The Mitochondrial Respiratory Chain Is Partially Organized in a Supercomplex Assembly
2004; Elsevier BV; Volume: 279; Issue: 35 Linguagem: Inglês
10.1074/jbc.m405135200
ISSN1083-351X
AutoresCristina Bianchi, Maria Luisa Genova, Giovanna Parenti Castelli, Giorgio Lenaz,
Tópico(s)Metabolism and Genetic Disorders
ResumoThe model of the respiratory chain in which the enzyme complexes are independently embedded in the lipid bilayer of the inner mitochondrial membrane and connected by randomly diffusing coenzyme Q and cytochrome c is mostly favored. However, multicomplex units can be isolated from mammalian mitochondria, suggesting a model based on direct electron channeling between complexes. Kinetic testing using metabolic flux control analysis can discriminate between the two models: the former model implies that each enzyme may be rate-controlling to a different extent, whereas in the latter, the whole metabolic pathway would behave as a single supercomplex and inhibition of any one of its components would elicit the same flux control. In particular, in the absence of other components of the oxidative phosphorylation apparatus (i.e. ATP synthase, membrane potential, carriers), the existence of a supercomplex would elicit a flux control coefficient near unity for each respiratory complex, and the sum of all coefficients would be well above unity. Using bovine heart mitochondria and submitochondrial particles devoid of substrate permeability barriers, we investigated the flux control coefficients of the complexes involved in aerobic NADH oxidation (I, III, IV) and in succinate oxidation (II, III, IV). Both Complexes I and III were found to be highly rate-controlling over NADH oxidation, a strong kinetic evidence suggesting the existence of functionally relevant association between the two complexes, whereas Complex IV appears randomly distributed. Moreover, we show that Complex II is fully rate-limiting for succinate oxidation, clearly indicating the absence of substrate channeling toward Complexes III and IV. The model of the respiratory chain in which the enzyme complexes are independently embedded in the lipid bilayer of the inner mitochondrial membrane and connected by randomly diffusing coenzyme Q and cytochrome c is mostly favored. However, multicomplex units can be isolated from mammalian mitochondria, suggesting a model based on direct electron channeling between complexes. Kinetic testing using metabolic flux control analysis can discriminate between the two models: the former model implies that each enzyme may be rate-controlling to a different extent, whereas in the latter, the whole metabolic pathway would behave as a single supercomplex and inhibition of any one of its components would elicit the same flux control. In particular, in the absence of other components of the oxidative phosphorylation apparatus (i.e. ATP synthase, membrane potential, carriers), the existence of a supercomplex would elicit a flux control coefficient near unity for each respiratory complex, and the sum of all coefficients would be well above unity. Using bovine heart mitochondria and submitochondrial particles devoid of substrate permeability barriers, we investigated the flux control coefficients of the complexes involved in aerobic NADH oxidation (I, III, IV) and in succinate oxidation (II, III, IV). Both Complexes I and III were found to be highly rate-controlling over NADH oxidation, a strong kinetic evidence suggesting the existence of functionally relevant association between the two complexes, whereas Complex IV appears randomly distributed. Moreover, we show that Complex II is fully rate-limiting for succinate oxidation, clearly indicating the absence of substrate channeling toward Complexes III and IV. Considerable information exists on the structure at atomic resolution of most of the transmembrane protein complexes forming the mitochondrial respiratory chain; there is, however, still little direct information on the supramolecular organization of the enzymatic complexes in the inner mitochondrial membrane. Two extreme models for their arrangement in the membrane are conceivable: the model of a random organization of the individual respiratory complexes and that of a supercomplex assembly formed by stable association between proteins.The original solid-state model of Chance and Williams (1Chance B. Williams G.R. Nature. 