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Control of OXPHOS efficiency by complex I in brain mitochondria Tiziana Cocco, Consiglia Pacelli, Paola Sgobbo, Gaetano Villani ∗ Department of Medical Biochemistry, Biology & Physics, University of Bari, Piazza G. Cesare, 70124 Bari, Italy Received 20 April 2007; received in revised form 28 June 2007; accepted 8 August 2007 Available online 17 September 2007
Abstract In the present work we have analysed the efficiency (P/O ratio) of energy production by oxidative phosphorylation (OXPHOS) in rat brain, liver and heart mitochondria. This study has revealed tissue-specific differences in the mean values of P/O ratios and ATP production rates. A marked dependence of the P/O ratio on the respiration rates has been observed with complex I (NADH:ubiquinone oxidoreductase), but not with complex II (succinate dehydrogenase) respiratory substrates. The physiological impact of the P/O variations with complex I substrates has been further confirmed by extending the analysis to brain mitochondria from three independent groups of animals utilized to study the effects of dietary treatments on the age-related changes of OXPHOS. The general site-specificity of the rate-dependent P/O variability indicates that the decoupling, i.e. decreased coupling between electron transfer and proton pumping, is likely to be mostly due to slip of mitochondrial complex I. These findings suggest an additional mechanism for the pivotal role played by the energy-conserving respiratory complex I in the physiological and adaptive plasticity of mitochondrial OXPHOS. © 2007 Elsevier Inc. All rights reserved. Keywords: Brain; Mitochondria; Oxidative phosphorylation (OXPHOS); Complex I; P/O ratio; Aging; Antioxidant dietary treatment
1. Introduction Mitochondrial oxidative phosphorylation (OXPHOS) represents the major source of ATP in mammalian cells relying on aerobic energy metabolism. The mitochondrial respiratory complex I (NADH:ubiquinone oxidoreductase), complex III (ubiquinol:ferricytochrome c oxidoreductase) and complex IV (cytochrome c oxidase) build up a transmembrane electrochemical potential (H+ or p) by coupling their electron transfer activities to H+ -translocation from the matrix (negative, N) to the outer (positive, P) side of the inner mitochondrial membrane. The electrochemical gradient is then utilized backward for ATP synthesis by the OXPHOS complex V (ATP synthase). The coupling mechanism between electron transfer and proton pumping activities of the mitochondrial respiratory
Abbreviations: COX, cytochrome oxidase; OXPHOS, oxidative phosphorylation; NAC, N-acetylcysteine ∗ Corresponding author. Tel.: +39 080 5448534; fax: +39 080 5448538. E-mail address:
[email protected] (G. Villani). 0197-4580/$ – see front matter © 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.neurobiolaging.2007.08.002
complexes has been the object of intense research since many years and several models have been proposed to explain the peculiar properties of this process which remains, however, still unsolved (Hosler et al., 2006; Papa et al., 2006; Wikstrom, 2004). Related to this topic, a specific controversial aspect concerns the possible physiological variability of the energy conservation efficiency of the redox-driven proton pumps. The yield of mitochondrial ATP production by OXPHOS can be influenced by several factors among which uncoupling (leak) and decoupling (slip) play a major role in the modulation of the protonmotive force (Brown, 1992; Harper and Brand, 1993; Kadenbach, 2003; Luvisetto et al., 1991; Murphy, 1989; Murphy and Brand, 1987; Pietrobon et al., 1983). Uncoupling is mostly due to modifications of the permeability properties of the mitochondrial inner membrane, resulting in an increased leak of protons, caused by physical changes in the composition and structure of the lipid bilayer as well as by the presence of uncoupling agents and/or proteins (UCPs) (Krauss et al., 2005; Lowell and Spiegelman, 2000). Therefore, uncoupling manifests itself as a general (i.e. not site-specific)
