Lead accumulation in photosynthetic Euglena gracilis depends on polyphosphates and calcium.

Worldwide increasing levels of lead in water systems require the search for efficient ecologically friendly strategies to remove it. Hence, lead accumulation by the free-living algae-like Euglena gracilis and its effects on cellular growth, respiration, photosynthesis, chlorophyll, calcium, and levels of thiol- and phosphate-molecules were analyzed. Photosynthetic cells were able to accumulate 4627 mg lead/kgDW after 5 days of culture with 200 µM Pb2+. Nevertheless, exposure to 50, 100 and 200 µM Pb2+ for up to 8 days did not modify growth, viability, chlorophyll content and oxygen consumption/production. Enhanced biosynthesis of thiol molecules and polyphosphates, i.e. the two canonical metal ion chelation mechanisms in E. gracilis, was not induced under such conditions. However, in cells cultured in the absence of phosphate, lead accumulation and polyphosphate content markedly decreased, while culturing in the absence of sulfate did not modify the accumulation of this metal. In turn, the total amount of intracellular calcium slightly increased as the amount of intracellular lead increased, whereas under Ca2+ deficiency lead accumulation doubled. Therefore, the results indicated that E. gracilis is highly resistant to lead through mechanisms mediated by polyphosphates and Ca2+ and can in fact be classified as a lead hyperaccumulator microorganism.

Nickel accumulation by the green algae-like Euglena gracilis.

Nickel accumulation and nickel effects on cellular growth, respiration, photosynthesis, ascorbate peroxidase (APX) activity, and levels of thiols, histidine and phosphate-molecules were determined in Euglena gracilis. Cells incubated with 0.5-1mM NiCl2 showed impairment of O2 consumption, photosynthesis, Chl a+b content and APX activity whereas cellular integrity and viability were unaltered. Nickel accumulation was depressed by Mg2+ and Cu2+, while Ca2+, Co2+, Mn2+ and Zn2+ were innocuous. The growth half-inhibitory concentrations for Ni2+ in the culture medium supplemented with 2 or 0.2mM Mg2+ were 0.43 or 0.03mM Ni2+, respectively. Maximal nickel accumulation (1362mg nickel/Kg DW) was achieved in cells exposed to 1mM Ni2+ for 24h in the absence of Mg2+ and Cu2+; accumulated nickel was partially released after 72h. GSH polymers content increased or remained unchanged in cells exposed to 0.05-1mM Ni2+; however, GSH, cysteine, γ-glutamylcysteine, and phosphate-molecules all decreased after 72h. Histidine content increased in cells stressed with 0.05 and 0.5mM Ni2+ for 24h but not at longer times. It was concluded that E. gracilis can accumulate high nickel levels depending on the external Mg2+ and Cu2+ concentrations, in a process in which thiols, histidine and phosphate-molecules have a moderate contribution.

Increased synthesis of α-tocopherol, paramylon and tyrosine by Euglena gracilis under conditions of high biomass production.

AIMS: To analyse the production of different metabolites by dark-grown Euglena gracilis under conditions found to render high cell growth. METHODS AND RESULTS: The combination of glutamate (5 g l(-1) ), malate (2 g l(-1) ) and ethanol (10 ml l(-1) ) (GM + EtOH); glutamate (7·15 g l(-1) ) and ethanol (10 ml l(-1) ); or malate (8·16 g l(-1) ), glucose (10·6 g l(-1) ) and NH(4) Cl (1·8 g l(-1) ) as carbon and nitrogen sources, promoted an increase of 5·6, 3·7 and 2·6-fold, respectively, in biomass concentration in comparison with glutamate and malate (GM). In turn, the production of α-tocopherol after 120 h identified by LC-MS was 3·7 ± 0·2, 2·4 ± 0·1 and 2 ± 0·1 mg [g dry weight (DW)](-1) , respectively, while in the control medium (GM) it was 0·72 ± 0·1 mg (g DW)(-1) . For paramylon synthesis, the addition of EtOH or glucose induced a higher production. Amino acids were assayed by RP-HPLC; Tyr a tocopherol precursor and Ala an amino acid with antioxidant activity were the amino acids synthesized at higher concentration. CONCLUSIONS: Dark-grown E. gracilis Z is a suitable source for the generation of the biotechnologically relevant metabolites tyrosine, α-tocopherol and paramylon. SIGNIFICANCE AND IMPACT OF THE STUDY: By combining different carbon and nitrogen sources and inducing a tolerable stress to the cell by adding ethanol, it was possible to increase the production of biomass, paramylon, α-tocopherol and some amino acids. The concentrations of α-tocopherol achieved in this study are higher than others reported previously for Euglena, plant and algal systems. This work helps to understand the effect of different carbon sources on the synthesis of bio-molecules by E. gracilis and can be used as a basis for future works to improve the production of different metabolites of biotechnological importance by this organism.

