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• W2167645466 abstract "Metabolic control analysis was applied to intact HepG2 cells. The effect on the control coefficient of cytochrome c oxidase (CcOX) over cell respiration of both the electrical (Δψ) and chemical (ΔpH) component of the mitochondrial transmembrane proton electrochemical gradient (ΔμH+) was investigated. The overall O2 consumption and specific CcOX activity of actively phosphorylating cells were titrated with cyanide under conditions in which Δψ and ΔpH were selectively modulated by addition of ionophores. In the absence of ionophores, CcOX displayed a high control coefficient (CIV = 0.73), thus representing an important site of regulation of mitochondrial oxidative phosphorylation. A high control coefficient value (CIV = 0.85) was also measured in the presence of nigericin, i.e. when Δψ is maximal, and in the presence of nigericin and valinomycin (CIV = 0.77), when ΔμH+ is abolished. In contrast, CcOX displayed a markedly lower control coefficient (CIV = 0.30) upon addition of valinomycin, when Δψ is converted into ΔpH. These results show that Δψ is responsible for the tight control of CcOX over respiration in actively phosphorylating cells. Metabolic control analysis was applied to intact HepG2 cells. The effect on the control coefficient of cytochrome c oxidase (CcOX) over cell respiration of both the electrical (Δψ) and chemical (ΔpH) component of the mitochondrial transmembrane proton electrochemical gradient (ΔμH+) was investigated. The overall O2 consumption and specific CcOX activity of actively phosphorylating cells were titrated with cyanide under conditions in which Δψ and ΔpH were selectively modulated by addition of ionophores. In the absence of ionophores, CcOX displayed a high control coefficient (CIV = 0.73), thus representing an important site of regulation of mitochondrial oxidative phosphorylation. A high control coefficient value (CIV = 0.85) was also measured in the presence of nigericin, i.e. when Δψ is maximal, and in the presence of nigericin and valinomycin (CIV = 0.77), when ΔμH+ is abolished. In contrast, CcOX displayed a markedly lower control coefficient (CIV = 0.30) upon addition of valinomycin, when Δψ is converted into ΔpH. These results show that Δψ is responsible for the tight control of CcOX over respiration in actively phosphorylating cells. Cytochrome c oxidase (CcOX), 3The abbreviations used are: CcOXcytochrome c oxidaseTMPDN,N,N′,N′-tetramethyl-p-phenylenediamine. the final electron acceptor of the mitochondrial respiratory chain, catalyzes the four-electron reduction of O2 to H2O, while actively pumping protons from the matrix into the intermembrane space (1.Brunori M. Giuffrè A. Sarti P. J. Inorg. Biochem. 2005; 99: 324-336Crossref PubMed Scopus (107) Google Scholar, 2.Brzezinski P. Gennis R.B. J. Bioenerg. Biomembr. 2008; 40: 521-531Crossref PubMed Scopus (229) Google Scholar). The electron transfer and H+ translocation across the mitochondrial inner membrane that are catalyzed by CcOX and by the other respiratory chain complexes generate and maintain the transmembrane proton electrochemical gradient (ΔμH+), comprising the electrical component, i.e. the membrane potential (Δψ), and the chemical proton gradient (ΔpH). According to the classical chemiosmotic theory (3.Nicholls D.G. Ferguson S.J. Bioenergetics. 3rd Ed. Academic Press, London2002Google Scholar), the ΔμH+ is used by F0F1-ATP synthase (complex V) to synthesize ATP from ADP and phosphate. The activity of the respiratory complexes is down-regulated by ΔμH+ and mitochondria oscillate between two respiratory energetic states, namely state 3, with ATP synthesis occurring at the expense of ΔμH+ during active oxidative phoshorylation, and state 4, i.e. the resting state, as originally defined (4.Chance B. Williams G.R. J. Biol. Chem. 1955; 217: 409-427Abstract Full Text PDF PubMed Google Scholar). cytochrome c oxidase N,N,N′,N′-tetramethyl-p-phenylenediamine. A number of pathological states have been correlated to mitochondrial dysfunction and alteration of ΔμH+, with uncontrolled production of free radicals and/or lack of energy (5.Kadenbach B. Ramzan R. Vogt S. Trends Mol. Med. 2009; 15: 139-147Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 6.Hüttemann M. Lee I. Pecinova A. Pecina P. Przyklenk K. Doan J.W. J. Bioenerg. Biomembr. 