Gnaiger 2003 Adv Exp Med Biol

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Gnaiger E (2003) Oxygen conformance of cellular respiration. A perspective of mitochondrial physiology.

» Adv Exp Med Biol 543:39-55. PMID: 14713113, Bioblast pdf

Gnaiger Erich (2003) Adv Exp Med Biol

Abstract: Oxygen pressure declines from normoxic air-level to the microenvironment of mitochondria where cytochrome c oxidase (CIV) reduces oxygen to water at oxygen levels as low as 0.3 kPa (2 Torr; 3 μM; 1.5 % air saturation). Intracellular hypoxia is defined as (1) local oxygen pressure below normoxic reference states, or (2) limitation of mitochondrial respiration by oxygen levels below kinetic saturation, resulting in oxyconformance. High-resolution respirometry provides the methodology to measure mitochondrial and cellular oxygen kinetics in the relevant low oxygen range <1 kPa (7.5 mmHg; 9-10 μM; 5 % air saturation). Respiration of isolated heart mitochondria follows hyperbolic oxygen kinetics with half-saturating oxygen pressure, p50, of 0.04 kPa (0.3 Torr; 0.4 μM) in the ADP-stimulated state OXPHOS. Thus mitochondrial respiration proceeds at 90 % of its hyperbolic maximum at the p50 of myoglobin, suggesting the possibility of a small but significant oxygen limitation even under normoxia in active muscle. Any impairment of oxygen delivery, therefore, induces oxyconformance. In addition, a shift of mitochondrial oxygen kinetics to the right, particularly by competitive inhibition of CIV by NO, causes a further depression of respiration and a compensatory increase of local oxygen pressure. Above 1 kPa, mitochondrial oxygen uptake increases above hyperbolic saturation, which is probably due to oxygen radical production rather than the kinetics of CIV. In cultured cells, the pronounced oxygen uptake above mitochondrial saturation at air-level oxygen pressure cannot be inhibited by rotenone and antimycin A, amounting to > 20% of ROUTINE respiration in fibroblasts. Biochemical models of oxyconformance of CIV are evaluated relative to patterns of intracellular oxygen distribution in the tissue and enzyme turnover in vivo, considering the kinetic effects of CIV excess capacity on flux through the mitochondrial electron transfer system. Keywords: Oxygen kinetics, Cytochrome c oxidase, Mitochondrial respiratory control, Oxygen limitation, Hypoxia

O2k-Network Lab: AT Innsbruck Gnaiger E

Cited by

Gnaiger 2020 BEC MitoPathways

Gnaiger E (2020) Mitochondrial pathways and respiratory control. An introduction to OXPHOS analysis. 5th ed. Bioenerg Commun 2020.2.
  • Komlódi T, Sobotka O, Gnaiger E (2021) Facts and artefacts on the oxygen dependence of hydrogen peroxide production using Amplex UltraRed. Bioenerg Commun 2021.4. https://doi:10.26124/BEC:2021-0004
  • Komlódi T, Gnaiger E (2022) Discrepancy on oxygen dependence of mitochondrial ROS production - review. MitoFit Preprints 2022 (in prep).

Updated terminology

Caption text
Term Updated (2020) Publication (2003)
cytochrome c oxidase CIV COX
CI Complex I complex I
R ROUTINE respiration routine respiration
P OXPHOS State 3
ETS electron transfer system electron transport chain
Rox residual oxygen consumption non-COX respiration
noncoupled uncoupled
amol·s-1·x-1 pmol·s-1·Mx-1 pmol·s-1·10-6 cells
O2k Oroboros Oxygraph-2k OROBOROS Oxygraph
mtIM mitochondrial inner membrane inner mitochondrial membrane


  • A synkinetic systems approach is required to explain tissue-specific differences in mitochondrial oxygen affinity, which is a function of the properties of the electron transport {transfer} pathway [25, 26]. The excess capacity of COX {CIV} ensures that this enzyme operates far from its limiting turnover capacity even at maximum activity of the respiratory chain {system}. When the excess capacity of COX {CIV} is reduced, then COX {CIV} is pushed to increasing turnover at identical rates of mitochondrial respiration. As a consequence, the mitochondrial p50 declines {Correction: The oxygen affinity declines, but the mitochondrial p50 increases instead of declining}. Downregulation of cytochrome c oxidase activity, therefore, increases the degree of oxyconformance in the low-oxygen range (Figure 1).

