 2008 http://www.mitophysiology.org/index.php?id=mip-textbook Mitochondrial Respiratory Physiology. Mitochondrial respiratory control: Electron transport system, oxidative phosphorylation and leak – ETS, OXPHOS and LEAK. Erich Gnaiger Medical University Innsbruck, Austria erich.gnaiger@i-med.ac.at

  2. H+ H+ H+ c aa3 bc1 I II O2 S F NADH F 1 Excess capacity Excess capacity: Insurance against a specific enzymatic injury. 2 H+ H+ Q 1 Biochemical threshold: Cellular function is buffered against a specific enzymatic defect. Threshold 0.0 0.5 1.0 0 Excess Capacity and Biochemical Threshold Pathway flux Enzymatic defect

  3. H+ H+ H+ c aa3 bc1 I II O2 S F NADH F 1 H+ H+ Q Excess Capacity and Biochemical Threshold Different excess capacities imply tissue-specific (in)sensitivity to enzymatic defects in: • genetic mitochondrial disorders • aging • ischemia-reperfusion injury • degenerative diseases

  4. c KCN 1.00 • Isolated step in intact isolated mitochondria: • TMPD+Ascorbate • Cyanide titration 0.75 0.50 0.25 0 10 20 0.00 H+ Antimycin A Cytochrome c Oxidase KCN Titration aa3 F1 TMPD Ascorbate O2 ADP ATP H+ Rel. inhibition of COX KCN concentration [µM]

  5. c 1.00 2.5 2.0 0.75 Excess capacity 1.5 0.50 1.0 0.25 0.5 0 10 20 0.00 0.25 0.50 0.75 1.00 0.0 0.00 0.5 mM TMPD + 2 mM ascorbate: 2-fold relative COX capacity Electron Transport Chain and Cytochrome c Oxidase Excess capacity H+ H+ H+ I aa3 bc1 Q F DH 1 O2 ADP ATP NADH Glutamate+Malate H+ H+ Rel. inhibition of COX Flux/Complex I TMPD+Asc Glu+Mal KCN concentration [µM] Rel. inhibition of COX

  6. H+ H+ H+ c IV III aa3 bc1 I II S F O2 NADH ADP ATP F 1 H+ H+ Q Electron Transport System Which metabolic state represents electron transport capacity? A. Definition of ETS capacity. B. Measurement in mitochondria and permeabilized cells. C. Measurement in intact cells.

  7. c Conventional Protocol Derived from Bioenergetics Electron Transport Chain H+ H+ H+ I aa3 bc1 Q F DH 1 O2 ADP ATP NADH H+ H+ Bioenergetic paradigm (1): Respiratory capacity in State 3, feeding electrons specifically into complex I

  8. c II FADH2 Conventional Protocol Derived from Bioenergetics Electron Transport Chain H+ H+ aa3 bc1 Q F 1 O2 ADP ATP H+ H+ Bioenergetic paradigm (1): Respiratory capacity in State 3, feeding electrons specifically into complex I,or complex II

  9. c II FADH2 Conventional Protocol Derived from Bioenergetics Electron Transport Chain H+ H+ aa3 bc1 Q F 1 O2 ADP ATP H+ H+ Bioenergetic paradigm (1): Respiratory capacity in State 3, feeding electrons specifically into complex I,or complex II (rotenone+succinate) Then we are surprised to find ...

  10. FCCP c I DH NADH H+ 3 2 1 0 Intact versus Permeabilized Cells H+ H+ H+ aa3 bc1 Q II F 1 Uncoupled O2 Succinate Coupled ADP ATP GM H+ Permeabilized Intact In permeabilized cells, State 3 respiration(Glutamate+Malate) is short of representing respiratory capacity ofintact uncoupledcells. Respiration /GMADP Fibroblasts NIH3T3 GMADP ROUTINE uncoupl. State 3 Cell

  11. H+ H+ H+ c aa3 bc1 I II O2 S F NADH F 1 H+ H+ Q But low COX excess in intact cells„raises the critical issue of how accurately the data obtained with isolated mitochondria reflect the in vivo situation“. • Villani, Attardi (1997) PNAS 94: 1166. Contoversy on Isolated Mitochondria COX excess capacity is high in isolated mitochondria, with corresponding phenotypic threshold. • Letellier et al (1994) Biochem. J. 302: 171. • Gnaiger et al (1998) BBA 1365: 249 • Rossignol et al (2003) Biochem. J. 370: 751. • Antunes et al (2004) PNAS 101: 16774.