1955; 176: 250-254Crossref PubMed Scopus (197) Google Scholar) changed gradually because the oxidative phosphorylation enzymes were found functionally active when isolated as individual complexes (2Hatefi Y. Haavik A.G. Rieske J.S. J. Biol. Chem. 1962; 237: 1676-1680Abstract Full Text PDF PubMed Google Scholar) and was substituted by the model of enzyme complexes individually dissolved in the lipid bilayer, as formulated in a systematic way by Hackenbrock et al. (3Hackenbrock C.R. Chazotte B. Gupte S.S. J. Bioenerg. Biomembr. 1986; 18: 331-368Crossref PubMed Scopus (278) Google Scholar) in their Random Collision Model of mitochondrial electron transport. Despite the acceptance of the idea that electron transfer in mitochondrial membranes depends on random collisions between small diffusing molecules (coenzyme Q and cytochrome c) and complexes (I–IV) independently embedded in the bilayer (4Höchli M. Hackenbrock C.R. Proc. Natl. Acad. Sci. U. S. A. 1976; 73: 1636-1640Crossref PubMed Scopus (60) Google Scholar), the concept of a solid-state organization was never abandoned since preferential associations between specific complexes were occasionally described (2Hatefi Y. Haavik A.G. Rieske J.S. J. Biol. Chem. 1962; 237: 1676-1680Abstract Full Text PDF PubMed Google Scholar, 5Ozawa T. Nishikimi M. Suzuki H. Tanaka M. Shimomura Y. Ozawa T. Papa S. Bioenergetics: Structure and Function of Energy-trasducing Systems. Japan Scientific Societies Press, Tokyo1987: 101-119Google Scholar). Stable supercomplexes of Complexes III and IV were isolated from several bacteria, e.g. from Paracoccus denitrificans (6Berry E.A. Trumpower B.L. J. Biol. Chem. 1985; 260: 2458-2467Abstract Full Text PDF PubMed Google Scholar, 7Stroh A. Anderka O. Pfeiffer K. Yagi T. Finel M. Ludwig B. Schägger H. J. Biol. Chem. 2004; 279: 5000-5007Abstract Full Text Full Text PDF PubMed Scopus (195) Google Scholar), thermophilic Bacillus PS3 (8Sone N. Sekimachi M. Kutoh E. J. Biol. Chem. 1987; 262: 15386-15391Abstract Full Text PDF PubMed Google Scholar), thermoacidophilic archeon Sulfolobus (9Iwasaki T. Matsuura K. Oshima T. J. Biol. Chem. 1995; 270: 30881-30892Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar), and Corynebacterium glutamicum (10Niebisch A. Bott M. J. Biol. Chem. 2003; 278: 4339-4346Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar).Recently, Schägger and colleagues (11Schägger H. IUBMB Life. 2001; 52: 119-128Crossref PubMed Scopus (163) Google Scholar, 12Schägger H. Pfeiffer K. J. Biol. Chem. 2001; 276: 37861-37867Abstract Full Text Full Text PDF PubMed Google Scholar) produced new evidence of stoichiometric assemblies of individual complexes, in yeast and in mammalian mitochondria, and suggested a model of the respiratory chain (the respirasome) based on direct channeling between complexes and not on random collisions. In particular, blue native PAGE of digitonin-solubilized mitochondria from Saccharomyces cerevisiae revealed two bands with apparent masses of ∼750 and 1000 kDa containing the subunits of Complexes III and IV, as assigned after two dimensional SDS-PAGE followed by N-terminal protein sequencing (13Schägger H. Pfeiffer K. EMBO J. 2000; 19: 1777-1783Crossref PubMed Scopus (1011) Google Scholar). Similar interactions of supercomplexes were investigated in bovine heart mitochondria; Complex I–III interactions were apparent from the presence of a I1III2 complex that was found further assembled into two major supercomplexes (I1III2IV2 and I1III2IV4) comprising different copy numbers of Complex IV. The existence of respirasome-like supercomplexes was also reported for higher plant mitochondria on blue native gels upon gentle solubilizations; between 50 and 90% of Complex I is assembled with dimeric Complex III into the stable I1III2 supercomplex (14Eubel H. Jänsch L. Braun H.P. Plant Physiol. 2003; 133: 1-13Crossref Scopus (273) Google Scholar).In addition to biochemical or ultrastructural approaches, functional analysis is a powerful source of information on the lateral organization of protein complexes of the respiratory chain, and it has been widely used to discriminate between “solid” and “liquid” states. On the basis of the kinetics of electron transfer reactions involving lipid soluble quinones, Kröger and Klingenberg (15Kröger A. Klingenberg M. Eur. J. Biochem. 