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decay of the mitochondrial energy conservation efficiency. On the other hand, decoupling of specific redox-driven proton pumps derives from “slippage” events that could be caused by the activation of intramolecular electron transfer routes not associated with proton translocation, or by mechanistic/kinetic alterations of the coupling pathways/reactions. This situation would appear as a site-specific decrease of the H+ /e− stoichiometry and of its associated ATP production efficiency. While several examples of physiological uncoupling of mitochondrial OXPHOS can be found (Kadenbach, 2003), it is not yet established whether or not OXPHOS efficiency can be naturally modulated by decoupling mechanism(s). In particular, slip of proton pumps has been proposed to occur at high values of the electron transfer rates and/or of the protonmotive force (Luvisetto et al., 1991; Murphy and Brand, 1987; Pietrobon et al., 1983), with these two parameters being functionally related to each other. A classical way to study the efficiency of mitochondrial OXPHOS is based on the measurement of the P/O ratio, namely the ATP produced per oxygen atom reduced by the respiratory chain. Also in this case, the mechanistic values of the P/O ratios, are still in question and have been recently reviewed (Hinkle, 2005; see also Lee et al., 1996). At any event, the analysis of the P/O ratio represents a more direct and physiological approach to the study of OXPHOS efficiency, as compared with the measurements of the H+ /e− stoichiometries of the proton pumps. The P/O ratio has also been shown to change by modulating the electron flux through the respiratory complexes (Fitton et al., 1994; Hinkle and Yu, 1979). At a more general level of energy metabolism, the trade-off between yield and rate of ATP production has been recently discussed in terms of its involvement in evolutionary transition from unicellular to multicellular undifferentiated heterotrophic organisms and in their environmental competition for food utilization (Pfeiffer et al., 2001). In the present work we have analysed the rate dependence of the P/O ratio in brain, heart and liver mitochondria from young (2 months) rats. Tissue-specific differences in the ATP production efficiency (P/O ratio) have been observed with complex I but not complex II substrates, with the variability of the P/O ratio correlating with the mitochondrial respiratory rate. The analysis has been then extended to brain mitochondria isolated from rats belonging to three additional independent groups utilized as animal models of different physiological studies. The rate-dependent variability of the P/O ratio by complex I substrates was remarkably present, within the same tissue, in brain mitochondria from old (28 months) rats and old rats subjected to a long-term (16 months) dietary treatment with the antioxidant N-acetylcysteine (NAC) (Cocco et al., 2005) or, respectively, to a long-term (16 months) hypocaloric diet.
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2. Experimental procedures 2.1. Animals and diet Animals (male Wistar rats), purchased from Harlan, Italy, were housed two to three animals per cage and maintained on a 12 h light/dark cycle in a temperature controlled (22 ± 1 ◦ C) room. Treatment with modified diets started at 12 months of age and continued until sacrifice. Young (2 months) and old (28 months) control rats were fed ad libitum with standard maintenance pellets, while the NAC-treated group was fed ad libitum with pellets containing 0.3% (w/w) of Nacetylcysteine (NAC) as reported by Miquel et al. (1995). The hypocaloric regimen followed the ‘every-other-day (EOD) feeding’ method, which is equivalent to a reduction of caloric intake to about 60% as compared with the ad libitum fed age-matched controls (Goodrick et al., 1983). All experiments were performed in accordance with local and national guidelines covering animal experimentation. Animals, not showing macroscopic evidence of pathologies, were sacrificed by decapitation and tissues were rapidly removed, rinsed free of blood and placed in ice-cold mitochondrial isolation buffer. Pooled tissues from two to three rats were used for each mitochondrial preparation. 