2008; 40: 445-456Crossref PubMed Scopus (175) Google Scholar). The metabolic control analysis (MCA) (7.Kacser H. Burns J.A. Symp. Soc. Exp. Biol. 1973; 27: 65-104PubMed Google Scholar, 8.Heinrich R. Rapoport T.A. Eur. J. Biochem. 1974; 42: 97-105Crossref PubMed Scopus (186) Google Scholar) is a key tool to estimate, under different metabolic conditions, the relative contribution of each respiratory complex to the control of the respiratory chain electron flux, and in turn of oxidative phosphorylation. MCA has been used to estimate, both in isolated mitochondria and intact cells, the control coefficient of each respiratory complex; a value that has been shown to vary depending on tissues and on metabolic conditions leading to distinct mitochondrial energization states (9.Rossignol R. Letellier T. Malgat M. Rocher C. Mazat J.P. Biochem. J. 2000; 347: 45-53Crossref PubMed Scopus (171) Google Scholar, 10.Mazat J.P. Rossignol R. Malgat M. Rocher C. Faustin B. Letellier T. Biochim. Biophys. Acta. 2001; 1504: 20-30Crossref PubMed Scopus (79) Google Scholar). Using the MCA approach, a large CcOX control over respiration has been observed in human hepatoma HepG2 cells (11.Piccoli C. Scrima R. Boffoli D. Capitanio N. Biochem. J. 2006; 396: 573-583Crossref PubMed Scopus (79) Google Scholar, 12.Villani G. Attardi G. Proc. Natl. Acad. Sci. U.S.A. 1997; 94: 1166-1171Crossref PubMed Scopus (175) Google Scholar). Piccoli et al. (11.Piccoli C. Scrima R. Boffoli D. Capitanio N. Biochem. J. 2006; 396: 573-583Crossref PubMed Scopus (79) Google Scholar) have recently addressed the role of ΔμH+ on the control coefficient of CcOX (CIV), performing the investigation under nonphosphorylating conditions, i.e. in state 4. They found a low CIV value in the presence of either oligomycin, blocking ATP synthase, or oligomycin plus valinomycin, converting Δψ into ΔpH. The relative effect of Δψ and ΔpH on the activity of CcOX, has been extensively investigated on the isolated enzyme. Experiments carried out using CcOX reconstituted into artificial phospholipid vesicles (13.Sarti P. Malatesta F. Antonini G. Vallone B. Brunori M. J. Biol. Chem. 1990; 265: 5554-5560Abstract Full Text PDF PubMed Google Scholar, 14.Brunori M. Sarti P. Colosimo A. Antonini G. Malatesta F. Jones M.G. Wilson M.T. EMBO J. 1985; 4: 2365-2368Crossref PubMed Scopus (42) Google Scholar) proved that, if the electron delivery to the enzyme is not limiting, as in the presence of excess reducing substrates, CcOX activity is controlled predominantly by Δψ, rather than by H+ re-equilibration, but see also Refs. 15.Nicholls P. Cooper C.E. Wrigglesworth J.M. Biochem. Cell Biol. 1990; 68: 1128-1134Crossref PubMed Scopus (11) Google Scholar, 16.Gregory L. Ferguson-Miller S. Biochemistry. 1989; 28: 2655-2662Crossref PubMed Scopus (40) Google Scholar, 17.Capitanio N. De Nitto E. Villani G. Capitanio G. Papa S. Biochemistry. 1990; 29: 2939-2945Crossref PubMed Scopus (24) Google Scholar. In the present work, we have extended previous studies in actively respiring HepG2 cells to further investigate the relative effect of Δψ and ΔpH on the control coefficient of CcOX, but under phosphorylating state 3 conditions (11.Piccoli C. Scrima R. Boffoli D. Capitanio N. Biochem. J. 2006; 396: 573-583Crossref PubMed Scopus (79) Google Scholar), i.e. in the absence of oligomycin. We have shown that in intact cells, where the ATP synthase is not impaired by inhibitors, the metabolic control exerted by CcOX over the respiratory chain depends on Δψ. Our results also suggest that the depressing effect over respiration of ΔμH+ affects the distribution of control among the individual steps of the oxidative phosphorylation system. Sodium