1973; 34: 358-368Crossref PubMed Scopus (258) Google Scholar) proposed that ubiquinone (coenzyme Q (CoQ)) 1The abbreviations used are: CoQ, coenzyme Q or ubiquinone; FMN, flavin mononucleotide; SMP, submitochondrial particles.1The abbreviations used are: CoQ, coenzyme Q or ubiquinone; FMN, flavin mononucleotide; SMP, submitochondrial particles. in mitochondria is a homogeneous pool that shuttles electrons from dehydrogenases to Complex III. Previous results in our laboratory, taking into account the high diffusion coefficient of the quinone (16Lenaz G. Fato R. J. Biomembr. Bioenerg. 1986; 18: 369-401Crossref PubMed Scopus (41) Google Scholar), favored a liquid state model of the mitochondrial membrane, implying collisional processes not limited by diffusion, but we did not exclude a more clustered organization of the respiratory complexes (17Lenaz G. FEBS Lett. 2001; 509: 151-155Crossref PubMed Scopus (58) Google Scholar, 18Bianchi C. Fato R. Genova M.L. Parenti Castelli G. Lenaz G. Biofactors. 2003; 18: 3-9Crossref PubMed Scopus (40) Google Scholar). In mitochondria from S. cerevisiae under conditions of approximately physiological ionic strength, neither ubiquinone nor cytochrome c displays pool behavior, indicating that, at least in yeast, the mitochondrial respiratory chain complexes form one functional respiratory unit (19Boumans H. Grivell L.A. Berden J.A. J. Biol. Chem. 1998; 273: 4872-4877Abstract Full Text Full Text PDF PubMed Scopus (135) Google Scholar).Flux control analysis (20Kacser A. Burns J.A. Biochem. Soc. Trans. 1979; 7: 1149-1160Crossref PubMed Scopus (322) Google Scholar, 21Moreno-Sanchez R. Bravo C. Westerhoff H.V. Eur. J. Biochem. 1999; 264: 427-433Crossref PubMed Scopus (24) Google Scholar) represents another approach that can bring new insights on the structure of the respiratory chain. If a metabolic pathway is composed of distinct enzymes, the extent to which each enzyme is rate-controlling may be different, and the sum of all the flux control coefficients for the different enzymes should be equal to unity. On the other hand, in a supercomplex, the metabolic pathway would behave as a single enzyme unit, and inhibition of any one of the enzyme components would elicit the same flux control. In particular, in a system in which the respiratory chain is totally dissociated from other components of the oxidative phosphorylation apparatus (i.e. ATP synthase, membrane potential, and carriers), such as open non-phosphorylating submitochondrial particles (SMP), the existence of a supercomplex would elicit a flux control coefficient near unity at any of the respiratory complexes, and the sum of all coefficients would be above 1 (22Kholodenko N.B. Westerhoff H.V. FEBS Lett. 1993; 320: 71-74Crossref PubMed Scopus (85) Google Scholar).We have addressed the problem in SMP and in permeabilized mitochondria from beef heart using metabolic flux control analysis in NADH oxidase and succinate oxidase systems. The results favor the idea of a preferential association of Complex I and Complex III, whereas the other respiratory complexes appear to be functionally independent.EXPERIMENTAL PROCEDURESChemicals—Most chemicals were obtained from Sigma. Carboxin was purchased from Supelco, Bellefonte, PA. Mucidin (strobilurin A) was a kind gift from Dr. F. Nerud of the Academy of Sciences in Prague, Czech Republic.Preparation of Mitochondria and Submitochondrial Particles—Bovine heart mitochondria particles and SMP were prepared by a large scale procedure essentially as described elsewhere (23Smith A. Methods Enzymol. 1967; 10: 81-86Crossref Scopus (468) Google Scholar, 24Boveris A. Methods Enzymol. 