2.2. Isolation of brain cortex non-synaptic mitochondria Free non-synaptic cerebral cortex mitochondria were isolated essentially as in Nagy and Delgado-Escueta (1984). All steps were carried out at 0–4 ◦ C. Brain cortex was washed with SHE medium (0.32 M sucrose, 5 mM K-Hepes, pH 7.4, 0.1 mM EDTA, 0.2 mM phenylmethylsulphonyl fluoride (PMSF from Sigma)) and homogenized in 10 volumes of the same medium. The homogenized suspension was centrifuged at 1200 × g for 4 min and the resulting supernatant was then centrifuged for 10 min at 18,000 × g to obtain the crude mitochondrial pellet and the cytosolic fraction as supernatant. The mitochondrial pellet was resuspended in 1 ml of SHE medium, diluted with 7.5 ml of 8.5% Percoll/sucrose solution (8.5% (v/v) Percoll in 0.25 M sucrose, 5 mM KHepes, pH 7.4, 0.1 mM EDTA) and finally layered into centrifuge tubes onto a preformed two-step discontinuous (8 ml of 10%, 8 ml of 20%) Percoll/sucrose gradient. After centrifugation at 22,000 × g for 25 min, the “free” mitochondria were obtained as a pellet at the bottom of the tube. The pellet was washed three times with SHE medium and one time in BSA medium (0.32 M sucrose, 5 mM K-Hepes, pH 7.4, 0.1 mM EDTA, 0.05% fatty acid free bovine serum albumin, BSA) and finally resuspended in SHE buffer at a concentration of 8–10 mg protein/ml. 2.3. Isolation of heart mitochondria Heart mitochondria were isolated by differential centrifugation, essentially as previously described (Di Paola et al., 2000). The final pellet was resuspended in 0.25 M
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sucrose, 10 mM Tris–Cl, pH 7.4, 0.25 mM phenylmethylsulphonyl fluoride, 10 M EGTA at a concentration of 20–25 mg protein/ml. This preparative procedure mostly results in the isolation of the subsarcolemmal fraction of heart mitochondria (Fannin et al., 1999). 2.4. Isolation of liver mitochondria Liver tissue was minced and homogenized in 10 volumes of isolation medium containing 0.25 M sucrose, 10 mM K-Hepes, pH 7.4, 1 mM EGTA, 0.25 mM phenylmethylsulphonyl fluoride. The homogenate was centrifuged at 1200 × g for 10 min. The resulting supernatant was centrifuged at 9500 × g for 10 min and the pellet, resuspended in the same buffer, was centrifuged at 14,000 × g for 10 min. The pellet was washed gently to remove any light or looselypacked damaged mitochondria, resuspended in the isolation buffer and centrifuged again as above. The final pellet was resuspended in isolation medium at a protein concentration of 50–60 mg/ml. All the centrifugation steps were carried out at 0–4 ◦ C. 2.5. Protein measurement Mitochondrial protein concentrations were determined according to the Bradford method (Bradford, 1976) using bovine serum albumin as standard.
7.4, 30 mM Tris–Cl, 10 M EGTA. Rat liver mitochondria were suspended at a final concentration of 0.25 mg/ml in a medium containing 0.25 M sucrose, 50 mM Hepes, pH 7.4, 10 mM K-phosphate buffer, 4 mM MgCl2 and 1 mM EGTA. For all tissues the assay mixture was supplemented with 0.3 mM P1 ,P5 -di(adenosine-5 )pentaphosphate (Ap5 A from Sigma) in order to inhibit the adenylate kinase activity. Respiration (state II) was started by the addition of pyruvate (10 mM)/malate (5 mM), in the case of brain and heart mitochondria, glutamate (5 mM)/malate (2.5 mM) for liver mitochondria, or succinate (10 mM) as complex II substrate for rotenone (1 g/ml)-inhibited mitochondria. State III respiration was induced by the addition of 250 and 100 M Mg++ -ADP for complexes I and II substrates, respectively. State IV respiration was reached when all the added ADP was finished. The P/O ratio was calculated by dividing the nmoles of added ADP by the natoms of oxygen consumed during the ADP-stimulated (state III) respiration (Chance and Williams, 1955). 2.7. Analysis of cytochrome content The mitochondrial aa3 cytochrome content was calculated, from dithionite-reduced minus ferricyanide-oxidized difference spectra performed in a Beckman DU7400, using a millimolar extinction coefficient (1 cm light path) of 24 for the absorbance differences at the wavelength pair of 605–630 nm.