ascorbate, N,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD), nigericin, valinomycin, and oligomycin were from Sigma. The human hepatoma cell line HepG2 was from ATCC. Dulbecco's modified Eagle's medium, trypsin, EDTA, penicillin, streptomycin, pyruvate, and fetal bovine serum were from Invitrogen. The HepG2 cell line was maintained in culture with Dulbecco's modified Eagle's medium supplemented with 10% (v/v) fetal bovine serum, 100 units ml−1 penicillin, 100 μg ml−1 streptomycin, 1 mm pyruvate, and 2 mm l-glutamine. Prior to the experiments, cells were detached by trypsinization, collected by centrifugation, and suspended into the medium equilibrated at 37 °C, for counting and assessing cell viability by Trypan Blue exclusion. After trypsinization and centrifugation, cells were washed twice, suspended at a final density of 4.5 to 6.5 × 106 cells ml−1 into buffer solution (25 mm HEPES, pH 7.2, 118 mm NaCl, 4.8 mm KCl, 1.2 mm MgSO4, 1.2 mm KH2PO4, and 20 mm glucose, pre-equilibrated at 37 °C) and used immediately for experimentation. Aliquots of the cell suspension were distributed into the 1.5-ml chambers of a dual chamber standalone high resolution respirometer (Oxygraph-2k, Oroboros Instruments) to monitor the endogenous cell O2 consumption and, in parallel, the CcOX specific activity at 37 °C. When indicated, ionophores were used at the lowest concentration causing maximal stimulation of respiration (i.e. 33 nm valinomycin or 100 nm nigericin or a combination of both substances). Cyanide sensitivity was assayed by sequential addition of the inhibitor (using 80 mm or 8 mm NaCN stock solutions). According to Refs. 11.Piccoli C. Scrima R. Boffoli D. Capitanio N. Biochem. J. 2006; 396: 573-583Crossref PubMed Scopus (79) Google Scholar, 12.Villani G. Attardi G. Proc. Natl. Acad. Sci. U.S.A. 1997; 94: 1166-1171Crossref PubMed Scopus (175) Google Scholar, and 18.Villani G. Attardi G. Methods Cell Biol. 2007; 80: 121-133Crossref PubMed Scopus (20) Google Scholar, under all conditions, the specific activity of CcOX was measured in the presence of 20 nm antimycin A, using as substrates excess ascorbate (10 mm) and nonsaturating amounts of TMPD (0.2 mm), thus not at Vmax. At the concentration used, TMPD did not significantly reduces mitochondrial Δψ, as assessed using the fluorimetric dye JC1 in cell suspensions (data not shown). Based on the metabolic control analysis (19.Kacser H. Burns J.A. Biochem. Soc. Trans. 1979; 7: 1149-1160Crossref PubMed Scopus (325) Google Scholar, 20.Moreno-Sánchez R. Bravo C. Westerhoff H.V. Eur. J. Biochem. 1999; 264: 427-433Crossref PubMed Scopus (24) Google Scholar, 21.Kholodenko B.N. Westerhoff H.V. FEBS Lett. 1993; 320: 71-74Crossref PubMed Scopus (88) Google Scholar), the control coefficient of CcOX (CIV) was calculated from the cyanide-titration profile of the global cell respiration and the CcOX specific activity, according to the following equation, CIV=S1/S2(Eq. 1) where S1 = (dJ/dI)I→0 and S2 = (dv/dI)I→0, J is the global O2 consumption rate, v is the specific activity of CcOX, and I is the cyanide concentration. The experimental error of CIV (ϵC(IV)) has been evaluated as follows, εC(IV)=CIV(ε1S1)2+(ε2S2)2(Eq. 2) where ϵ1 and ϵ2 are the S.D. of S1 and S2. The effect of ΔμH+ on the metabolic control of cytochrome c oxidase over cell respiration was investigated by performing cyanide titrations of both the endogenous respiration (global activity) and the O2 consumption sustained by the sole CcOX (specific activity) in intact HepG2 cells. To gather information on the relative effect of the electrical and the chemical component of ΔμH+, measurements were carried out in the absence and presence of ionophores specifically collapsing either Δψ (valinomycin) or ΔpH (nigericin). Data reported in Table 1 indicate that, in the absence of ionophores, cell respiration sustained by endogenous substrates proceeds at a rate of 62.8 ± 20.8 pmol O2·s−1·106 cells−1 that is almost completely (≥95%) abolished by 1 mm NaCN or 20 nm antimycin A (data not shown). This rate is only