1984; 105: 429-435Crossref PubMed Scopus (417) Google Scholar). The SMP were open membranes devoid of substrate permeability barriers as shown by the lack of stimulation of cytochrome c-dependent activities by detergents.Enzyme Assays—NADH-coenzyme Q reductase activity was measured in a dual wavelength spectrophotometer (V550 extended model, Jasco Europe, Cremella-LC, Italy) at 25 °C and using decylubiquinone as acceptor (25Ventura B. Genova M.L. Bovina C. Formiggini G. Lenaz G. Biochim. Biophys. Acta. 2002; 1553: 249-260Crossref PubMed Scopus (85) Google Scholar). Other enzyme activities were assayed as described elsewhere but at the same temperature and buffer conditions. In particular, aerobic NADH oxidation and ubiquinol-cytochrome c reductase activity were determined spectrophotometrically according to (25Ventura B. Genova M.L. Bovina C. Formiggini G. Lenaz G. Biochim. Biophys. Acta. 2002; 1553: 249-260Crossref PubMed Scopus (85) Google Scholar) except that low amounts of membrane suspensions were employed (40 μg of protein/ml and 2 μg of protein/ml, respectively). Ferrous cytochrome c for cytochrome oxidase activity was prepared as in Ref. 26Genova M.L. Castelluccio C. Fato R. Parenti Castelli G. Merlo Pich M. Formiggini G. Bovina C. Marchetti M. Lenaz G. Biochem. J. 1995; 311: 105-109Crossref PubMed Scopus (81) Google Scholar. All activities involving cytochrome c were assayed by monitoring the absorbance change of cytochrome c upon reduction or oxidation (molar absorption coefficient = 19.1 mm–1 cm–1 at 550–540 nm). Succinate-ubiquinone reductase activity was measured indirectly by following the ubiquinone-dependent reduction of 2,6-dichloroindophenol (27Hatefi Y. Stiggall D.L. Methods Enzymol. 1978; 53: 21-27Crossref PubMed Scopus (165) Google Scholar) in the presence of 25 μg of protein/ml of membrane suspension, previously activated by incubating with 0.5 m potassium succinate for 5 min at 37 °C and in the absence of exogenous quinone due to its partial competition with respect to the specific inhibitor of Complex II, carboxin (28Matsson M. Hederstedt L. J. Bioenerg. Biomembr. 2001; 33: 99-105Crossref PubMed Scopus (60) Google Scholar). The rate of aerobic succinate oxidation in the membrane suspension was assayed polarographically (29Estabrook R.W. Methods Enzymol. 1967; 10: 41-47Crossref Scopus (1888) Google Scholar), by means of a thermostatically controlled oxygraph apparatus (Oroboros Oxygraph, Paar, Graz, Austria) equipped with a Clark-type electrode and a rapid mixing device.Titration Curves, Determination of Flux Control Coefficients, and Threshold Plots—Metabolic flux control analysis was performed by titrating the whole respiratory chain activity (global flux) and its single steps with inhibitors of the individual complexes, i.e. rotenone for Complex I, carboxin for Complex II, mucidin for Complex III, and KCN for Complex IV. The choice of mucidin was dictated by its specific inhibition of the steady-state activity of Complex III, resulting in a hyperbolic relationship between activity and the amount of inhibitor (30Von Jagow G. Gribble G.W. Trumpower B.L. Biochemistry. 1986; 25: 775-780Crossref PubMed Scopus (57) Google Scholar). It has long been observed that titration with the commonly used antimycin A gives a non-linear inhibition of ubiquinol-cytochrome c oxidoreductase due to the functional asymmetry in the bc1 complex dimer and to a stimulatory effect upon binding of one molecule of antimycin per dimer at low inhibitor/enzyme ratios (31Bechmann G. Weiss H. Rich P.R. Eur. J. Biochem. 1992; 208: 315-325Crossref PubMed Scopus (29) Google Scholar, 32Covian R. Gutierrez-Cirlos E.B. Trumpower B.L. J. Biol. Chem. 2004; 279: 15040-15049Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar), whereas myxothiazol can also inhibit Complex I (33Degli Esposti M. Ghelli A. Crimi M. Estornell E. Fato R. Lenaz G. Biochem. Biophys. Res. Commun. 1993; 190: 1090-1096Crossref PubMed Scopus (90) Google Scholar). The concentration of rotenone and mucidin, as ethanolic stock solutions, was checked spectrophotometrically (30Von Jagow G. Gribble G.W. Trumpower B.L. Biochemistry. 1986; 25: 775-780Crossref PubMed Scopus (57) Google Scholar, 34Singer T.P. Methods Enzymol. 