2.6. Measurement of oxygen consumption rates
3. Results
The respiratory activity of freshly prepared mitochondria was measured polarographically in a Rank Brothers oxygraph (Bottisham Cambridge, England), at 25 ◦ C. Rat brain mitochondria were suspended at a final concentration of 0.2 mg/ml in a medium containing 300 mM mannitol, 0.2 mM EDTA, 10 mM K-phosphate buffer, pH 7.4, 10 mM KCl, 10 mM Tris–Cl. Rat heart mitochondria were suspended at a final concentration of 0.25 mg/ml in a medium containing 75 mM sucrose, 50 mM KCl, 2 mM KH2 PO4 , pH
An important bioenergetic parameter to be determined, when dealing with mitochondrial metabolism, is the efficiency of OXPHOS. This is usually measured as coupling of mitochondrial respiration to ATP production, namely as P/O ratio. The most classic methodology utilizes an oxygen electrode and takes advantage of the transition of the mitochondrial respiration from the so-called state IV (or state II) rate (in absence of ADP) to the faster state III rate upon addition of a discrete amount of ADP. In this way, the P/O
Fig. 1. Tissue-specific ATP production capacity and efficiency by mitochondrial OXPHOS. P/O ratios (nmole ATP/natom O) (A), ADP-stimulated (state III) respiration rates (B) and ATP production rates (C) were measured in the presence of NAD-dependent substrates (open bars) or succinate (gray bars). The P/O ratios were calculated from the traces of polarographic experiments. The ATP production rates were calculated by multiplying the individual P/O ratio values by the double of the corresponding ADP-stimulated (state III) respiration rates. The values reported are the means ± S.D. of five different experiments carried out on brain, heart and liver mitochondria from young (2 months old) rats. The tissue-specific differences were statistically significant (P < 0.05, two-way ANOVA and Bonferroni’s t-test) with the exception of the P/O ratio by succinate and of the P/O ratio by NAD-dependent substrates of heart vs. liver mitochondria. For other details see under Section 2.
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ratio is calculated by dividing the nmoles of added ADP by the natoms of oxygen consumed during the ADP-stimulated (state III) respiration (Chance and Williams, 1955). The P/O ratio mean values obtained for brain, liver and heart mitochondria isolated from young (2 months) rats are shown in Fig. 1. This analysis revealed tissue-specific differences for the P/O ratios measured in the presence of NAD-dependent respiratory substrates with brain, heart and liver mitochondria exhibiting P/O ratios of 2.0, 2.8 and 2.6, respectively. No difference could be observed when comparing the P/O ratios obtained with the same mitochondria respiring on succinate (Fig. 1A). This situation does not reflect the relative tissue-specific properties of the overall mitochondrial energy production rate. In fact, the total mitochondrial ATP power output depends not only on the OXPHOS energy conservation efficiency, but also on the capacity of the respiratory chain under phosphorylating conditions (state III respiration rates). As shown in Fig. 1B, the mitochondrial state III respiratory fluxes by NAD-dependent substrates were markedly different in the three tissues with brain, heart and liver mitochondria showing respiratory rates of 176, 99 and 57 nmole O2 × min−1 × mg(protein)−1 , respectively. A similar trend was also observed when succinate was used as respiratory substrate with brain, heart and liver mitochondria exhibiting respiratory rates of 197, 158 and 78 nmole O2 × min−1 × mg(protein)−1 , respectively. The ATP production rates of brain, heart and liver mitochondria from young rats are shown in Fig. 1C. Brain mitochondria, having the lowest P/O ratio values, but the highest respiratory rate with complex I substrates, result to be the organelles with the highest OXPHOS rate (715 nmole ATP × min−1 × mg(protein)−1 ), followed by heart and liver mitochondria (557 and respectively). 296 nmole ATP × min−1 × mg(protein)−1 , The OXPHOS rates calculated for the succinate-respiring mitochondria were very similar to those obtained with complex I substrates. On the basis of the tissue-specific differences shown in Fig. 1, we wanted to correlate the energy conservation effi-
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Fig. 2. Dependence of P/O ratios on respiratory fluxes in brain, heart and liver mitochondria from young (2 months old) rats. State III (phosphorylating conditions) respiration rates were obtained by addition of 250 or 100 M ADP for complex I (open symbols) or complex II (closed symbols) substrates in brain (, 䊉), heart (, ) and liver (, ) mitochondria. Each symbol represents an independent experiment and the oxygen consumption rate refers to the ADP-coupled (state III) respiratory rate corresponding to the relative P/O measurement. The determination was done each time in duplicate and found to be reproducible within the same mitochondrial preparation.