  12. Controversy Living cells vs isolated mitochondria Bioenergetic paradigm (2) of substrate/uncoupler combinations which yield maximum flux in: • Intact cells: • Villani and Attardi (1997) PNAS • Permeabilized muscle fibers: • Kunz et al (2000) JBC • Isolated mitochondria: Rasmussen et al (2001) AJP

  13. 360 Rot FCCP Oligo-mycin 270 180 Respiration[pmol·s-1·10-6] Routine Ama 90 0 But intact cells do not have uncoupled mitochondria ! 0 20 40 60 80 Time [min] Oxidative Phosphorylation in Top Gear Gold standard to assess maximum aerobic capacity in cultured cells: • Uncoupled flux • Villani, Attardi (1997) PNAS 94: 1166

  14. Oxidative Phosphorylation in Top Gear – Mitochondrial Physiology Gold standard to assess maximum aerobic capacity in humans: • VO2 max Electron Transport Coupled to ATP Synthesis

  15. H+ H+ H+ c aa3 bc1 I II O2 S F NADH F 1 H+ H+ Q OXPHOS and Respiratory Capacity Oxidative Phosphorylation: Coupling O2 ATP ATP H+ H+ O2 Δp

  16. H+ H+ c aa3 bc1 I O2 Glycolysis GpDH Gp PDH OXPHOS H+ MDH NADH IDH Q GDH F 4 : 1 1 ODH ADP Succinate H+ H+ II II β-Oxidation SDH ETF Convergent Convergent Linear Coupled p. 24 Mitochondrial Pathways Convergent Redox and ET System www.oroboros.at/index.php?id=mipnet-publications

  17. H+ H+ H+ c IV III aa3 bc1 I II F 1 O2 S F NADH ADP ATP H+ H+ Q Question 1 ETS How do we measure mitochondrial electron transport capacity? • Mitochondria • Intact cells

  18. H+ H+ c aa3 bc1 O2 I II F 1 S F 2- 2- Pi Pi H+ H+ Q II MitoPathways Succinate + Rotenone Oxaloacetate2- NADH Malate2- Fumarate2- FADH2 Succinate2- Succinate2- www.oroboros.at/index.php?id=mipnet-publications

  19. H+ H+ H+ c aa3 bc1 I II O2 S F NADH F 1 H+ H+ 2- 2- Pi Pi Q MitoPathways Pyruvate+Malate+Succinate, PMS Pyruvate- H+ Pyruvate- NADH Malate2- H+ Acetyl-CoA Oxaloacetate2- H+ Citrate3- NADH Malate2- Malate2- HCO3- NADH 2-Oxoglutarate2- Fumarate2- NADH Malate2- CO2 FADH2 Succinate2- Succinate2- www.oroboros.at/index.php?id=mipnet-publications

  20. H+ H+ H+ c aa3 bc1 I O2 NADH F 1 2- 2- Pi Pi H+ H+ Q MitoPathways Pyruvate+Malate, PM Pyruvate- H+ Pyruvate- NADH Malate2- H+ Acetyl-CoA Oxaloacetate2- H+ Citrate3- NADH Malate2- Malate2- HCO3- NADH 2-Oxoglutarate2- Fumarate2- Complex II is not active in respiration on pyruvate + malate. NADH FADH2 Succinate2- CO2 Malate2- www.oroboros.at/index.php?id=mipnet-publications

  21. 2- 2- Pi Pi + NH4 MitoPathways Glutamate+Malate+Succinate, GMS Glutamate- H+ Malate2- Glutamate- H+ Oxaloacetate2- Aspartate- NADH Malate2- 2-Oxoglutarate2- NADH Fumarate2- Malate2- NADH CO2 FADH2 Glutamate- Glutamate- H+ Succinate2- H+ Succinate2- www.oroboros.at/index.php?id=mipnet-publications

  22. CI Substrates Permeab. GMN +ADP +c +uncoupler GM+Dig ADP 250 F F 200 c 150 100 50 0 200 160 CI CI CI 120 80 LEAK OXPHOS ETS 40 0 0:20 0:30 0:40 0:50 1:00 1:10 High-Resolution Respirometry in Permeabilized Cells Cytochrome c test: Intact mitochondrial outer membrane O2 Flow per cells O2Concentration Time [h:min] endogen. ROUTINE

  23. PC GMN +D +c +u +S +Rot +Ama Rot F S Ama GM+Dig D S1 250 F F 200 c 150 100 50 0 200 160 CI CI CI CI+II CII 120 80 LEAK OXPHOS ETS 40 0 0:20 0:30 0:40 0:50 1:00 1:10 High-Resolution Respirometry in Permeabilized Cells ETS capacity with CI+II substrates CeR O2 Flow per cells O2Concentration Time [h:min] endogen. ROUTINE