slightly stimulated in the presence of 100 nm nigericin, whereas it increases by ∼2–3-fold upon addition of 33 nm valinomycin alone or in combination with nigericin (132.7 ± 55.1 and 182.3 ± 69.1 pmol O2·s−1·106 cells−1, respectively). No significant changes were observed at the level of specific activity regardless of the presence of ionophores.TABLE 1Rate of oxygen consumption (±S.D.) in HepG2 cells in the absence and in the presence of ionophores. Independent experiments number, n ≥ 10.Endogenous respirationCcOX activitypmol O2·s−1·106 cells−1pmol O2·s−1·106 cells−1Control62.8 ± 20.8151.6 ± 30.5+Valinomycin132.7 ± 55.1135.5 ± 20.3+Nigericin70.3 ± 18.3126.7 ± 34.8+Valinomycin and nigericin182.3 ± 69.1125.9 ± 30.5 Open table in a new tab Fig. 1 shows typical sets of data acquired with untreated intact HepG2 cells (top panels) and in the presence of valinomycin (bottom panels). As expected, upon sequential additions of cyanide to the respiring cells, the O2 consumption rate decreases in a concentration dependent manner. In the absence of ionophores, both the global (Fig. 1A) and the specific activity (Fig. 1B) are affected by cyanide to a similar extent and particularly in the low cyanide concentration range (0–16 μm), critical for estimation of the CcOX control coefficient (CIV). In particular, at 16 μm NaCN (Fig. 1, addition d), the global and the CcOX specific residual activities are comparable (88 and 83% of the corresponding initial values, respectively), strongly indicating that the control coefficient approaches the unity under these conditions. In the presence of valinomycin, converting Δψ into ΔpH, the global activity is less sensitive to cyanide than the CcOX specific activity (compare panels C and D in Fig. 1); i.e. at 16 μm NaCN, the residual global activity is up to 95%, whereas the specific activity of CcOX is ∼81%. In other words, low concentration values of cyanide, while inhibiting CcOX, are still insufficient to significantly affect the endogenous respiration of valinomycin-treated cells, pointing to a low control coefficient of the enzyme (Table 2).TABLE 2Flux control coefficients of CcOX over endogenous respiration in HepG2 cells in the absence and in the presence of ionophores. Error analysis is under “Experimental Procedures.”Control coefficient (CIV)Control0.73 ± 0.06+Valinomycin0.30 ± 0.03+Nigericin0.85 ± 0.07+Valinomycin and nigericin0.77 ± 0.10 Open table in a new tab The effect of valinomycin is better appreciated in Fig. 2, summarizing the results of cyanide titration experiments carried out with untreated cells (A) or after addition of valinomycin (B), nigericin (C), or both the ionophores (D). For each condition, the residual global and CcOX specific activities (open and closed circles, respectively), measured in the presence of increasing amounts of cyanide, are expressed as percentages of the uninhibited respiration rate and reported as a function of the inhibitor concentration. The data were obtained by averaging the results of a series of titration experiments similar to those reported in Fig. 1. Fig. 2 clearly shows that the data points corresponding to the global and the CcOX specific activities markedly diverge only in valinomycin-treated cells (panel B, when Δψ is converted into ΔpH). Indeed, under that condition, the CcOX specific activity (closed circles) is more sensitive to cyanide than the global one (open circles), whereas under all the other conditions explored, the initial slopes of the curves are similar. As a result, the flux control coefficient, calculated as the ratio of the initial slope in the curve profile of the global and specific CcOX activities (see Eq. 1), is low in the presence of valinomycin, whereas it takes higher values approaching unity in the other cases (cf. Table 2). This finding strongly suggests that Δψ increases the control coefficient of CcOX over cell respiration. The role of ΔpH on the control coefficient of CcOX was investigated by carrying out cyanide titrations in the presence of nigericin, a K+/H+ antiporter, which