Cell Respiration Under Hypoxia: Facts and Artefacts in Mitochondrial Oxygen Kinetics

Conference paper
Part of the Advances in Experimental Medicine and Biology book series (AEMB, volume 662)


When oxygen supply to tissues is limiting, mitochondrial respiration and ATP production are compromised. To assess the bioenergetic consequences under normoxia and hypoxia, quantitative evaluation of mitochondrial oxygen kinetics is required. Using high-resolution respirometry, the “apparent Km” for oxygen or p50 of respiration in 32D cells was determined at 0.05 ± 0.01 kPa (0.4 mmHg, 0.5 μM, 0.25% air saturation). Close agreement with p50 of isolated mitochondria indicates that intracellular gradients are small in small cells at routine activity. At intracellular pO2 <2 kPa (15 mmHg, 10% air saturation) in various tissues under normoxia, respiration is limited by >2% with a p50 of 0.05 kPa. Over-estimation of p50 at 0.4 kPa (3 mmHg) would imply significant (>17%) oxygen limitation of respiration under intracellular normoxia. Based on a critical review, we conclude that p50 ranges from 0.01 to 0.10 kPa in mitochondria and small cells in the absence of inhibitors of cytochrome c oxidase, whereas experimental artefacts explain the controversial >200-fold range of p50 in the literature on mitochondrial oxygen kinetics.