  24. H+ H+ H+ H+ H+ c c IV III aa3 bc1 aa3 bc1 I I II F 1 O2 O2 S F NADH NADH ADP ATP H+ H+ II S F H+ Q Q F 1 FADH2 H+ H+ Reference State ETS Maximum electron transport capacity is obtained with convergent CI+II electron input. 1 CI+II:

  25. H+ H+ H+ H+ H+ c c IV III aa3 bc1 aa3 bc1 I I II F 1 O2 O2 S F NADH NADH ADP ATP H+ H+ H+ Q Q F 1 H+ H+ Q-Junction Ratio ETS With CI substrates, respiration is limited to 0.70 of ETS capacity. 0.70 CI: 1 CI+II:

  26. H+ H+ H+ H+ H+ c c IV III aa3 bc1 aa3 bc1 I II F 1 O2 O2 S F NADH ADP ATP H+ H+ II S F Q Q F 1 FADH2 H+ H+ Q-Junction Ratio ETS With CII substrates, respiration is limited to 0.36 of ETS capacity. 1 CI+II: 0.36 CII:

  27. H+ H+ H+ H+ H+ c c IV III aa3 bc1 aa3 bc1 I I II F 1 O2 O2 S F NADH NADH ADP ATP H+ H+ II S F H+ Q Q F 1 FADH2 H+ H+ Q-Junction Ratio ETS Convergent CI+II electron input exerts an additive effect in human fibroblasts. 0.70 CI: 1 CI+II: 0.36 CII:

  28. ? ? ? Pyruvate Succinate Malate CII H+ Isocitrate CI Glutamate 3-Hydroxyacyl CoA Fatty acyl CoA 3-Glycerol phosphate Electron Transport: from Chain to System www.oroboros.at/index.php?id=mipnet-publications • Electron Transport Chain • Electron Transport System, ETS Uncoupler H+ H+ c CIV aa3 CIII bc1 Q NADH O2 H+ Convergent Electron Flux and the Q-junction

  29. The most frequent misnomer in bioenergetics: Electron Transport Chain

  30. ETS E

  31. H+ H+ H+ c IV III aa3 bc1 I II F 1 O2 S F NADH ADP ATP H+ H+ Q Question 1 ETS How do we measure mitochondrial electron transport capacity? • Mitochondria • Intact cells

  32. Uncoupling Oligomycin FCCP 250 200 150 100 50 0 LEAK ETS 200 160 0:00 0:30 1:00 1:30 2:00 2:30 3:00 120 80 40 0 High-Resolution Respirometry in Intact Cells Fibroblasts NIH3T3 Respiration [pmol∙s-1∙10-6 cells] O2 Concentration Cells Time [h:min] ROUTINE Gnaiger E (2008) In: Mitochondrial Dysfunction in Drug-Induced Toxicity. (Dykens JA, Will Y, eds) John Wiley.

  33. 250 200 150 100 0 50 100 150 200 250 50 0 Mitochondrial Pathways and Q-Junction ETS CI+II: Glutamate+Malate+Succinate uncoupled ETS capacities were identical in intact and permeabilized cells, with convergent electron flow through Complexes I and II (CI+II e-input). Perm. cells [pmol∙s-1∙10-6 cells] Intact cells [pmol∙s-1∙10-6 cells]

  34. L/R R/E L/E Identical ETS Capacity in Permeabilized and Intact Cells A: Permeabilized Cells B: Intact Cells Control Coupling ROUTINE ETS LEAK ETS R E L E uncoupler Oligomycin Culture medium CI+II combined 0.32 0.29 0.29 1 0.09 1 0.09 186 199 pmol∙s-1∙10-6 cells

  35. Uncoupling FCCP 250 200 150 100 50 O2 ATP 0 ETS 200 160 0:00 0:30 1:00 1:30 2:00 2:30 3:00 H+ 120 H+ State 3? 80 ADP 40 OXPHOS Capacity ? 0 High-Resolution Respirometry in Intact Cells Fibroblasts NIH3T3 Oligomycin Respiration [pmol∙s-1∙10-6 cells] O2 Concentration Cells Time [h:min] ROUTINE LEAK

  36. H+ H+ H+ c IV III aa3 bc1 I II F 1 O2 S F NADH ADP ATP H+ H+ Q Question 2 OXPHOS How do we measure OXPHOS capacity? • Mitochondria • In intact cells ?