ciency and the capacity of the mitochondrial respiratory chain by plotting the respiration rates versus the corresponding P/O ratios for each independent mitochondrial preparation. As illustrated in Fig. 2, a rate dependence of the P/O ratio was evident with complex I, but not with complex II respiratory substrates. The points relative to the measurements carried out in the presence of NAD-dependent substrates were best-fitted by a polynomial curve rather than by a linear equation. The question arises as to the same tissue can show, under different circumstances, a “physiological” variation of the P/O ratio that could be associated with relative changes in the respiratory fluxes. For this purpose, we have extended the analysis to brain mitochondria isolated from animals utilized to study the effects of dietary treatments on the age-related decay of mitochondrial bioenergetic properties. Indeed, different P/O ratio mean values with NAD-dependent substrates
Fig. 3. Rate dependence of P/O ratios by complex I and complex II respiratory substrates in brain mitochondria. The experimental conditions are the same as in Fig. 2 for complex I (A) and complex II (B) substrates. Symbols refer to brain mitochondria from young (), old (), NAC-treated old () and hypocaloricregimen-subjected old (♦) rats. Each symbol represents an independent experiment and the oxygen consumption rate refers to the ADP-coupled (state III) respiration rate corresponding to the relative P/O measurement. The determination was done each time in duplicate and found to be reproducible within the same mitochondrial preparation.
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Fig. 4. Rate dependence of complex I-specific P/O ratio. The P/O ratios have been calculated, for each mitochondrial preparation, by subtracting the value measured with succinate to the one measured with complex I substrates and plotted vs. the corresponding respiration rates by complex I substrates. Symbols refer to brain (), heart ( ) and liver (×) mitochondria from young rats and to brain mitochondria from old (), NAC-treated old () and hypocaloric-regimen-subjected old (♦) rats.
have been obtained for brain mitochondria isolated from old rats and old rats treated with a long-term diet containing the antioxidant N-acetylcysteine (Cocco et al., 2005) as well as for old rats subjected to a long-term hypocaloric diet (our unpublished data). The plots reported in Fig. 3 confirm, even within the same tissue under different physiological conditions, the specificity of the rate-dependent variability of the P/O ratio for complex I substrates (Fig. 3A) and the absence of any significant relationship between flux and efficiency in the case of mitochondria respiring on succinate (Fig. 3B). The site-specificity of the rate-dependent variability of the P/O ratio becomes more evident by considering, for each mitochondrial preparation, the P/O ratio obtained by subtracting the value measured with succinate to the one measured with complex I substrates (Fig. 4). The range of the ratedependent variability of the P/O ratio for the mitochondrial complex I practically spans the theoretical unit contributed by this proton pump. Furthermore, we have measured the heme aa3 content so as to express the respiratory rates as COX turnover numbers and to plot them versus their corresponding P/O ratios. As shown in Fig. 5A, the P/O ratio depends on COX turnover rates by complex I substrates in a very similar way as its H+ /e− stoichiometry does (Capitanio et al., 1996), reaching maximal values at relatively low enzyme turnovers and then gradually decreasing when the respiratory flux forces the enzyme activity at higher rates. Also in this case, as expected on the basis of the previous data, the rate dependence was practically absent when using succinate as respiratory substrate (Fig. 5B). It should be also mentioned that similar results were also obtained by plotting the P/O ratios versus the bc1 electron transfer rate (data not shown).
Fig. 5. Dependence of P/O ratio on COX intrinsic activity. COX turnover numbers (COX T.N.) were calculated by dividing the individual state III respiration rates relative to each P/O measurement (expressed as nequiv. e− × s−1 × mg(protein)−1 ) by the corresponding aa3 cytochrome content (nmole aa3 /mg(protein)). Values for complex I (A) or complex II (B) respiratory substrates refer to brain (), heart ( ) and liver (×) mitochondria from young rats and to brain mitochondria from old (), NAC-treated old () and hypocaloric-regimen-subjected old (♦) rats.