Selected quotes

  • The high affinity of cytochrome c oxidase for oxygen implies independence of mitochondrial respiration of oxygen over a wide range of oxygen levels, which gives rise to the paradigm of “oxygen regulation“, although “kinetic oxygen saturation” describes more accurately the underlying mechanism. In contrast, various degrees of oxyconformance are observed in cells [2, 9, 28, 33, 36]. Biochemical and physiological approaches are required to separate the primary kinetic mechanisms from secondary effects of oxygen sensing, signalling, gene expression and protein synthesis or degradation. Modern trends in mitochondrial bioenergetics integrate (1) molecular and enzyme kinetic properties of the membrane proteins constituting the electron transfer system, particularly the proton pumps such as cytochrome c oxidase [70], (2) synkinetic properties of the mitochondrial metabolic network involved in the control of flux and energetic efficiency [26, 27], and (3) the regulatory role of mitochondrial signalling in the cell and of intracellular conditions in the tissue.
  • Several apparent paradoxes have emerged in the physiology and pathology of hypoxia, such as the oxygen, lactate, efficiency, and diving paradoxes [32]. While some have been rationalized and solved, others remain hot spots of current research. Another apparent paradox on hypoxia arises in studies of the bioenergetics of isolated and cultured cells, where respiration, contractile performance or protein synthesis are apparently oxygen limited at partial pressures at or above normoxic tissue levels. Such extended oxygen conformance deviates from the “regulatory” pattern or oxygen independence of mitochondrial respiration to <1 kPa (7.5 mmHg [28]).
  • Compared with ambient oxygen pessure of 20 kPa (150 mmHg), oxygen levels are low within active tissues and are under tight control by microcirculatory adjustments to match oxygen supply and demand. Alveolar normoxia of 13 kPa (100 mmHg) contrasts with a corresponding 1 to 5 kPa (10 to 40 mmHg) extracellular pO2 in solid organs such as heart, brain, kidney and liver [19].
  • .. respiration inhibited by antimycin A and particularly by cyanide cannot simply be interpreted as non-mitochondrial respiration. Caution is required since cyanide is not specific for cytochrome c oxidase but is a direct inhibitor of other oxidases, such as urate oxidase [56] and inhibits the heme-containing catalase [20].
  • Diffusion limitation is further aggravated in permeabilized fiber bundles with a radius of 35 up to 200 µm [38, 52]. For comparison, 200 µm away from the nearest blood vessel, the pO2 drops from 1.9 kPa (14 mmHg) to zero in tumors with relatively low aerobic capacity [30].
  • Relative to isolated mitochondria, a staggering 100-fold increase of the extracellular p50 is measured in heavily stirred permeabilized fiber bundles prepared from rat heart and soleus muscle [39], in which case oxyconformance extends up to air saturation in terms of a monophasic hyperbolic oxygen dependence (Fig. 6).
  • Increased diffusion distances are in line with the distinct kinetic responses to external oxygen, when highly oxygen-independent fibroblasts and endothelial cells are compared with oxyconforming cardiomyocytes and fiber bundles (Figs. 4 and 6), spanning a 0.1·106-fold volume range (Table 1).
  • It remains to be defined, how low the pO2 needs to be set in the incubation medium to provide a “normoxic” environment for embryonic cardiomyocytes.
  • The adaptive mechanisms of metabolic downregulation in hypometabolic states of hypoxia [31], however, are more clearly appreciated by relating physiological and biochemical control mechanisms to the diversity of oxygen regimes and metabolic challenges met by various types of mitochondria, cells, tissues and organisms.