1979; 55: 454-462Crossref PubMed Scopus (75) Google Scholar). The inhibition curves were obtained with a non-linear regression fitting procedure performed on all the raw titration values of every set of experimental data using the application program SigmaPlot (35Software Jandel Scientific SigmaPlot Scientific Graphing Software. Jandel Scientific Corp., San Rafael, CA1995Google Scholar).The control coefficients (Cvi) were calculated from the measured percent changes in the enzyme activities upon the addition of a small concentration of specific inhibitors (25Ventura B. Genova M.L. Bovina C. Formiggini G. Lenaz G. Biochim. Biophys. Acta. 2002; 1553: 249-260Crossref PubMed Scopus (85) Google Scholar). Thus, Cvi = (dJ/dI)I=0/(dvi/dI)I=0, i.e. the ratio of the initial slope of the inhibition curve of the global flux (J) to the initial slope of the inhibition curve of the individual step (vi). Because of this kind of mathematical analysis, the flux control coefficients shown in this report could not be in the form of mean values with standard errors. However, as a gauge of the accuracy of the data, it can be emphasized that the correlation coefficient (r2) of each fitted curve was higher than 0.97, indicating the tight closeness of the regression to the experimental values. Threshold plots were derived from the titration curves by drawing the percent rate of the global activity as a function of the inhibition percentage of the single step activity for the same inhibitor concentration.RESULTSFlux Control Analysis for the Respiratory Complexes Involved in Aerobic NADH Oxidation—Both the aerobic NADH oxidation (global activity) and the specific activity of Complex I, III, and IV (individual steps) have been studied in bovine heart submitochondrial particles by titration with specific inhibitors. The stepwise inhibition using rotenone is shown in Fig. 1, top. The titration curve profile is similar for both NADH oxidase and NADH-decylubiquinone oxidoreductase activity. As shown in the expanded region on the left, the two curves are quite superimposable in their initial portion; therefore, the corresponding straight tangents for rotenone concentration tending to zero exhibit an almost parallel course with a slope of –0.87 and –0.95, respectively. The flux control coefficient (Cvi) obtained by the ratio of the initial slopes is 0.91 (Table I).Table IFlux control coefficients (Cvi) of the complexes involved in NADH oxidation by different segments of the respiratory chain in bovine heart mitochondria and SMPStepNADH oxidase activityNADH-cytochrome c oxidoreductase activity (SMP)BHMaBovine heart mitochondria.SMPComplex I1.060.911.12Complex III0.900.610.57Complex IV0.260.06NAbNot applicable.a Bovine heart mitochondria.b Not applicable. Open table in a new tab The flux control exerted by Complex III over aerobic NADH oxidation was determined using mucidin to progressively inhibit the enzyme activity; the two sets of experimental data (Fig. 1, middle) show a similar hyperbolic profile, and the fitted titration curves are very close in their initial slopes. This property accounts for the high control coefficient obtained for Complex III (Table I).A much lower flux control coefficient was measured for Complex IV (Table I). The effect of KCN inhibition on the global flux (i.e. NADH oxidase activity) was weaker than that on Complex IV activity alone at the same concentrations of the inhibitor. By inspection of the plots in Fig. 1, bottom, it can be observed that the initial slopes of the two inhibition curves strongly diverge. The low control coefficient of cytochrome oxidase is maintained under conditions of either high or low ionic strength (data not shown), indicating that the mode of diffusion of cytochrome c (in three dimensions at high ionic strength and in two dimensions at low ionic strength (3Hackenbrock C.R. Chazotte B. Gupte S.S. J. Bioenerg. Biomembr. 1986; 18: 331-368Crossref PubMed Scopus (278) Google Scholar)) does not affect the low extent of control exerted by the terminal enzyme of the respiratory chain in bovine heart SMP.Further analysis of the metabolic flux control of Complex I and Complex III over the respiratory chain was performed by studying the variation of the NADH-cytochrome c oxidoreductase activity in KCN-inhibited SMP in the presence of the specific inhibitors of the two mentioned complexes. In that case also (Fig. 2), where the experimental system is simplified by the absence of the terminal segment of the respiratory chain (i.e. cytochrome c oxidase), we found that the inhibition curves of the global activities are almost