  1. 1.
    Weibel ER, Taylor CR, Hoppeler H (1991) The concept of symmorphosis: a testable hypothesis of structure-function relationship. Proc Natl Acad Sci USA 88:10357–10361.PubMedCrossRefGoogle Scholar
  2. 2.
    Gnaiger E, Lassnig B, Kuznetsov AV et al. (1998) Mitochondrial oxygen affinity, respiratory flux control and excess capacity of cytochrome c oxidase. J Exp Biol 201:1129–1139.PubMedGoogle Scholar
  3. 3.
    Gnaiger E, Steinlechner-Maran R, Méndez G et al. (1995) Control of mitochondrial and cellular respiration by oxygen. J Bioenerg Biomembr 27:583–596.PubMedCrossRefGoogle Scholar
  4. 4.
    Takahashi E, Endoh H, Doi K (2000) Visualization of myoglobin-facilitated mitochondrial O2 delivery in a single isolated cardiomyocyte. Biophys J 78:3252–3259.PubMedCrossRefGoogle Scholar
  5. 5.
    Takahashi E (2008) Anoxic cell core can promote necrotic cell death in cardiomyocytes at physiological extracellular PO2. Am J Physiol Heart Circ Physiol 294:H2507–H2515.PubMedCrossRefGoogle Scholar
  6. 6.
    Gnaiger E (2003) Oxygen conformance of cellular respiration. A perspective of mitochondrial physiology. Adv Exp Med Biol 543:39–55.PubMedGoogle Scholar
  7. 7.
    Chance B, Williams GR (1955) Respiratory enzymes in oxidative phosphorylation. III. The steady state. J Biol Chem 217:409–427.PubMedGoogle Scholar
  8. 8.
    Puchowicz MA, Varnes ME, Cohen BH et al. (2004) Oxidative phosphorylation analysis: assessing the integrated functional activity of human skeletal muscle mitochondria-case studies. Mitochondrion 4:377–385.PubMedCrossRefGoogle Scholar
  9. 9.
    Gnaiger E, ed (2007) Mitochondrial pathways and respiratory control. OROBOROS MiPNet Publications, Innsbruck.Google Scholar
  10. 10.
    Chance B (1965) Reaction of oxygen with the respiratory chain in cells and tissues. J Gen Physiol 49:163–195.PubMedCrossRefGoogle Scholar
  11. 11.
    Gnaiger E (2001) Bioenergetics at low oxygen: dependence of respiration and phosphorylation on oxygen and adenosine diphosphate supply. Respir Physiol 128:277–297.PubMedCrossRefGoogle Scholar
  12. 12.
    Steinlechner-Maran R, Eberl T, Kunc M et al. (1996) Oxygen dependence of respiration in coupled and uncoupled endothelial cells. Am J Physiol 271:C2053–C2061.Google Scholar
  13. 13.
    Gnaiger E, Lassnig B, Kuznetsov AV, Margreiter R (1998) Mitochondrial respiration in the low oxygen environment of the cell. Effect of ADP on oxygen kinetics. Biochim Biophys Acta 1365:249–254.PubMedCrossRefGoogle Scholar
  14. 14.
    Hütter E, Renner K, Pfister G, Stöckl P et al. (2004) Senescence-associated changes in respiration and oxidative phosphorylation in primary human fibroblasts. Biochem J 380:919–928.PubMedCrossRefGoogle Scholar
  15. 15.
    Gnaiger E (2008) Polarographic oxygen sensors, the oxygraph, and high-resolution respirometry to assess mitochondrial function. In: Dykens JA, Will Y (ed) Mitochondrial dysfunction in drug-induced toxicity. Wiley, New York.Google Scholar
  16. 16.
    Renner K, Amberger A, Konwalinka G et al. (2003) Changes of mitochondrial respiration, mitochondrial content and cell size after induction of apoptosis in leukemia cells. Biochim Biophys Acta 1642:115–123.PubMedCrossRefGoogle Scholar
  17. 17.
    Schindler FJ (1967) Determination of oxygen affinity. Methods Enzymol 10:629–634.CrossRefGoogle Scholar
  18. 18.
    Rumsey WL, Schlosser C, Nuutinen EM et al. (1990) Cellular energetics and the oxygen dependence of respiration in cardiac myocytes isolated from adult rat. J Biol Chem 265:15392–15402.PubMedGoogle Scholar
  19. 19.
    Cornish-Bowden A (1995) Fundamentals of enzyme kinetics. Portland Press, London.Google Scholar
  20. 20.
    Troppmair J, Rapp UR (2003) Raf and the road to cell survival: a tale of bad spells, ring bearers and detours. Biochem Pharmacol 66:1341–1345.PubMedCrossRefGoogle Scholar
  21. 21.
    Sugano T, Oshino N, Chance B (1974) Mitochondrial functions under hypoxic conditions. The steady states of cytochrome c reduction and of energy metabolism. Biochim Biophys Acta 347:340–358.PubMedCrossRefGoogle Scholar
  22. 22.