NF-kappa B is required for the development of tumor spheroids.

Tumor cells cultured in three-dimensional models provide a more realistic and biologically meaningful analysis of the initial phases of cancer development and drug resistance. Several studies have demonstrated that culture of cancer cells in three dimensions induces cellular resistance to a variety of anti-neoplastic drugs by poorly understood mechanisms. The role of the transcription factor NF-kappaB and inhibitors of apoptosis proteins (IAPs) in the onset and development of drug resistance during tumor spheroid growth has not been established. In this work, we found a significant increase in the activity and expression of NF-kappaB and its downstream target XIAP (X-linked IAP) in cancer cells grown as multi-cellular tumor spheroids. Blocking XIAP expression with RNA interference markedly increased the sensitivity of cancer tumor spheroid cells toward anti-neoplastic drugs, indicating a role for IAPs in establishing drug resistance. In turn, inhibition of NF-kappaB by negative dominants suppressed spheroid formation, whereas overexpression of the upstream kinase IkappaBKbeta increased their growth and resistance. The present data suggested that NF-kappaB and its downstream target XIAP were essential for the growth and drug resistance of small avascular tumor.

The Pb-hyperaccumulator aquatic fern Salvinia minima Baker, responds to Pb(2+) by increasing phytochelatins via changes in SmPCS expression and in phytochelatin synthase activity.

The relationship between accumulation of Pb(2+) and the activation of chelation and metal sequestration mechanisms mediated by phytochelatins (PC) was analyzed in the Pb(2+) hyperaccumulator aquatic fern Salvinia minima, after exposure to 40microM Pb(NO(3))(2). The tissue accumulation pattern of lead and the phytochelatin biosynthesis responses were analyzed in both, S. minima submerged root-like modified fronds (here named "roots"), and in its aerial leaf-like fronds ("leaves"). S. minima roots accumulated a significantly higher concentrations of Pb(+2) than leaves did. Accumulation of Pb(2+) in roots was bi-phasic with a first uptake phase reached after 3h exposure and a second higher uptake phase reached after 24h exposure. In leaves, a single delayed, smaller uptake phase was attained only after 9h of exposure. In roots lead accumulation correlated with an increased phytochelatin synthase (PCS) activity and an enhanced PC production. A higher proportion of polymerized PC(4) was observed in both tissues of exposed S. minima plants relative to unexposed ones, although a higher concentration of PC(4) was found in roots than in leaves. PCS activity and Pb(2+) accumulation was also higher in roots than in leaves. The expression levels of the S. minima PCS gene (SmPCS), in response to Pb(2+) treatment, were also evaluated. In S. minima leaves, the accumulation of Pb(2+) correlated with a marked increase in expression of SmPCS, suggesting a transcriptional regulation in the PCS activation and PC accumulation in this S. minima tissue. However, in roots, the basal expression of SmPCS was down-regulated after Pb(2+) treatment. This fact did not correlate with the later but strong increase in both, PCS activity and PC production; suggesting that the PC biosynthesis activation in S. minima roots occurs only by post-translational activation of PCS. Taken together, our data suggest that the accumulation of PC in S. minima is a direct response to Pb(2+) accumulation, and phytochelatins do participate as one of the mechanism to cope with Pb(2+) of this Pb-hyperaccumulator aquatic fern.

Simultaneous Cd2+, Zn2+, and Pb2+ uptake and accumulation by photosynthetic Euglena gracilis.

The ability of Euglena gracilis to simultaneously remove and accumulate Zn2+, Cd2+, and Pb2+ from culture up- to media was evaluated. E. gracilis was able to remove up to 80% of the Cd2+ present in the medium when cultured with 20 or 50 microM CdCl2. Higher external Cd2+ concentrations increased Cd2+ accumulation per cell but decreased cell growth, thus decreasing the capacity of the cell culture to remove Cd2+. E. gracilis removed 70% to 80% of the Zn2+ present in the medium when cultured with 5 to 50 microM ZnSO4. Zn2+ did not affect Cd2+ removal capacity. E. gracilis was much less efficient in removing Pb2+ (<15%) when cultured with 100 or 200 microM Pb(NO3)2. Moreover, Pb2+ decreased the efficiency to remove Cd2+, but it did not affect Zn2+ removal. Cd2+ induced a generalized increase in the cellular thiol compounds, including phytochelatins, and Pb2+ had an additive effect only at 200 microM. Zn2+ did not stimulate phytochelatin synthesis. Cd2+ and Pb2+ colocated in the same cytosolic high-molecular-weight fraction. Because Pb2+ is a weak phytochelatin inducer, competition between Pb2+ and Cd2+ for transportation across the plasma membrane and binding to phytochelatins and other thiol compounds is proposed to explain the detrimental effects of Pb2+ on the Cd2+ removal capacity of E. gracilis.