superimposable with the corresponding titration curves of Complex I and Complex III specific activity, showing the same behavior as in Fig. 1, top and middle. Moreover, comparable values were determined for the corresponding flux control coefficients (Table I).Fig. 2Flux control over NADH-cytochrome c oxidoreductase activity in bovine heart submitochondrial particles. The plots on the right side show the stepwise inhibition of Complex I (—□—), Complex III (—○—), and NADH-cytochrome c oxidoreductase activity (—▿—) by titration with rotenone (top) and mucidin (bottom), respectively. The data points are cumulative of repeated experiments; symbols were omitted in the gray area of the plots on the right and were drawn for clearness in the expanded region shown on the left.View Large Image Figure ViewerDownload (PPT)It may be argued that the control coefficients in SMP, which are open vesicles or membrane sheets of very small size, could be affected by the number of complexes of the same nature existing in each single membrane fragment or that sonication for preparing submitochondrial particles might induce artificial results. For that reason, we have performed experiments in non-sonicated bovine heart mitochondria after rupturing the membrane barriers to substrates by repeated freezing and thawing. The flux control coefficients in bovine heart mitochondria (Table I) indicate that both Complex I and Complex III are largely rate-controlling upon NADH oxidation, whereas the level of control exerted by Complex IV is extremely low. The results are very similar to those obtained in SMP, suggesting that the size of the membrane fragments is non-influent on the control coefficients.Flux Control Analysis for the Respiratory Complexes Involved in Aerobic Succinate Oxidation—The succinate oxidase activity of the respiratory chain is catalyzed by the integrated redox activity of Complex II, Complex III, and Complex IV. We have performed the analysis of the metabolic flux control of those three enzymes by the stepwise inhibition with carboxin, mucidin, and potassium cyanide, respectively, in submitocondrial particles and in mitochondria from bovine heart.Fig. 3, top, shows the great similarity between the titration curve of succinate oxidase and the hyperbolic curve of Complex II alone (succinate-2,6-dichloroindophenol oxidoreductase activity) in SMP. The initial profile of both inhibition curves is steep, and the corresponding straight tangents for carboxin concentration tending to zero are almost coincident; in that case, the flux control coefficient approaches the unity (Table II).Fig. 3Flux control over succinate oxidase activity in bovine heart submitochondrial particles. The plots on the right side show the stepwise inhibition of Complex II (——), Complex III (—○—), Complex IV (—⋄—), and succinate oxidase activity (—▴—) by titration with carboxin (top), mucidin (middle), and potassium cyanide (bottom), respectively. The data points are cumulative of repeated experiments; symbols were omitted in the gray area of the plots on the right and were drawn for clearness in the expanded region shown on the left.View Large Image Figure ViewerDownload (PPT)Table IIFlux control coefficients (Cvi) of the respiratory complexes involved in succinate oxidation in bovine heart mitochondria and SMPStepSuccinate oxidase activityBHMaBovine heart mitochondria.SMPComplex II0.880.98Complex III0.340.04Complex IV0.200.12a Bovine heart mitochondria. Open table in a new tab The experimental data and the fitted titration curves of Complex III, Complex IV, and the respiratory activity measured with succinate in SMP (Fig. 3, middle and bottom) show instead that very small changes occur in the full pathway metabolic flux when low amounts of mucidin or cyanide are employed, whereas important modifications can be induced in the specific activity of the two individual enzyme complexes by using the same concentrations of inhibitors. According to the metabolic control theory, it can be asserted that Complex III and Complex IV exhibit a very low control over aerobic succinate oxidation in bovine heart submitochondrial particles (Cvi equal to 0.04 and 0.12, respectively). Quantification of the contribution of the various steps