    Verkhovsky MI, Morgan JE, Puustinen A, Wikström M (1996) Kinetic trapping of oxygen in cell respiration. Nature 380:268–270.PubMedCrossRefGoogle Scholar
  23. 23.
    Riistama S, Puustinen A, García-Horsman A et al. (1996) Channelling of dioxygen into the respiratory enzyme. Biochim Biophys Acta 1275:1–4.PubMedCrossRefGoogle Scholar
  24. 24.
    Hütter E, Renner K, Jansen-Dürr P, Gnaiger E (2002) Biphasic oxygen kinetics of cellular respiration and linear oxygen dependence of antimycin A inhibited oxygen consumption. Mol Biol Rep 29:83–87.PubMedCrossRefGoogle Scholar
  25. 25.
    Pecina P, Gnaiger E, Zeman J et al. (2004) Decreased affinity for oxygen of cytochrome-c oxidase in Leigh syndrome caused by SURF1 mutations. Am J Physiol Cell Physiol 287:C1384–C1388.PubMedCrossRefGoogle Scholar
  26. 26.
    Laisk A, Oja V, Eichelmann H (2007) Kinetics of leaf oxygen uptake represent in planta activities of respiratory electron transport and terminal oxidases. Physiol Plant 131:1–9.PubMedCrossRefGoogle Scholar
  27. 27.
    Gnaiger E, Mendez G, Hand SC (2000) High phosphorylation efficiency and depression of uncoupled respiration in mitochondria under hypoxia. Proc Natl Acad Sci USA 97:11080–11085.PubMedCrossRefGoogle Scholar
  28. 28.
    St-Pierre J, Tattersall GJ, Boutilier RG (2000) Metabolic depression and enhanced O2 affinity of mitochondria in hypoxic hypometabolism. Am J Physiol Regul Integr Comp Physiol 279:R1205–R1214.PubMedGoogle Scholar
  29. 29.
    Mik EG, Stap J, Sinaasappel M et al. (2006) Mitochondrial PO2 measured by delayed fluorescence of endogenous protoporphyrin IX. Nature Methods 3:939–945.PubMedCrossRefGoogle Scholar
  30. 30.
    Longmuir IS (1957) Respiration rate of rat-liver cells at low oxygen concentrations. Biochem J 65:378–382.PubMedGoogle Scholar
  31. 31.
    Mendez G, Gnaiger E (1994) How does oxygen pressure control oxygen flux in isolated mitochondria? A methodological approach by high-resolution respirometry and digital data analysis. In Gnaiger E, Gellerich FN, Wyss M (ed) What is controlling life? Modern Trends BioThermoKinetics, Innsbruck University Press, Innsbruck.Google Scholar
  32. 32.
    Costa LE, Mendez G, Boveris A (1997) Oxygen dependence of mitochondrial function measured by high-resolution respirometry in long-term hypoxic rats. Am J Physiol 273:C852–C858.PubMedGoogle Scholar
  33. 33.
    Palacios-Callender M, Quintero M, Hollis VS et al. (2004) Endogenous NO regulates superoxide production at low oxygen concentrations by modifying the redox state of cytochrome c oxidase. Proc Natl Acad Sci USA 101:7630–7635.PubMedCrossRefGoogle Scholar
  34. 34.
    Hollis VS, Palacios-Callender M, Springett RJ et al. (2003) Monitoring cytochrome redox changes in the mitochondria of intact cells using multi-wavelength visible light spectroscopy. Biochim Biophys Acta 1607:191–202.PubMedCrossRefGoogle Scholar
  35. 35.
    Presley T, Kuppusamy P, Zweier JL, Ilangovan G (2006) Electron paramagnetic resonance oximetry as a quantitative method to measure cellular respiration: a consideration of oxygen diffusion interference. Biophys J 91:4623–4631.PubMedCrossRefGoogle Scholar
  36. 36.
    Gnaiger E, Forstner H (1983) Polarographic Oxygen Sensors. Aquatic and Physiological Applications. Springer, Berlin.Google Scholar
  37. 37.
    Cole RC, Sukanek PC, Wittenberg JB, Wittenberg BA (1982) Mitochondrial function in the presence of myoglobin. J Appl Physiol 53:1116–1124.PubMedGoogle Scholar
  38. 38.
    Hoffman DL, Salter JD, Brookes PS (2007) Response of mitochondrial reactive oxygen species generation to steady-state oxygen tension: implications for hypoxic cell signaling. Am J Physiol Heart Circ Physiol 292:H101–H108.PubMedCrossRefGoogle Scholar
  39. 39.
    Brookes PS, Kraus DW, Shiva S et al. (2003) Control of mitochondrial respiration by NO, effects of low oxygen and respiratory state. J Biol Chem 278:31603–31609.PubMedCrossRefGoogle Scholar
  40. 40.
    Kreutzer U, Jue T (1995) Critical intracellular O2 in myocardium as determined by 1H nuclear magnetic resonance signal of myoglobin. Am J Physiol 268:H1675–H1681.PubMedGoogle Scholar
