Textbook on Mitochondrial Physiology:

Mitochondrial Respiratory Control

Erich Gnaiger

Daniel Swarovski Research Laboratory, Department of General and Transplant Surgery, Medical University Innsbruck, Austria; erich.gnaiger@i-med.ac.at

(C) 2009 Copyright remains with the author.

Refer to: Gnaiger E (2009) Mitochondrial respiratory control. In: Textbook on Mitochondrial Physiology (Gnaiger E, ed) Mitochondr. Physiol. Soc. electronic ed: www.mitophysiology.org.

    Oxidative phosphorylation (OXPHOS) is a key element of bioenergetics, extensively studied to resolve mechanisms of energy transduction and respiratory control in the electron transport system (ETS).  Electron transport capacity is quantified as oxygen consumption in uncoupled mitochondria or cells (ETS; State E).  In contrast, maximum ADP-stimulated respiration is a measure of OXPHOS capacity (P; State 3).  P/E ratios yield an index of OXPHOS limitation by the phorsphorylation system.

    Respiratory steady states have been defined by Chance and Williams (1955) according to a protocol for oxygraphic experiments with isolated mitochondria, for studies of mitochondrial respiratory control [1].  As simple as they may appear, measurements of respiratory States (1-2-3-4) and respiratory control ratios (RCR) need to be well designed and concepts require clarification in the context of modern literature [2,3].

    In the absence of ADP, respiration compensates mainly for proton leak (LEAK respiration, L), thus maintaining a high mitochondrial membrane potential. The L/E flux control ratio was 0.10±0.01 SD (N=5) in fibroblast mitochondria respiring on glutamtame+malate+succinate (GMS [2]).  At a L/E ratio of 0.10, the LEAK respiration is 10% of ETS capacity, indicating tight coupling of mitochondria in permeabilized cells.  The L/E ratio (0.09±0.02 SD; N=18) was identical in intact cells, evaluated by inhibition by oligomycin (L) and stimulation by uncoupler (E).  Conventionally, ADP stimulation is expressed by the respiratory control ratio (RCR = State 3/State 4), which is frequently used as an index of coupling for diagnosis of mitochondrial defects.  Compared to the L/E ratio of 0.10, the State 4/State 3 (L/P) ratio with glutamate+malate was 0.25 (RCR=4.0).  RCR yields a severe underestimation of coupling, since the L/P ratio relates LEAK respiration to OXPHOS (rather than uncontrolled maximum ETS) capacity.  The RCR is useful only in the limiting case when the P/E ratio is 1.0 and ETS capacity is not limited by substrate supply or ETS defects [3].

    With proper expression of normalized respiratory fluxes (the inverse of the conventional RCR), a linear relationship exists between P/O ratios and ‘RCR’ [4]. Changes of the ‘RCR’ may not merely be caused by uncoupling, but frequently are caused by alterations of catalytic OXPHOS capacities, including the phosphorylation system [3,4]. Interpretation of ‘RCR’, therefore, is complicated in cases of multiple mitochondrial defects.  For experimental examples on measurements of P/O ratios [4] and respiratory and uncoupling control ratios, see ref. [6-8].

    Coupled OXPHOS flux was 0.50±0.09 of ETS capacity in permeabilized NIH3T3 fibroblasts respiring on glutamate+malate+succinate (GMS), reflecting control of the phosphorylation system over OXPHOS in this human cell line [2].  Electrons flow to oxygen from Complex I or II with three or two coupling sites.  Compared to ETS capacity in intact cells [5], conventional State 3 respiration in permeabilized cells was only 0.38±0.06 with ADP and glutamate+malate.  ETS capacities were identical in intact and permeabilized uncoupled cells, however, with convergent electron flow to the Q-junction from glutamate+malate+succinate through Complexes I and II (CI+II e-input [2]).  Convergent CI+II e-input provides the relevant basis for quantifying enzymatic thresholds and excess capacities of individual steps of OXPHOS, and for evaluation of mitochondrial defects.  Convergent CI+II e-input corresponds to operation of the tricarboxylic acid cycle and mitochondrial substrate supply in vivo and yields novel insights into the physiological diversity of mitochondria from various tissues.  Multiple substrate-uncoupler-inhibitor titration (SUIT) protocols and advanced OXPHOS flux control analysis extend the diagnostic potential of mitochondrial physiology in health and disease [3,9,10].

