1. Department of Biochemistry, University of Cambridge Nitric oxide and other gas interactions with mitochondria Guy Brown www.mitophysiology.org/index.php?id=mip-textbook

2. NADH QH 2 c IV III I NO CO HCN H 2 S O 2 H 2 O out in Mitochondrial inner membrane Gases bind to the oxygen binding site of mitochondrial complex IV, blocking energy production, in the same way as hypoxia. This may be one way in which our cells regulate their energy production. It may also be one way in which our body kills pathogens. However, it may also kill our cells in disease.

3. Path of electrons through Complex IV Electrons pass from c > Cu A > a > a 3 /Cu B. Consumes 90% of our oxygen. Major generator of proton motive force. Figure from Lehninger: Principles of Biochemistry

4. Gas binding to complex IV N O Binuclear centre consists of heme a 3 and Cu B. O 2 binds only when both Fe & Cu reduced. NO & CO bind when Fe reduced (Fe 2+ ). HCN & H 2 S bind when Fe oxidised (Fe 3+ ).

5. Vascular relaxation Synaptic modulation Anti-inflammatory Induction of HO-1 causes small inhibition of cellular respiration at low O 2. Probably insignificant in vivo due to CO binding to haemoglobin. haem CO biliverdin Fe 2+ haem oxygenase sGC or Cyt Ox? low sensitivity CO – CARBON MONOXIDE O2O2

6. Synaptic modulation Estimated 5  M in brain & 1  M in blood, but may be bound (e.g. to metHb). Cellular respiration is half inhibited by about 10-50  M cyanide Probably insignificant in vivo. ? HCN H2O2H2O2 Peroxidase in neutrophils & neurons? catalase Cyt Ox? HCN – HYDROGEN CYANIDE

7. Estimated 1-10  M in aorta. Cellular respiration is half inhibited by 10-30  M. Lower concentrations are rapidly oxidised by mitochondria. High concentration induce suspended animation state. cysteine H2SH2S cystathionine beta-synthase (CBS) or cystathionine gamma lyase (CSE) K ATP channels Cyt Ox? H 2 S – HYDROGEN SULPHIDE Vascular relaxation Synaptic modulation Heart contractility

8. InhibitorMechanism K i k on (M -1 s -1 )k off (s -1 ) HCNNon- competitive 200 nM2 x 10 3 5 x 10 -4 H 2 SNon- competitive 200 nM10 4 10 -3 COCompetitive with O 2 200 nM10 5 2 x 10 -2 NOCompetitive with O 2 0.2 nM10 8 2 x 10 -2 Cooper C.E. & Brown G.C. (2008) The inhibition of mitochondrial cytochrome oxidase by the gases carbon monoxide, nitric oxide, hydrogen cyanide and hydrogen sulfide: J. Bioenergetics & Biomembranes

9. InhibitorMechanism K i Light sensitive In vivo nM HCNNon- competitive 200 nMNo< 1000 H 2 SNon- competitive 200 nMNo< 1000 COCompetitive with O 2 200 nMYes< 1000 NOCompetitive with O 2 0.2 nMYes< 100

10. NADH QH 2 c IV III I HCN H 2 S O 2 H 2 O out in Mitochondrial inner membrane Summary NO & CO inhibition is rapidly reversed by O 2, light or Hb. HCN & H 2 S inhibition is slow & insensitive to O 2, light or Hb. It is unclear whether gas inhibition is significant in vivo. NO CO H2SH2S

11. NADH QH 2 c IV III I NO O 2 H 2 O out in Mitochondrial inner membrane Nitric oxide (NO) and its derivatives have 3 major effects on mitochondria: 1.NO inhibition of cytochrome oxidase 2.SNO inactivation of complex I 3.ONOO - activation of permeability transition. SNO e-e- e-e- NO ONOO - cGMP PTP

12. eNOS nNOS iNOS mtNOS? NO Ca 2+ ˜ P Ca 2+ Cytokines, pathogens Sources of nitric oxide (NO)

13. NO Fe 2+ -NO Iron nitrosyl ONOO - Peroxynitrite NO 2 RSNO S-nitrosothiol Fe 2+ O2-O2- O2O2 RS. Targets/reactions of nitric oxide (NO) N2O3N2O3 nitration R-NO 2 + nitrosation