Keywords: Oxia terms


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Bioblast links: Hypoxia, normoxia, hyperoxia - >>>>>>> - Click on [Expand] or [Collapse] - >>>>>>>
AerobicoxThe aerobic state of metabolism is defined by the presence of oxygen (air) and therefore the potential for oxidative reactions (ox) to proceed, particularly in oxidative phosphorylation (OXPHOS). Aerobic metabolism (with involvement of oxygen) is contrasted with anaerobic metabolism (without involvement of oxygen): Whereas anaerobic metabolism may proceed in the absence or presence of oxygen (anoxic or oxic conditions), aerobic metabolism is restricted to oxic conditions. Below the critical oxygen pressure, aerobic ATP production decreases.
AnaerobicAnaerobic metabolism takes place without the use of molecular oxygen, in contrast to aerobic metabolism. The capacity for energy assimilation and growth under anoxic conditions is the ultimate criterion for facultative anaerobiosis. Anaerobic metabolism may proceed not only under anoxic conditions or states, but also under hyperoxic and normoxic conditions (aerobic glycolysis), and under hypoxic and microxic conditions below the limiting oxygen pressure.
AnoxiaanoxIdeally the terms anoxia and anoxic (anox, without oxygen) should be restricted to conditions where molecular oxygen is strictly absent. Practically, effective anoxia is obtained when a further decrease of experimental oxygen levels does not elicit any physiological or biochemical response. The practical definition, therefore, depends on (i) the techiques applied for oxygen removal and minimizing oxygen diffusion into the experimental system, (ii) the sensitivity and limit of detection of analytical methods of measuring oxygen (O2 concentration in the nM range), and (iii) the types of diagnostic tests applied to evaluate effects of trace amounts of oxygen on physiological and biochemical processes. The difficulties involved in defining an absolute limit between anoxic and microxic conditions are best illustrated by a logarithmic scale of oxygen pressure or oxygen concentration. In the anoxic state (State 5), any aerobic type of metabolism cannot take place, whereas anaerobic metabolism may proceed under oxic or anoxic conditions.
Critical oxygen pressurepcThe critical oxygen pressure, pc, is defined as the partial oxygen pressure, pO2, below which aerobic catabolism (respiration or oxygen consumption) declines significantly. If anaerobic catabolism is activated simultaneously to compensate for lower aerobic ATP generation, then the limiting oxygen pressure, pl, is equal to the pc. In many cases, however, the pl is substantially lower than the pc.
HyperoxiahyperoxHyperoxia is defined as environmental oxygen pressure above the normoxic reference level. Cellular and intracellular hyperoxia is imposed on isolated cells and isolated mitochondria at air-level oxygen pressures which are higher compared to cellular and intracellular oxygen pressures under tissue conditions in vivo. Hyperoxic conditions may impose oxidative stress and may increase maximum aerobic performance.
HypoxiahypoxHypoxia (hypox) is defined in respiratory physiology as the state when insufficient O2 is available for respiration, compared to environmental hypoxia defined as environmental oxygen pressures below the normoxic reference level. Three major categories of hypoxia are (1) environmental hypoxia, (2) physiological tissue hypoxia in hyperactivated states (e.g. at VO2max) with intracellular oxygen demand/supply balance at steady state in tissues at environmental normoxia, compared to tissue normoxia in physiologically balanced states, and (3) pathological tissue hypoxia including ischemia and stroke, anaemia, chronic heart disease, chronic obstructive pulmonary disease, severe COVID-19, and obstructive sleep apnea. Pathological hypoxia leads to tissue hypoxia and heterogenous intracellular anoxia. Clinical oxygen treatment ('environmental hyperoxia') may not or only partially overcome pathological tissue hypoxia.
Intracellular oxygenpO2,iPhysiological, intracellular oxygen pressure is significantly lower than air saturation under normoxia, hence respiratory measurements carried out at air saturation are effectively hyperoxic for cultured cells and isolated mitochondria.
Limiting oxygen pressureplThe limiting oxygen pressure, pl, is defined as the partial oxygen pressure, pO2, below which anaerobic catabolism is activated to contribute to total ATP generation. The limiting oxygen pressure, pl, may be substantially lower than the critical oxygen pressure, pc, below which aerobic catabolism (respiration or oxygen consumption) declines significantly.
MicroxiamicroxMicroxia (deep hypoxia) is obtained when trace amounts of O2 exert a stimulatory effect on respiration above the level where metabolism is switched to a purely anaerobic mode.
Normoxia is a reference state, frequently considered as air-level oxygen pressure at sea level (c. 20 kPa in water vapor saturated air) as environmental normoxia. Intracellular tissue normoxia is variable between organisms and tissues, and intracellular oxygen pressure is frequently well below air-level pO2 as a result of cellular (mainly mitochondrial) oxygen consumption and oxygen gradients along the respiratory cascade. Oxygen pressure drops from ambient normoxia of 20 kPa to alveolar normoxia of 13 kPa, while extracellular normoxia may be as low as 1 to 5 kPa in solid organs such as heart, brain, kidney and liver. Pericellular pO2 of cells growing in monolayer cell cultures may be hypoxic compared to tissue normoxia when grown in ambient normoxia (95 % air and 5 % CO2) and a high layer of culture medium causing oxygen diffusion limitation at high respiratory activity, but pericellular pO2 may be effectively hyperoxic in cells with low respiratory rate with a thin layer of culture medium (<2 mm). Intracellular oxygen levels in well-stirred suspended small cells (5 - 7 mm diameter; endothelial cells, fibroblasts) are close to ambient pO2 of the incubation medium, such that matching the experimental intracellular pO2 to the level of intracellular tissue normoxia requires lowering the ambient pO2 of the medium to avoid hyperoxia.
» Oxygen, dioxygen, O2
» Intracellular oxygen
» Oxygen pressure
» Oxygen solubility
» Gas pressure
» pascal
» Pressure
» Barometric pressure
» Concentration
Related keyword lists
» Keywords: Oxygen signal
» Keywords: Concentration and pressure