converts the mitochondrial ΔpH into Δψ; the titration profiles of both the global and specific activities in Fig. 2C and the corresponding flux coefficient values are comparable with those acquired in the untreated cells. The abolishment of ΔμH+ by the simultaneous addition of nigericin and valinomycin to the cell suspension was also achieved. As shown in Fig. 2D, under these conditions, CcOX exerts a high metabolic control over respiration, based on the finding that the specific CcOX activity and the endogenous cell respiration display similar cyanide sensitivity, particularly in the low cyanide concentration range. Fig. 3 shows the threshold plots relative to our data; the residual endogenous cell respiration rate at any given cyanide concentration is plotted as a function of the percentage of cyanide-inhibited CcOX in the sample. Two different threshold profiles have been observed: the profile for valinomycin-treated cells has a markedly curved shape, and 50% inhibition of CcOX by cyanide results in only 15–20% decrease of the global flux. Under all the other conditions (i.e. untreated cells or both in the presence of nigericin alone and in combination with valinomycin), the profiles appear significantly less curved. Particularly, under fully uncoupled conditions, the CcOX inhibition correlates almost linearly with that of the global respiratory flux. Therefore, in untreated cells, the CcOX control over cell respiration is fairly high, whereas when Δψ is converted into ΔpH, the CcOX control is markedly reduced. MCA applied to isolated mitochondria showed that the control of the respiratory rate is distributed among several steps, depending on fluxes and availability of the mitochondrial substrates (10.Mazat J.P. Rossignol R. Malgat M. Rocher C. Faustin B. Letellier T. Biochim. Biophys. Acta. 2001; 1504: 20-30Crossref PubMed Scopus (79) Google Scholar). Besides the respiratory chain complexes, proton leakage, phosphate carrier, pyruvate carrier, ATP synthase, and adenine nucleotide carrier have been found to exert some control over oxidative phosphorylation. The control exerted by the electron transport chain complexes increases, however, when evaluated in experimental systems closer to in vivo conditions (22.Kunz W.S. Kudin A. Vielhaber S. Elger C.E. Attardi G. Villani G. J. Biol. Chem. 2000; 275: 27741-27745Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 23.Telford J.E. Kilbride S.M. Davey G.P. J. Biol. Chem. 2009; 284: 9109-9114Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar) and in intact cells (11.Piccoli C. Scrima R. Boffoli D. Capitanio N. Biochem. J. 2006; 396: 573-583Crossref PubMed Scopus (79) Google Scholar, 12.Villani G. Attardi G. Proc. Natl. Acad. Sci. U.S.A. 1997; 94: 1166-1171Crossref PubMed Scopus (175) Google Scholar, 24.Villani G. Greco M. Papa S. Attardi G. J. Biol. Chem. 1998; 273: 31829-31836Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar, 25.Villani G. Attardi G. Free Radic. Biol. Med. 2000; 29: 202-210Crossref PubMed Scopus (104) Google Scholar). The higher control coefficient value in cells rather than in isolated mitochondria (11.Piccoli C. Scrima R. Boffoli D. Capitanio N. Biochem. J. 2006; 396: 573-583Crossref PubMed Scopus (79) Google Scholar) and the low inhibition threshold level (20% for several human cell lines (25.Villani G. Attardi G. Free Radic. Biol. Med. 2000; 29: 202-210Crossref PubMed Scopus (104) Google Scholar)) measured for CcOX under near physiological conditions point to a tight control of the enzyme in intact cells (25.Villani G. Attardi G. Free Radic. Biol. Med. 2000; 29: 202-210Crossref PubMed Scopus (104) Google Scholar). Because CcOX activity is reportedly influenced by the proton electrochemical gradient (see below), we have focused our attention on the role of ΔμH+ and its electrochemical components (Δψ and ΔpH) on the activity of CcOX and the control of the enzyme over cell respiration in whole HepG2 cells. The main result presented is that, under near physiological conditions, the control of CcOX over cell respiration predominantly depends on the electrical component (Δψ) of the proton electrochemical gradient. This finding is fully consistent with a large body of previous spectroscopic (1.Brunori M. Giuffrè A. Sarti P. J. Inorg. Biochem. 