14. Ca 2+ /Calmodulin Phosphorylation eNOS nNOS Cytokines Pathogens Inflammation iNOS Soluble guanylate cyclase Smooth muscle relaxation Neuromodulation Platelet aggregation ? Low, transient levels High, sustained levels Function of NO: “The double-edged sword” Pathogen & Host: Cell Death/Cytostasis

15. Ca 2+ /Calmodulin Phosphorylation eNOS nNOS Cytokines Pathogens Inflammation iNOS Soluble guanylate cyclase Mitochondria? Pathogen & Host: Cell Death/Cytostasis Low, transient levels High, sustained levels Function of NO: “The double-edged sword” Smooth muscle relaxation Neuromodulation Platelet aggregation

16. Ca 2+ /Calmodulin Phosphorylation eNOS nNOS Cytokines Pathogens Inflammation iNOS Soluble guanylate cyclase Mitochondria? Pathogen & Host: Cell Death/Cytostasis Low, transient levels High, sustained levels Function of NO: “The double-edged sword” Smooth muscle relaxation Neuromodulation Platelet aggregation Biogenesis Protection

17. Brown, G. C. (2007) Nitric oxide and mitochondria. Front. Biosci. 12, 1024-33.

18. NO effects on mitochondria: Inhibition of respiration O 2 -, H 2 O 2 & ONOO - production Mitochondrial permeability transition

19. Cytochrome oxidase is inhibited by NO Inhibition is rapid. Inhibition is potent. Inhibition is reversible when NO gone. Inhibition reversible by light. Inhibition at oxygen binding site in competition with O 2. Brown, G. C. & Cooper, C. E. (1994) Nanomolar concentrations of nitric oxide reversibly inhibit synaptosomal respiration by competing with oxygen at cytochrome oxidase. FEBS Lett. 356, 295-298.

20. NO inhibition of cytochrome oxidase is competitive with O 2, raising the K M of respiration into the physiological range. Brown, G. C. & Cooper, C. E. (1994) Nanomolar concentrations of nitric oxide reversibly inhibit synaptosomal respiration by competing with oxygen at cytochrome oxidase. FEBS Lett. 356, 295-298.

21. eNOS regulates cellular respiration and its sensitivity to oxygen in cultured endothelial cells NO may be a physiological regulator of respiration and its affinity for oxygen Clementi, E., Brown, G. C., Foxwell, N. & Moncada, S. (1999) On the mechanism by which vascular endothelial cells regulate their oxygen consumption. Proc. Natl. Acad. Sci. USA 96, 1559-1562.

22. Oxyhemoglobin Cells Oxygen trace NO trace 2 min 0.4  M NO 50  M O 2 Aortic endothelial cells activated with LPS+IFN  produce NO from iNOS that continuously inhibits respiration until reversed by oxyhaemoglobin. Borutaite, V., Matthias, A., Harris, H., Moncada, S. & Brown, G. C. (2001) Reversible inhibition of cellular respiration by nitric oxide in vascular inflammation. Am. J. Physiol. 281, H2256-H2260.

23. The oxygen consumption of aortic endothelial cells is inhibited by LPS/IFN  -induced iNOS induction, and reversed by the NO scavenger haemoglobin Borutaite, V., Matthias, A., Harris, H., Moncada, S. & Brown, G. C. (2001) Reversible inhibition of cellular respiration by nitric oxide in vascular inflammation. Am. J. Physiol. 281, H2256-H2260.

24. Activated astrocytes reversibly inhibit cellular respiration via NO Brown, G. C., Bolanos, J. P., Heales, S. J. R. & Clark, J. B. (1995) Nitric oxide produced by activated astrocytes rapidly and reversibly inhibits cellular respiration. Neuroscience Lett. 193, 201-204.

25. Cytokine-activated macrophages express iNOS and reversibly inhibit the respiration of co-incubated cells. NO iNOS MACROPHAGE TUMOUR CELL O2O2 Brown, G. C., Foxwell, N. & Moncada, S. (1998) Transcellular regulation of cell respiration by nitric oxide generated by activated macrophages. FEBS Lett.439, 321-324.

26. I II III IV ATPase Outer membrane Inner membrane mitochondria Death? Apoptosis or Necrosis?

27. NO sensitizes isolated aorta to hypoxia-induced necrosis Time, hours LDH activity,  mol/min/mg wet weight Borutaite, V., Moncada, S. & Brown, G. C. (2005) Nitric oxide from inducible nitric oxide synthase sensitizes the inflamed aorta to hypoxic damage via respiratory inhibition. Shock 23, 319-323.