  41. 41.
    Richardson RS, Leigh JS, Wagner PD, Noyszewski EA (1999) Cellular PO2 as a determinant of maximal mitochondrial O2 consumption in trained human skeletal muscle. J Appl Physiol 87:325–331.PubMedGoogle Scholar
  42. 42.
    Marcinek DJ, Ciesielski WA, Conley KE, Schenkman KA (2003) Oxygen regulation and limitation to cellular respiration in mouse skeletal muscle in vivo. Am J Physiol Heart Circ Physiol 285:H1900–H1908.PubMedGoogle Scholar
  43. 43.
    Wittenberg BA, Wittenberg JB (1985) Oxygen pressure gradients in isolated cardiac myocytes. J Biol Chem 260:6548–6554.PubMedGoogle Scholar
  44. 44.
    Benard G, Rossignol R (2008) Ultrastructure of the mitochondrion and its bearing on function and bioenergetics. Antiox Redox Signaling 10:1313–1342.CrossRefGoogle Scholar
  45. 45.
    Gjedde A, Johannsen P, Cold GE, Ostergaard L (2005) Cerebral metabolic response to low blood flow: possible role of cytochrome oxidase inhibition. J Cereb Blood Flow Metab 25:1183–1196.PubMedCrossRefGoogle Scholar
  46. 46.
    Molé PA, Chung Y, Tran TK et al. (1999) Myoglobin desaturation with exercise intensity in human gastrocnemius muscle. Am J Physiol 277:R173–R180.PubMedGoogle Scholar
  47. 47.
    Mik EG, Johannes T, Zuurbier CJ et al. (2008) In vivo mitochondrial oxygen tension measured by a delayed fluorescence lifetime technique. Biophys J 95:3977–3990.PubMedCrossRefGoogle Scholar
  48. 48.
    Arthur PG, Ngo C-T, Moretta P, Guppy M (1999) Lack of oxygen sensing by mitochondria in platelets. Eur J Biochem 266:215–219.PubMedCrossRefGoogle Scholar
  49. 49.
    Kwast KE, Burke PV, Staahl BT, Poyton RO (1999) Oxygen sensing in yeast: evidence for the involvement of the respiratory chain in regulating the transcription of a subset of hypoxic genes. Proc Natl Acad Sci USA 96:5446–5451.PubMedCrossRefGoogle Scholar
  50. 50.
    Fukuda R, Zhang H, Kim J-W et al. (2007) HIF-1 regulates cytochrome oxidase subunits to optimize efficiency of respiration in hypoxic cells. Cell 129:111–122.PubMedCrossRefGoogle Scholar
  51. 51.
    Barzu O, Satre M (1970) Determination of oxygen affinity of respiratory systems using oxyhemoglobin as oxygen donor. Anal Biochem 36:428–433.PubMedCrossRefGoogle Scholar
  52. 52.
    Degn H, Wohlrab H (1971) Measurement of steady-state values of respiration rate and oxidation levels of respiratory pigments at low oxygen tensions. A new technique. Biochim Biophys Acta 245:347–355.CrossRefGoogle Scholar
  53. 53.
    Robiolio M, Rumsey WL, Wilson DF (1989) Oxygen diffusion and mitochondrial respiration in neuroblastoma cells. Am J Physiol 256:C1207–C1213.PubMedGoogle Scholar
  54. 54.
    Steinlechner R, Eberl T, Margreiter R, Gnaiger E (1994) Oxygen dependence of cellular respiration in endothelial cells: a sensitive toxicological test. In Gnaiger E, Gellerich FN, Wyss M (ed) What is controlling life? 50 years after Erwin Schrödinger’s What is Life? Innsbruck University Press, Innsbruck.Google Scholar
  55. 55.
    Mertens S, Noll T, Spahr R et al. (1990) Energetic response of coronary endothelial cells to hypoxia. Am J Physiol 258:H689–H694.PubMedGoogle Scholar
  56. 56.
    Wilson DF, Rumsey WL, Green TJ, Vanderkooi J (1988) The oxygen dependence of mitochondrial oxidative phosphorylation measured by a new optical method for measuring oxygen concentration. J Biol Chem 263:2712–2718.PubMedGoogle Scholar
  57. 57.
    Jones CI, Han Z, Presley T et al. (2008) Endothelial cell respiration is affected by the oxygen tension during shear exposure: role of mitochondrial peroxynitrite. Am J Physiol Cell Physiol 295:C180–C191.PubMedCrossRefGoogle Scholar

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© Springer Science+Business Media, LLC 2010

Authors and Affiliations

  1. 1.Department of General and Transplant Surgery, D. Swarovski Research LaboratoryMedical University of InnsbruckInnsbruckAustria
  2. 2.D. Swarovski Research Laboratory, Department of General and Transplant SurgeryMedical University of InnsbruckInnsbruckAustria
  3. 3.OROBOROS INSTRUMENTSInnsbruckAustria

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