2005; 99: 324-336Crossref PubMed Scopus (107) Google Scholar, 13.Sarti P. Malatesta F. Antonini G. Vallone B. Brunori M. J. Biol. Chem. 1990; 265: 5554-5560Abstract Full Text PDF PubMed Google Scholar, 14.Brunori M. Sarti P. Colosimo A. Antonini G. Malatesta F. Jones M.G. Wilson M.T. EMBO J. 1985; 4: 2365-2368Crossref PubMed Scopus (42) Google Scholar, 26.Antonini G. Malatesta F. Sarti P. Brunori M. J. Biol. Chem. 1991; 266: 13193-13202Abstract Full Text PDF PubMed Google Scholar) and electrometric (27.Verkhovsky M.I. Tuukkanen A. Backgren C. Puustinen A. Wikström M. Biochemistry. 2001; 40: 7077-7083Crossref PubMed Scopus (47) Google Scholar, 28.Bloch D. Belevich I. Jasaitis A. Ribacka C. Puustinen A. Verkhovsky M.I. Wikström M. Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 529-533Crossref PubMed Scopus (177) Google Scholar) measurements carried out using CcOX reconstituted into artificial phospholipid vesicles. Those experiments provide a possible mechanistic explanation of how the membrane potential controls the activity of the enzyme: the rate-limiting step in the CcOX catalytic cycle, i.e. the internal electron transfer from heme a to the oxidized heme a3/CuB site, was shown to be coupled to both a vectorial H+ uptake from the mitochondrial N-phase and H+ translocation to the P-phase, synchronous with the redox reaction (28.Bloch D. Belevich I. Jasaitis A. Ribacka C. Puustinen A. Verkhovsky M.I. Wikström M. Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 529-533Crossref PubMed Scopus (177) Google Scholar). The electron transfer process is thus electrogenic and it consistently slows down in the presence of a suitable ΔΨ (≥180 mV in COV, (13.Sarti P. Malatesta F. Antonini G. Vallone B. Brunori M. J. Biol. Chem. 1990; 265: 5554-5560Abstract Full Text PDF PubMed Google Scholar)). Altogether, these findings support the original proposal by Brunori et al. (3.Nicholls D.G. Ferguson S.J. Bioenergetics. 3rd Ed. Academic Press, London2002Google Scholar, 14.Brunori M. Sarti P. Colosimo A. Antonini G. Malatesta F. Jones M.G. Wilson M.T. EMBO J. 1985; 4: 2365-2368Crossref PubMed Scopus (42) Google Scholar) that ΔΨ stabilizes CcOX in a state characterized by a lower rate of internal electron transfer and thus a lower turnover rate. This stabilization is removed by valinomycin, selectively converting ΔΨ into ΔpH and allows the enzyme to turn over more rapidly (molecular basis of the respiratory control ratio in CcOX vesicles). Consistently, we show that the addition of valinomycin to HepG2 cells decreases the control coefficient from CIV = 0.73 to CIV = 0.30, indicating that Δψ contributes to the control exerted by CcOX in the cell. This finding might appear in contrast with the report of Piccoli et al. (11.Piccoli C. Scrima R. Boffoli D. Capitanio N. Biochem. J. 2006; 396: 573-583Crossref PubMed Scopus (79) Google Scholar), which showed minor effects of valinomycin on the CcOX control coefficient evaluated in the same cell line. The discrepancy, however, is only apparent because those experiments, differently from the present work, were carried out in the presence of the ATP synthase inhibitor oligomycin, i.e. in state 4 condition. Under these conditions, ΔμH+ is likely maximal, and most of the control over respiration is exerted by the H+ back leak into the matrix rather than by CcOX or by any other respiratory chain complex (11.Piccoli C. Scrima R. Boffoli D. Capitanio N. Biochem. J. 2006; 396: 573-583Crossref PubMed Scopus (79) Google Scholar, 29.Brand M.D. Hafner R.P. Brown G.C. Biochem. J. 1988; 255: 535-539PubMed Google Scholar); therefore, the collapse of Δψ by valinomycin cannot significantly affect the control exerted by CcOX. Conversely, our data show that, in actively phosphorylating cells, Δψ is responsible for the CcOX tight