Eric Gnaiger

Oxidative Phosphorylation in Top Gear. Lessons from Mitochondrial Physiology and Implications for Functional Diagnosis.

Erich Gnaiger

Innsbruck Medical University, Dept. Transplant Surgery, D. Swarovski Research Laboratory, A-6020 Innsbruck, Austria. - Este endereço de e-mail está sendo protegido de spam, você precisa de Javascript habilitado para vê-lo

Oxidative phosphorylation (OXPHOS) is a key element of bioenergetics, extensively studied to resolve the mechanisms of energy transduction in the electron transport chain and analyze various modes of mitochondrial respiratory control. OXPHOS flux control is exerted by (i) coupling of electron transport to proton translocation and ATP synthesis, (ii) catalytic capacities of respiratory complexes, carriers and transporters, (iii) kinetic regulation by ADP, oxygen and various substrates feeding electrons into the respiratory chain, and (iv) specific inhibitors such as NO. Electrons flow to oxygen along a linear thermodynamic cascade from either Complex I with three coupling sites, or from Complex II with two coupling sites. These branches of electron transport are separated by using either NADH-linked substrates or the classical succinate/rotenone combination, to analyze site-specific H+/e and P/O ratios or defects of specific respiratory complexes in functional diagnosis. The experimental separation of branched electron transport is common to the extent of establishing a bioenergetic paradigm in studies of OXPHOS. This bioenergetic paradigm is extended by a novel perspective of mitochondrial physiology emerging from a series of studies based on high-resolution respirometry (OROBOROS Oxygraph-2k).

      In NIH3T3 fibroblasts, uncoupled oxygen flux in intact cells was 2.5-fold higher than coupled respiration (state 3), measured in digitonin permeabilized cells with saturating ADP and Complex I substrates (glutamate+malate) [1]. This raised doubts on conventional respiratory protocols which do not reflect the high electron transport capacity of cells in vivo. The apparent discrepancy between intact cells and mitochondria was resolved by combining concepts of mitochondrial physiology and bioenergetics: (i) Parallel electron input through respiratory Complexes I and II (CI+II e-input; with glutamate+malate+succinate) exerted an additive effect on respiratory flux, increasing coupled respiration 1.3-fold. (ii) Since mitochondrial oxidation is coupled to phosphorylation in living cells, uncoupled respiration represents OXPHOS capacity only in cases when the phosphorylation system does not exert control over coupled respiration. When the flux control coefficient of any component of the phosphorylation system (adenylate nucleotide translocase, phosphate carrier, ATP synthase) is not zero, however, then maximum OXPHOS capacity can be estimated only in the coupled state. In fibroblasts, coupled respiration with parallel CI+II e-input was indeed limited by the phosphorylation system, as shown by a 2-fold stimulation of respiration by uncoupling with FCCP. Complete agreement was established between unpermeabilized and permeabilized cells when measured in comparable uncoupled states with parallel CI+II e-input [1].

      An identical pattern was observed in permeabilized human skeletal muscle fibers [2], consistent with published results on isolated mitochondria [3]. Compared to fibroblasts, stimulation of coupled flux by parallel CI+II e-input was increased from 1.3- to 1.6-fold, while the effect of uncoupling was correspondingly less. In mouse heart, finally, the stimulatory effect of parallel CI+II e-input was even more pronounced (2-fold), while control by the phosphorylation system was diminished almost completely [4].

      In summary, these results on the operation of OXPHOS in top gear resolve discrepancies between intact cells and mitochondria. The additive effect of parallel electron input into Complexes I+II indicates a high down-stream excess capacity of respiratory chain complexes including cytochrome c oxidase (COX). Parallel CI+II e-input exerts the most pronounced stimulatory effect on coupled respiration when the phosphorylation system exerts low flux control. Parallel CI+II e-input corresponds to the operation of the citric acid cycle and mitochondrial substrate supply in vivo, and thus extends conventional respiratory protocols of bioenergetics in studies of mitochondrial physiology applied for the diagnosis of respiratory control in health and disease. Importantly, by establishing the reference state of maximum coupled respiration, parallel CI+II e-input provides the proper basis for (i) quantifying excess capacities of Complexes III and IV, (ii) interpreting flux control by various components such as the phosphorylation system or COX (particularly under the control of NO), and (iii) for evaluation of specific enzymatic defects in the context of mitochondrial respiratory physiology and pathology.

1. Naimi A, Garedew A, Troppmair J, Boushel R, Gnaiger E (2005) Limitation of aerobic metabolism by the phosphorylation system and mitochondrial respiratory capacity of fibroblasts in vivo. The coupled reference state and reinterpretation of the uncoupling control ratio. Mitochondr. Physiol. Network 10.9: 55-57. www.mitophysiology.org.

2.   Gnaiger E, Wright-Paradis C, Sondergaard H, Lundby C, Calbet JA, Saltin B, Helge J, Boushel R (2005) High-resolution respirometry in small biopsies of human muscle: correlations with body mass index and age. Mitochondr. Physiol. Network 10.9: 14-15. www.mitophysiology.org.

3. Rasmussen UF, Rasmussen HN, Krustrup P, Quistorff B, Saltin B, Bangsbo J (2001) Aerobic metabolism of human quadriceps muscle: in vivo data parallel measurements on isolated mitochondria. Am. J. Physiol. Endocrinol. Metab. 280: E301–E307.

4.   Lemieux H, Garedew A, Blier PU, Tardif J-C, Gnaiger E (2006) Temperature effects on the control and capacity of mitochondrial respiration in permeabilized fibers of the mouse heart (submitted).

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