28. 25  M O 2 5 min 1  M NO a bcd The oxygen consumption of aortic rings is inhibited and oxygen-dependent after iNOS induction by LPS+IFN  +LPS/IFN  LPS/IFN  +1400W Control Borutaite, V., Moncada, S. & Brown, G. C. (2005) Nitric oxide from inducible nitric oxide synthase sensitizes the inflamed aorta to hypoxic damage via respiratory inhibition. Shock 23, 319-323.

29. NO produced by iNOS sensitizes aorta to hypoxia Time, hours LDH,  mol/min/mg ww Borutaite, V., Moncada, S. & Brown, G. C. (2005) Nitric oxide from inducible nitric oxide synthase sensitizes the inflamed aorta to hypoxic damage via respiratory inhibition. Shock 23, 319-323.

30. iNOS NO IMMUNE CELL TISSUE CELL CELL DEATH ADAPTION /REACTION Mitochondrial Cytochrome oxidase Hypoxia  Relevant to: inflammation, sepsis, ischaemia, cancer, atheroschlerosis, neurodegeneration ? Glycolysis NO reversibly inhibits mitochondrial respiration at cytochrome oxidase. NO inhibition is competitive with O 2, raising the K m of respiration into physiological range. NO from iNOS may sensitise tissues to hypoxia.

31. Do NO and hypoxia synergise to kill neurons?

32. 100  M DETA/NO +MK801 CONTROL NO donor DETA/NO synergises with hypoxia (2% O 2 ) to induce ‘apoptosis’ in CGC neurons NORMOXIA HYPOXIA Mander, P., Borutaite, V., Moncada, S. & Brown G. C. (2005) Nitric oxide from glial iNOS sensitizes neurons to hypoxic death via mitochondrial respiratory inhibition. J. Neurosci. Res. 79, 208-215.

33. HYPOXIA ALONEHYPOXIA + DETA/NO HYPOXIA + DETA/NO + DEOXYGLUCOSE NO/HYPOXIA INDUCE NEURONAL DEATH

34. NO donors lower cellular ATP in presence of glucose, but completely deplete ATP in absence of glucose.

35. 100  M DETA/NO +MK801 100  M DETA/NO +DeoxyGlucose CONTROL DeoxyGlucose NO donor DETA/NO synergises with hypoxia (2% O 2 ) and deoxyglucose to induce necrosis in CGC neurons

36. LPS/IFN  +MK801 CONTROL LPS/IFN  +1400W NO from iNOS (induced by LPS/IFN  ) synergises with hypoxia (2% O 2 ) to induce necrosis in CGC neurons

37. NO completely but reversibly inhibits neuronal respiration at cytochrome oxidase NO from activated astrocytes reversibly inhibits neuronal respiration Bal-Price, A. & Brown, G. C. (2001) Inflammatory neurodegeneration mediated by nitric oxide from activated glia, inhibiting neuronal respiration, causing glutamate release and excitoxicity. J. Neuroscience 21, 6480-6491.

38. Hypoxia induces neuronal death via inhibiting cytochrome oxidase resulting in excitotoxity Glutamate Ca 2+ NEURON mito NMDAR GluT  HYPOXIA Important in stroke and dementia? NO

39. NO causes rapid depletion of ATP in neuronal but not in astrocytic cultures 0 20 40 60 80 100 ATP level (% of control) 52010 424 (min)(hrs) neuronal cultures 0 20 40 60 80 100 ATP level (% of control) 530141624 (min) (hrs) Time of exposure to NO donor DETA/NO (500  M) astrocyte culture

40. Rapid release of glutamate from neuronal cultures induced by an NO donor DETA/NO and respiratory inhibitor myxothiazol 0 4 8 12 16 20 Glutamate 1 5 1030 4 240 min hrs Time of exposure to DETA/NO   0 5 10 15 20 Glutamate 0 15 10 30 4 24 minhrs Time of exposure to myxothiazol   Release is rapid. Over concentration range as inhibits respiration. Other respiratory inhibitors (e.g. cyanide) cause release. Release greater at low O 2. Calcium and cGMP independent.

41. nNOS is activated by NMDA receptor and might contribute to hypoxic death by sensitising cytochrome oxidase NO Glutamate Ca 2+ mito NMDAR GluT  HYPOXIA nNOS

42. Summary: NO reversibly inhibits mitochondrial respiration at cytochrome oxidase. NO inhibition is competitive with O 2, raising the K m of respiration into physiological range. NO from iNOS may sensitise cells to hypoxic/ischaemic death. Glycolytic capacity determines sensitivity and form of cell death. Relevant in ischaemic, infectious, inflammatory and neurodegerative diseases.