control over respiration, whereas under the same condition, ΔpH seems irrelevant in this respect, as the addition of nigericin is essentially ineffective. The effect of valinomycin on the CIV appears fully consistent with the results of rapid mixing experiments carried out using CcOX vesicles (13.Sarti P. Malatesta F. Antonini G. Vallone B. Brunori M. J. Biol. Chem. 1990; 265: 5554-5560Abstract Full Text PDF PubMed Google Scholar, 14.Brunori M. Sarti P. Colosimo A. Antonini G. Malatesta F. Jones M.G. Wilson M.T. EMBO J. 1985; 4: 2365-2368Crossref PubMed Scopus (42) Google Scholar). According to the MCA theory, all of the control coefficients of the steps involved in an integrated process should sum to one (summation theorem (21.Kholodenko B.N. Westerhoff H.V. FEBS Lett. 1993; 320: 71-74Crossref PubMed Scopus (88) Google Scholar)). Thus, in the presence of valinomycin, in response to a decrease of the CcOX flux control, a compensatory increase of the control level of other steps is expected. The chemical H+ gradient (ΔpH) is expected to increase at the expense of the electrical component and to balance the dissipation of mitochondrial Δψ. Under these conditions, the respiratory complexes whose activity is more sensitive to ΔpH (30.Lorusso M. Cocco T. Minuto M. Capitanio N. Papa S. J. Bioenerg. Biomembr. 1995; 27: 101-108Crossref PubMed Scopus (11) Google Scholar, 31.Papa S. Lorusso M. Izzo G. Capuano F. Biochem. J. 1981; 194: 395-406Crossref PubMed Scopus (18) Google Scholar), i.e. those upstream CcOX, likely increase their control over respiration. When both ΔpH and Δψ collapse, as in the presence of both valinomycin and nigericin, the enzymes upstream CcOX may lose such control. Consistently, our and previous data (11.Piccoli C. Scrima R. Boffoli D. Capitanio N. Biochem. J. 2006; 396: 573-583Crossref PubMed Scopus (79) Google Scholar, 12.Villani G. Attardi G. Proc. Natl. Acad. Sci. U.S.A. 1997; 94: 1166-1171Crossref PubMed Scopus (175) Google Scholar) show that the abolishment of ΔμH+ leads to a high CIV over respiration and suggest that other factors, such as substrate concentration and level of effectors, may limit CcOX activity under these conditions (25.Villani G. Attardi G. Free Radic. Biol. Med. 2000; 29: 202-210Crossref PubMed Scopus (104) Google Scholar) and account for the high control over respiration. We observed two different types of threshold plots (Fig. 3). In the presence of valinomycin alone, the data show a plateau phase in which inhibition of up to 30–40% CcOX modestly affects the overall respiratory flux. Thus, when Δψ is converted into ΔpH, there is an excess of functional capacity of CcOX. Under all of the other conditions examined (control cells alone or plus nigericin in the presence/absence of valinomycin), a clear threshold is not detected consistent with a higher control of CcOX corresponding to a smaller threshold value (10.Mazat J.P. Rossignol R. Malgat M. Rocher C. Faustin B. Letellier T. Biochim. Biophys. Acta. 2001; 1504: 20-30Crossref PubMed Scopus (79) Google Scholar). To summarize, we have demonstrated that in intact cells under phosphorylating conditions (i.e. with fully active ATP synthase), CcOX activity and its control over oxidative phosphorylation are dependent on Δψ. The Δψ dependence suggests that specific mutations associated to a diminished CcOX efficiency and/or an altered Δψ/ΔpH balance (32.Namslauer I. Brzezinski P. Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 3402-3407Crossref PubMed Scopus (35) Google Scholar) may change the distribution of control in the respiratory metabolic pathway and therefore the specific tissue susceptibility to diseases." @default.
• W2167645466 created "2016-06-24" @default.
• W2167645466 creator A5001298745 @default.
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• W2167645466 date "2009-11-01" @default.
• W2167645466 modified "2023-10-12" @default.
• W2167645466 title "Control of Respiration by Cytochrome c Oxidase in Intact Cells" @default.
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