4. Discussion The efficiency of mitochondrial OXPHOS mostly depends on the proton permeability of the mitochondrial inner membrane, the catalytic properties of the ATP synthase and the H+ /e− stoichiometry of the three proton pumping respiratory complexes. In the present work we have carried out a comparative analysis of OXPHOS efficiency and capacity in rat heart, liver and brain mitochondria utilizing complex I (NADdependent) or complex II (succinate) respiratory substrates. Brain mitochondria show the lowest OXPHOS efficiency (Fig. 1A) and the highest respiration rate (Fig. 1B) with complex I substrates, as compared with heart and liver mitochondria. The final energy output, resulting from the combination of both OXPHOS efficiency and capacity (Fig. 1C), points out that brain mitochondria have the highest ATP production rate with complex I substrates, followed by heart and liver mitochondria. Although the above differences reflect intrinsic mitochondrial bioenergetic properties, the physiological maximal OXPHOS capacities would finally depend on the tissue-specific mitochondrial load and, there-
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fore, on the regulation of organelle biogenesis and turnover (Benard et al., 2006). A suggestive interpretation of the tissue-specific differences of the trade-off between yield and rate of mitochondrial OXPHOS could derive from the observation that the brain has a limited defense from reactive oxygen species (Floyd and Hensley, 2002; Kaushik and Kaur, 2003; Tian et al., 1998). In line with this situation, the preference of a higher rate, rather than a better yield, to fulfil the energy demand in brain mitochondria from young rats, as compared with heart and liver mitochondria isolated from the same animals, could represent a protective mechanism against the formation of the above damaging molecules. In fact, a higher respiratory rate would lower the intracellular oxygen tension, thus decreasing the chemical reaction(s) leading to free radical production (cf. Papa and Skulachev, 1997). However, an interesting observation coming out from the above analysis is the lack of any significant difference in the P/O ratio values among the three different mitochondrial populations when using succinate as respiratory substrate (Fig. 1A). On the other hand, a trend of tissue-specific differences in succinate-dependent respiration rates similar to the one obtained with complex I substrates could be still observed. These data, while ruling out any uncoupling effect possibly associated with the intrinsic properties of the different mitochondria, suggested us to carry out a more detailed correlative analysis between respiration rates and P/O ratios. The most striking finding of our work is the physiological rate-dependent variability of the P/O ratio with complex I, but not complex II substrates. The most immediate interpretation of these data would indicate complex I as the energy conservation site responsible for the rate-dependent regulation of the mitochondrial energy conservation efficiency (Fig. 4). Given the comparable range of variability of the respiration rates measured with the different substrates, the site-specificity of the changes of the P/O ratio would seem to rule out a dependence of the catalytic efficiency of the ATP synthase on the respiratory fluxes. Mitochondrial complex I has been shown to pump protons at a stoichiometry of 2H+ /e− (Galkin et al., 1999; Wikstrom, 1984), but only a limited amount of data has been so far produced on the proton translocation mechanism of this complex multisubunit enzyme (Brandt, 2006). However, a rate-dependent variability of the H+ /e− stoichiometry from 0 to 2 was previously reported for mitochondrial complex I (Capitanio et al., 1991a,b). Similarly, rate-dependent variations of the H+ /e− ratio were previously evidentiated in rat liver mitochondria respiring on succinate (Papa et al., 1991) and associated with the variable protonmotive activity of COX (Capitanio et al., 1991c, 1996; Papa et al., 1991). However, these data were obtained by in vitro manipulation of the electron transfer rates utilizing respiratory complex inhibitors, or modulating the concentration of specific electron donors and/or redox mediators. Furthermore, the H+ /e− stoichiometry was measured in the presence of the
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K+ -ionophore valinomycin and mostly under level-flow (i.e. negligible H+ ) conditions. We also have found a very similar correlation between complex IV (Fig. 5A) or complex III (data not shown) electron transfer rates and P/O ratio with complex I substrates. However, the calculation of the intrinsic activities of the respiratory complexes obtained by direct measurements of the heme contents in the mitochondrial preparations, would start from the assumption that the enzyme molecules would all work at the same time and rate, without taking into account the possible functional heterogeneity due to post-translational enzymatic and/or chemical modifications as well as from oxidative damages of protein structures. Moreover, one should be able to identify, within the overall enzyme pool, non-functional units deriving from partially assembled complexes and/or supercomplexes (D’Aurelio et al., 2006). Nevertheless, the internal absence of the rate dependence when using succinate as respiratory substrate (Fig. 5B; data not shown for complex III), would exclude a direct involvement of COX (or complex III) in the regulation of the P/O ratio. As an alternative interpretation, it should be considered that the P/O ratios have been measured during the state III respiration, condition under which a steady-state electrochemical gradient (H+ ) would exert its control on the rate-dependent intramolecular coupling steps between electron transfer and proton translocation. The possibility that the mitochondrial transmembrane H+ does not reach a threshold value above which this specific control takes place, could be supposed in the case of succinate-sustained respiration. In fact, under equivalent respiratory fluxes, the bypass of an energy conservation site with succinate would indeed result in a lower steady-state membrane potential, as compared with complex I substrates-dependent respiration. The extent of the electrochemical gradient would also depend on the relative abundance of redox-driven proton pumps and ATP synthase in the OXPHOS functional units. These last observations would be even more significant in light of the latest information on the assembly of the respiratory supercomplexes. Interestingly, complex II seems not to be a component of the supermolecular bioenergetic unit (Schagger and Pfeiffer, 2000; Wittig et al., 2006) whose assembly has been recently proposed to represent the key determinant of cellular respiration and, therefore, of mitochondrial OXPHOS (D’Aurelio et al., 2006). In this case, it could be suggested that the physiological ratedependent regulation of OXPHOS efficiency would also depend on the physical interaction between complexes I, III, IV and V favouring the substrate channeling and resulting in a localized (rather than a delocalized bulk-phase) equilibrium between energy-producing and energy-consuming systems. At any event, even a complete loss of the proton pumping by COX could not explain, by itself, the variability range of the P/O ratio observed in our experiments. In fact, the conversion of the redox energy in the transmembrane H+ by COX derives, in the first place,
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directly from the membrane anisotropy of reduction of O2 to H2 O, whereby electrons are donated by cytochrome c on the P side of the membrane and the substrate protons are taken up from the N aqueous phase (Mitchell and Moyle, 1970). Another important observation emerging from our analysis is that the mitochondrial OXPHOS efficiency with NAD-dependent respiratory substrates can vary in response to different physiological (aging) and/or environmental conditions (dietary treatments) within the same tissue (brain). It is noteworthy that the plots shown in Figs. 2–5 take into account the internal variations among single mitochondrial preparations (from each tissue) even within the same group of animals. The internal differences follow the general correlative trend, thus suggesting that they are likely to represent specific physiological variations probably due to a relatively small interindividual variability. Functional impairment of mitochondrial complex I has been associated with a variety of human diseases and in some cases the genetic defects in its subunits encoded by the nuclear or the mitochondrial genome have been identified (Janssen et al., 2006). However, the mitochondrial complex I deficiencies so far identified are related to specific defects in its biogenesis and/or electron transfer activity. On the other hand, very little is known on the existence of tissue-specific isoforms or expression control of complex I protein subunits (Carroll et al., 2006). It is interesting noting that the translation of the mitochondrial ND5 and ND3 genes has been previously shown to be distinctively regulated in rat brain synaptic endings during the postnatal development and maturation of the animal (Polosa and Attardi, 1991). Moreover, the understanding of the functional impact and the regulation of post-translational modification of mitochondrial complex I such as protein phosphorylation (Carroll et al., 2005; Chen et al., 2004; Palmisano et al., 2007), or oxidative (Beer et al., 2004)/nitrosative (Brown and Borutaite, 2004) stress-induced modifications, will be needed to gain further insight on the role of mitochondrial complex I in human physiopathology. In conclusion, the present work provides evidence of tissue-specific differences of mitochondrial OXPHOS capacity and efficiency with NAD-dependent substrates. The mitochondrial ATP production yield can change, within the same tissue (brain), in response to physiological and/or environmental variations. Therefore, we suggest that the physiological set up and the adaptive changes of mitochondrial OXPHOS efficiency could be mostly regulated by complex I, thus highlighting the pivotal role of this enzyme in cellular energy production by OXPHOS.
Disclosure statement The authors declare no conflict of interest. All experiments were performed in accordance with local and national guidelines covering animal experimentation.
Acknowledgements We thank Dr. Nazzareno Capitanio for critical reading of the manuscript. This work was supported by grants from National Research Project (PRIN 2000 no. MM05038851 and PRIN 2003 no. 2003064310) of the Italian Ministry for the University (MIUR).
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