43. I II III IV ATPase Outer membrane Inner membrane mitochondria O2-O2- peroxynitrite GS. S-nitroso-glutathione

44. NO inactivates complex I Incubation of cells with an NO donor (0.5mM DETA/NO) for hours results in inactivation of complex I and respiration. The inactivation is speeded by depleting cellular GSH with BSO. The inactivation is reversed by DTT, GSH methylester or light. The inactivation may be due to nitrosation of complex I thiol. DETA/NO +BSO -Hb +DTT, GSH or light Clementi, E., Brown, G. C., Feelisch, M. & Moncada, S. (1998) Persistent inhibition of cell respiration by nitric oxide: Crucial role of S-nitrosylation of mitochondrial complex I and protective action of glutathione. Proc. Natl. Acad. Sci. 95, 7631-7636.

45. NO + Ca 2+, peroxynitrite or S-nitrosothiols cause inhibition of complex I and this inhibition is reversed by light and thiols Control NO+Ca or NO donor +GSH+DTT +light Complex I activity, % of the control NO+Ca 2+ SNAP ONOO - Borutaite, V., Budriunaite, A. & Brown, G. C. (2000) Reversal of nitric oxide-, peroxynitrite- and S-nitrosothiol-induced inhibition of mitochondrial respiration or complex I activity by light and thiols. Biochim. Biophys. Acta 1459,405-412.

46. S-nitrosothiol inactivation of complex I reversibly increases H 2 O 2 production by mitochondria Rate of H 2 O 2 production, RFU/min/mg 0 5 10 15 20 25 ControlSNAP +light Rotenone Borutaite, V. & Brown, G. C. (2006) S-nitrosothiol inhibition of mitochondrial complex I causes a reversible increase in mitochondrial hydrogen peroxide production. Biochim. Biophys. Acta 1757, 562-6.

47. Brown, G. C. (2007) Nitric oxide and mitochondria. Front. Biosci. 12, 1024-33.

48. NO causes H 2 O 2 production in isolated mitochondria Control +1  M NO+4  M NO H 2 O 2, nmol/min/mg Borutaite, V. & Brown, G. C. (2003) Nitric oxide induces apoptosis via hydrogen peroxide, but necrosis via energy and thiol depletion. Free Rad. Biol. Med. 35, 1457-68.

49. NO-induced apoptosis is mediated by H 2 O 2 NO increases oxidative stress in cells (DCF). NO can induce apoptosis via H 2 O 2. Cells subsequently die by necrosis preceded by energy depletion. 0.0 0.1 0.2 0.3 +Ascorbate+NAC DETA/NO donor Control Caspase DEVDase activity * * +Catalase * Borutaite, V. & Brown, G. C. (2003) Nitric oxide induces apoptosis via hydrogen peroxide, but necrosis via energy and thiol depletion. Free Rad. Biol. Med. 35, 1457-68.

50. CrK VDACBAX I II III IV ATPase ANT Permeability transition pore CP Cyt.c Outer membrane Inner membrane mitochondria APAF1 APOPTOSIS CASPASE-3 CASPASE-9 cytosol I II GS. S-nitroso-glutathione O2-O2- peroxynitrite

51. Control NOC-18 GSNO ONOO - +CsA Cytochrome c, nmol/mg protein Nitrosothiols and peroxynitrite induce opening of permeability transition pore and release of cytochrome c in isolated mitochondria

52. ControlGSNO NOC-18 * DEVDase, nmol/min/mg Nitrosothiols-induced activation of caspases is blocked by cyclosporin A Borutaite, V. & Brown, G. C. (2003) Nitric oxide induces apoptosis via hydrogen peroxide, but necrosis via energy and thiol depletion. Free Rad. Biol. Med. 35, 1457-68.

53. NO actions on mitochondria relevant to cell death Respiratory inhibition at complexes I & IV. Stimulation of oxidant production. Induction of permeability transition. NO can induce cell death by each of these means. CrK VDAC BH 3 I II III IV ATPase ANT PTP CP MnSOD Aconitase Cyt.c Outer membrane Inner membrane

54. Anna Price Vilma Borutaite Collaborations: Salvador Moncada Emilio Clementi Aviva Tolkovsky Palwinder Mander Aiste Jekabsone