Keywords: Electron transport system, flux control ratio, uncoupling, leak, respiratory control ratio, multiple substrate-uncoupler-inhibitor tirations, Q-junction, fibroblasts.



References

  1. Chance B, Williams GR (1955) Respiratory enzymes in oxidative phosphorylation. III. The steady state. J. Biol. Chem. 217: 409-427.
  2. Gnaiger E (2008) Respiratory states and flux control ratios. In: Mitochondrial Pathways and Respiratory Control. OROBOROS MiPNet Publications, Innsbruck, 2nd ed: pp. 43-50.
  3. Gnaiger E (2009) Capacity of oxidative phosphorylation in human skeletal muscle. New perspectives of mitochondrial physiology. Int. J. Biochem. Cell Biol. doi:10.1016/j.biocel.2009.03.013
  4. Gnaiger E (2001) Bioenergetics at low oxygen: dependence of respiration and phosphorylation on oxygen and adenosine diphosphate supply. Respir. Physiol. 128: 277-297.
  5. Gnaiger E (2008) Polarographic oxygen sensors, the oxygraph and high-resolution respirometry to assess mitochondrial function. In: Mitochondrial Dysfunction in Drug-Induced Toxicity (Dykens JA, Will Y, eds) John Wiley (in press).
  6. Hütter E, Renner K, Pfister G, Stöckl P, Jansen-Dürr P, Gnaiger E (2004) Senescence-associated changes in respiration and oxidative phosphorylation in primary human fibroblasts. Biochem. J. 380: 919-928.
  7. Kuznetsov AV, Schneeberger S, Seiler R, Brandacher G, Mark W, Steurer W, Saks V, Usson Y, Margreiter R, Gnaiger E (2004) Mitochondrial defects and heterogeneous cytochrome c release after cardiac cold ischemia and reperfusion. Am. J. Physiol. Heart Circ. Physiol. 286: H1633–H1641.
  8. Stadlmann S, Rieger G, Amberger A, Kuznetsov AV, Margreiter R, Gnaiger E (2002) H2O2-mediated oxidative stress versus cold ischemia-reperfusion: mitochondrial respiratory defects in cultured human endothelial cells. Transplantation 74: 1800-1803.
  9. Boushel R, Gnaiger E, Schjerling P, Skovbro M, Kraunsøe R, Dela F (2007) Patients with Type 2 Diabetes have normal mitochondrial function in skeletal muscle. Diabetologia 50: 790-796.
  10. Aragonés J, Schneider M, Van Geyte K, Fraisl P, Dresselaers T, Mazzone M, Dirkx R, Zacchigna S, Lemieux H, Jeoung NH, Lambrechts D, Bishop T, Lafuste P, Diez-Juan A, K Harten S, Van Noten P, De Bock K, Willam C, Tjwa M, Grosfeld A, Navet R, Moons L, Vandendriessche T, Deroose C, Wijeyekoon B, Nuyts J, Jordan B, Silasi-Mansat R, Lupu F, Dewerchin M, Pugh C, Salmon P, Mortelmans L, Gallez B, Gorus F, Buyse J, Sluse F, Harris RA, Gnaiger E, Hespel P, Van Hecke P, Schuit F, Van Veldhoven P, Ratcliffe P, Baes M, Maxwell P, Carmeliet P (2008) Deficiency or inhibition of oxygen sensor Phd1 induces hypoxia tolerance by reprogramming basal metabolism. Nature Genetics 40: 170-180.



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