  37. 250 0:15 0:30 0:45 1:00 1:15 1:30 1:45 200 150 100 50 0 200 160 120 80 40 0 High-Resolution Respirometry in Permeabilized Cells OXPHOS capacity is less than ETS ADP-stimulated uncoupled ETS OXPHOS Omy Glutamate +Malate O2 Concentration ADP O2 Flow per cells Digitonin c ADP F Succinate FCCP Rot Time [h:min] CI CI+II CI+II CII

  38. OXPHOS P

  39. L/R L/P P/E R/E L/E L/E R/E R/P 0.58 0.29 Reserve capacity is overestimated 2-fold Flux Control Diagrams for Permeabilized and Intact Cells A: Permeabilized Cells B: Intact Cells Control Coupling ROUTINE LEAK OXPHOS ETS LEAK ETS P R L E L E ADP uncoupler uncoupler Oligomycin Glutamate+Malate+Succinate Culture medium CI+II combined 0.20 0.32 0.29 0.10 0.50 1 0.09 1 0.09 0.10

  40. H+ H+ H+ c IV III aa3 bc1 I II F 1 O2 S F NADH ADP ATP H+ H+ 0.50 O2 ATP P E Q H+ H+ OXPHOS The phosphorylation system exerts strong control over OXPHOS in human fibroblasts.

  41. H+ H+ H+ c IV III aa3 bc1 I II F 1 O2 S F NADH ADP ATP H+ H+ Q Question 3 LEAK How do we express respiratory coupling ratios? • Mitochondria • Intact cells

  42. 250 0:15 0:30 0:45 1:00 1:15 1:30 1:45 200 150 100 50 0 200 160 120 80 40 0 High-Resolution Respirometry in Permeabilized Cells L/Eratio but not L/Pratio reflects the relative LEAK. ADP-stimulated uncoupled ETS OXPHOS Omy Glutamate +Malate O2 Concentration ADP O2 Flow per cells Digitonin c ADP F Rot Succinate FCCP Time [h:min] CI CI CI+II CI+II CII

  43. ADP L/P P/E L/E 0.25 4.0 0.55 E Limitation by the phosphorylation system Respiratory Control Ratio (State 3/State 4) is the inverse L/P ratio ETS Capacity versus OXPHOS Capacity Coupling LEAK OXPHOS ETS P Control L E ADP uncoupler Substrate L P Glutamate +Malate Permeabilized Cells, NIH3T3 Fibroblasts

  44. ADP L/P P/E L/E 0.25 0.55 0.14 E L/E ratio expresses uncoupling 7.1 Respiratory Control Ratio should be the inverse L/E ratio ETS Capacity versus OXPHOS Capacity Coupling LEAK OXPHOS ETS P Control L E ADP uncoupler Substrate L P Glutamate +Malate Permeabilized Cells, NIH3T3 Fibroblasts

  45. 250 200 150 100 0 50 100 150 200 250 50 0 Mitochondrial Pathways and Q-Junction LEAK ETS capacities and LEAK respiration were identical in intact and permeabilized cells, with convergent electron flow through Complexes I and II (CI+II e-input) CI+II: GMSE CI+II: GMSL CI: GML Perm. cells [pmol∙s-1∙10-6 cells] CrE Intact cells [pmol∙s-1∙10-6 cells]

  46. LEAK L

  47. Mitochondrial Respiratory Control: The Q-Junction • Convergent e-input at the • Q-junction corresponds to the operation of the citric acid cycle. • The additive Q-junction effect and phosphorylation limitation of OXPHOS reveal an unexpected diversity of mitochondrial function. • Q-junction ratios: 0.97 to 0.5 www.oroboros.at/index.php?id=mipnet-publications

  48. Mitochondrial Respiratory Control: The Q-Junction p. 33 3. Interpretation of apparent excess capacities of ET complexes and of flux control coefficients is largely dependent on the metabolic reference state. Higher capacities with CI+II substrates explain apparent discrepancies between mitochondria and intact cells. www.oroboros.at/index.php?id=mipnet-publications

  49. Mitochondrial Respiratory Control: The Q-Junction p. 33 4. Interpretation of excess capacities of various components of the respiratory chain and of flux control coefficients is largely dependent on the metabolic reference state. Appreciation of the concept of the Q-junction will provide new insights into the functional design of the respiratory chain. www.oroboros.at/index.php?id=mipnet-publications

  50. Mitochondrial Respiratory Control: The Q-Junction p. 33 • The relation between membrane potential and flux is reversed when an increase in flux is effected by a change in substrate supply. • MultiSensor O2k: TPMP+ Mitochondrial Pathways and Respiratory Control. OROBOROS MiPNet Publ. 2007 www.oroboros.at/index.php?id=mipnet-publications

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