Publications: Tissue normoxia

Donnelly 2022 MitoFit Hypoxia2022-06-28Donnelly C, Schmitt S, Cecatto C, Cardoso LHD, Komlodi T, Place N, Kayser B, Gnaiger E (2022) The ABC of hypoxia – what is the norm. stress;RONS
DiProspero 2021 Toxicol In Vitro2021DiProspero TJ, Dalrymple E, Lockett MR (2021) Physiologically relevant oxygen tensions differentially regulate hepatotoxic responses in HepG2 cells. cellsHypoxia
Stepanova 2020 Methods Cell Biol2020Stepanova A, Galkin A (2020) Measurement of mitochondrial H2O2 production under varying O2 tensions. systemIsolated mitochondriaOxidative stress;RONS
Ast 2019 Nat Metab2019Ast T, Mootha VK (2019) Oxygen and mammalian cell culture: are we repeating the experiment of Dr. Ox? Nat Metab 1:858-860.
Keeley 2019 Physiol Rev2019Keeley TP, Mann GE (2019) Defining physiological normoxia for improved translation of cell physiology to animal models and humans.
Stepanova 2018 J Cereb Blood Flow Metab2018Stepanova A, Konrad C, Guerrero-Castillo S, Manfredi G, Vannucci S, Arnold S, Galkin A (2018) Deactivation of mitochondrial complex I after hypoxia-ischemia in the immature brain. J Cereb Blood Flow Metab 39:1790-802.RatNervous systemIsolated mitochondriaHypoxia
Stuart 2018 Oxid Med Cell Longev2018Stuart JA, Fonseca JF, Moradi F, Cunningham C, Seliman B, Worsfold CR, Dolan S, Abando J, Maddalena LA (2018) How Supraphysiological Oxygen Levels in Standard Cell Culture Affect Oxygen-Consuming Reactions. Oxid Med Cell Longev 2018:8238459.
Stepanova 2018 J Neurochem2018Stepanova A, Konrad C, Manfredi G, Springett R, Ten V, Galkin A (2018) The dependence of brain mitochondria reactive oxygen species production on oxygen level is linear, except when inhibited by antimycin A. J Neurochem 148:731-45.MouseNervous systemIsolated mitochondriaIschemia-reperfusion
Oxidative stress;RONS
Stepanova 2017 J Cereb Blood Flow Metab2017Stepanova A, Kahl A, Konrad C, Ten V, Starkov AS, Galkin A (2017) Reverse electron transfer results in a loss of flavin from mitochondrial complex I: Potential mechanism for brain ischemia-reperfusion injury. J Cereb Blood Flow Metab 37:3649-58.MouseNervous systemIsolated mitochondriaIschemia-reperfusion
Harrison 2015 J Appl Physiol2015Harrison DK, Fasching M, Fontana-Ayoub M, Gnaiger E (2015) Cytochrome redox states and respiratory control in mouse and beef heart mitochondria at steady-state levels of hypoxia.
HeartIsolated mitochondriaHypoxia
Carreau 2011 J Cell Mol Med2011Carreau A, El Hafny-Rahbi B, Matejuk A, Grillon C, Kieda C (2011) Why is the partial oxygen pressure of human tissues a crucial parameter? Small molecules and hypoxia.
Richardson 2006 J Physiol2006Richardson RS, Duteil S, Wary C, Wray DW, Hoff J, Carlier PG (2006) Human skeletal muscle intracellular oxygenation: the impact of ambient oxygen availability. J Physiol 571:415-24. doi: 10.1113/jphysiol.2005.102327HumanSkeletal muscleHypoxia
Pettersen 2005 Cell Prolif2005Pettersen EO, Larsen LH, Ramsing NB, Ebbesen P (2005) Pericellular oxygen depletion during ordinary tissue culturing, measured with oxygen microsensors. Cell Prolif 38:257-67.
Gnaiger 2003 Adv Exp Med Biol2003Gnaiger E (2003) Oxygen conformance of cellular respiration. A perspective of mitochondrial physiology.
Endothelial;epithelial;mesothelial cell
Intact cells
Permeabilized cells
Permeabilized tissue
Isolated mitochondria
Oxidase;biochemical oxidation
Gnaiger 2001 Respir Physiol2001Gnaiger E (2001) Bioenergetics at low oxygen: dependence of respiration and phosphorylation on oxygen and adenosine diphosphate supply.