to the control of succinate oxidase activity in permeabilized bovine heart mitochondria (Table II) has confirmed the same pattern of distribution for the control strengths in SMP.Threshold Plots—In Fig. 4, two types of threshold curves can be distinguished according to their profile: plots of the residual NADH oxidase activity as a function of percent inhibition of Complexes I and III are almost linear, both in SMP and in mitochondria, whereas the curves referring to Complex IV inhibition present a plateau phase followed by a steep breakage. This behavior, which can be explained in the framework of metabolic control analysis, stresses the fact that each of the first two sites of oxidative phosphorylation exerts a complete control in our experimental conditions, whereas no effect is observed on the respiratory rate (in the presence of NADH as substrate) until cytochrome oxidase has been inhibited up to 60% of its activity.Fig. 4Threshold plots of NADH oxidase activity in bovine heart submitochondrial particles (top) and mitochondria (bottom). Each point represents the percentage of residual activity as a function of percent inhibition of Complex I (left), Complex III (middle), and Complex IV (right) for the same inhibitor concentration. Freehand threshold plots were drawn through the data points.View Large Image Figure ViewerDownload (PPT)The combined control of Complex I and Complex III is evinced also from the flux analysis on the first segment of the respiratory chain that exhibits almost linear threshold plots of residual NADH-cytochrome c reductase activity for both complexes (Fig. 5). Among the three steps that carry out the oxidation of succinate by oxygen, the step that comes closest to be rate-limiting is succinate dehydrogenase, as revealed by inspection of the threshold plots in Fig. 6, since a clearly linear profile can be observed only for Complex II, whereas the threshold curves of Complex III and Complex IV are characterized by an evident breakage, indicating their weaker control capacities, whatever the sample of mitochondrial membrane preparation (see the legend for Fig. 6).Fig. 5Threshold plots of NADH-cytochrome c oxidoreductase activity in bovine heart submitochondrial particles. Each point represents the percentage of residual activity as a function of percent inhibition of Complex I (left) and Complex III (right) for the same inhibitor concentration. Freehand threshold plots were drawn through the data points.View Large Image Figure ViewerDownload (PPT)Fig. 6Threshold plots of succinate oxidase activity in bovine heart submitochondrial particles (top) and mitochondria (bottom). Each point represents the percentage of residual activity as a function of percent inhibition of Complex II (left), Complex III (middle), and Complex IV (right) for the same inhibitor concentration. Freehand threshold plots were drawn through the data points.View Large Image Figure ViewerDownload (PPT)DISCUSSIONThe occurrence in vivo of specific interactions among the respiratory complexes has long been suspected based on mounting evidence that these protein complexes can be isolated in stoichiometric supramolecular assemblies that are stable and, in some circumstances, functionally active. In their pioneering studies, Hatefi et al. (36Hatefi Y. Haavik A.G. Jurtshuk P. Biochim. Biophys. Acta. 1961; 52: 106Crossref PubMed Scopus (64) Google Scholar, 37Hatefi Y. Methods Enzymol. 1978; 53: 3Crossref PubMed Scopus (47) Google Scholar) isolated unresolved preparations of Complex I–III and Complex II–III by deoxycholateammonium acetate or -ammonium sulfate fractionation of mitochondria. Moreover, admixture of Complexes I and III resulted in reconstitution of NADH-cytochrome c reductase activity. The reconstituted Complex I–III was stable and very similar in composition to the preparation directly isolated from mitochondria. Maximal enzyme activity was achieved at a definite weight ratio of the two complexes, which corresponded to nearly equimolar amounts of flavin mononucleotide in Complex I and cytochrome c1 in Complex III (38Hatefi Y. Methods Enzymol. 1978; 53: 48-54Crossref PubMed Scopus (16) Google Scholar). In 1987, Ozawa et al. (5
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