Endothelial;epithelial;mesothelial cell
Intact cells
Isolated mitochondria
Oxidative stress;RONS
Gnaiger 2000 Proc Natl Acad Sci U S A2000Gnaiger E, Méndez G, Hand SC (2000) High phosphorylation efficiency and depression of uncoupled respiration in mitochondria under hypoxia.
LiverIsolated mitochondria
Gnaiger 1998 J Exp Biol1998Gnaiger E, Lassnig B, Kuznetsov AV, Rieger G, Margreiter R (1998) Mitochondrial oxygen affinity, respiratory flux control, and excess capacity of cytochrome c oxidase.
Endothelial;epithelial;mesothelial cell
Isolated mitochondria
Oxidase;biochemical oxidation
Intact cells
Gnaiger 1998 Biochim Biophys Acta1998Gnaiger 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 1365:249-54.RatHeart
Isolated mitochondria
Gnaiger 1995 J Bioenerg Biomembr1995Gnaiger E, Steinlechner-Maran R, Méndez G, Eberl T, Margreiter R (1995) Control of mitochondrial and cellular respiration by oxygen. J Bioenerg Biomembr 27:583-96.Human
Endothelial;epithelial;mesothelial cell
Isolated mitochondria
Intact cells
Gnaiger 1993 Transitions1993Gnaiger E (1993) Homeostatic and microxic regulation of respiration in transitions to anaerobic metabolism. In: The vertebrate gas transport cascade: Adaptations to environment and mode of life. Bicudo JEPW (ed), CRC Press, Boca Raton, Ann Arbor, London, Tokyo:358-70.Reptiles
Intact organism
Gnaiger 1991 Soc Exp Biol Seminar Series1991Gnaiger E (1991) Animal energetics at very low oxygen: Information from calorimetry and respirometry. In: Strategies for gas exchange and metabolism. Woakes R, Grieshaber M, Bridges CR (eds), Soc Exp Biol Seminar Series 44, Cambridge Univ Press, London:149-71.AnnelidsIntact organism
Gnaiger 1983 J Exp Zool1983Gnaiger E (1983) Heat dissipation and energetic efficiency in animal anoxibiosis. Economy contra power. J Exp Zool 228:471-90.Annelids
Skeletal muscleIntact organism
Abstracts: Tissue normoxia
Donnelly 2022 Abstract Bioblast20222.1. «10+5»
Donnelly Chris
Donnelly Chris, Schmitt S, Cecatto C, Cardoso L, Komlodi T, Place N, Kayser B, Gnaiger E (2022) The ABC of hypoxia – what is the norm. Bioblast 2022: BEC Inaugural Conference. In:
»MitoFit Preprint«
Oxidative stress;RONS
Gnaiger 2018 AussieMit2018Komlodi Timea, Sobotka Ondrej, Doerrier Carolina, Gnaiger Erich (2018) Mitochondrial H2O2 production is low under tissue normoxia but high at in-vitro air-level oxygen pressure - comparison of LEAK and OXPHOS states. AussieMit 2018 Melbourne AU.Mouse
Saccharomyces cerevisiae
Nervous system
Isolated mitochondria
Intact cells
Oxidative stress;RONS
Sobotka 2018 MiP20182018
Ondrej Sobotka
Measurement of ROS production under hypoxia and unexpected methodological pitfalls of Amplex UltraRed assay.
Saccharomyces cerevisiae
Nervous system
Isolated mitochondriaHypoxia
Komlodi 2017 MiP20172017
Timea Komlodi
H2O2 production under hypoxia in brain and heart mitochondria: does O2 concentration matter?
Nervous system
Isolated mitochondriaOxidative stress;RONS

Labels: MiParea: Respiration 

Organism: Human, Rat  Tissue;cell: Heart, Liver, Endothelial;epithelial;mesothelial cell, Fibroblast  Preparation: Permeabilized cells, Permeabilized tissue, Isolated mitochondria, Oxidase;biochemical oxidation, Intact cells 

Regulation: Oxygen kinetics  Coupling state: ROUTINE, OXPHOS  Pathway: N, ROX  HRR: Oxygraph-2k 

Tissue normoxia, BEC 2020.2, MitoFit 2021 AmR-O2, MitoFit2022Hypoxia, MitoFit 2022 ROS review