Phosphorylation of the spinach chloroplast 24 kDa RNA-binding protein (24RNP) increases its binding to petD and psbA 3' untranslated regions.

The chloroplast 24 kDa RNA binding protein (24RNP) from Spinacea oleracea is a nuclear encoded protein that binds the 3' untranslated region (3'UTR) of some chloroplast mRNAs and seems to be involved in some processes of mRNA metabolism, such as 3'UTR processing, maturation and stabilization. The 24RNP is similar to the 28RNP which is involved in the correct maturation of petD and psbA 3'UTRs, and when phosphorylated, decreases its binding affinity for RNA. In the present work, we determined that the recombinant 24RNP was phosphorylated in vitro either by an animal protein kinase C, a plant Ca(2+)-dependent protein kinase, or a chloroplastic kinase activity present in a protein extract with 3'-end processing activity in which the 24RNP is also present. Phosphorylation of 24RNP increased the binding capacity (B(max)) 0.25 time for petD 3'UTR, and three times for psbA 3'UTR; the affinity for P-24RNP only increased when the interaction with petD was tested. Competition experiments suggested that B(max), not K(d), might be a more important factor in the P-24RNP-3'UTR interaction. The data suggested that the 24RNP role in chloroplast mRNA metabolism may be regulated in vivo by changes in its phosphorylation status carried out by a chloroplastic kinase.

[Microbial interactions with heavy metals]. / Interacciones microbianas con metales pesados.

Living organisms are exposed in nature to heavy metals, commonly present in their ionized species. These ions exert diverse toxic effects on microorganisms. Metal exposure both selects and maintains microbial variants able to tolerate their harmful effects. Varied and efficient metal resistance mechanisms have been identified in diverse species of bacteria, fungi and protists. The study of the interactions between microorganisms and metals may be helpful to understand the relations of toxic metals with higher organisms such as mammals and plants. Some microbial systems of metal tolerance have the potential to be used in biotechnological processes, such as the bioremediation of environmental metal pollution or the recovery of valuable metals. In this work we analyze several examples of the interactions of different types of microbes with heavy metals; these cases are related either with basic research or with possible practical applications.

Role of protonatable groups of bovine heart bc(1) complex in ubiquinol binding and oxidation.

The pH dependence of the initial reaction rate catalyzed by the isolated bovine heart ubiquinol-cytochrome c reductase (bc1 complex) varying decylbenzoquinol (DBH) and decylbenzoquinone (DB) concentrations was determined. The affinity for DBH was increased threefold by the protonation of a group with pKa = 5.7 +/- 0.2, while the inhibition constant (Ki) for DB decreased 22 and 2.8 times when groups with pKa = 5.2 +/- 0.6 and 7.7 +/- 0.2, respectively, were protonated. This suggests stabilization of the protonated form of the acidic group by DBH binding. Initial rates were best fitted to a kinetic model involving three protonatable groups. The protonation of the pKa approximately 5.7 group blocked catalysis, indicating its role in proton transfer. The kinetic model assumed that the deprotonation of two groups (pKa values of 7.5 +/- 0.03 and approximately 9.2) decreases the catalytic rate by diminishing the redox potential of the iron-sulfur (Fe-S) cluster. The protonation of the pKa approximately 7.5 group also decreased the reaction rate by 80-86%, suggesting its role as acceptor of a proton from ubiquinol. The lack of effect on the Km for DBH when the pKa 7.5-7.7 group is deprotonated suggests that hydrogen bonding to this residue is not the main factor that determines substrate binding to the Qo site. The possible relationship of the pKa 5.2-5.7 and pKa 7.5-7.7 groups with Glu272 of cytochrome b and His161 of the Fe-S protein is discussed.

Metabolic changes induced by cold stress in rat liver mitochondria.

The mechanisms involved in the metabolic changes induced by cold stress in isolated rat liver mitochondria were studied. Respiration, ATP synthesis, and membrane potential as well as the contents of several metabolites were determined in liver mitochondria from cold-exposed rats. At different times of cold exposure, the force-flux relationships showed net variation in flux (enhanced respiration, diminished ATP synthesis) with no associated variation in force (H+ gradient); this suggested that decoupling rather than classical uncoupling was involved in the effects of cold stress. The flux control coefficient of the H+ leak on basal respiration was slightly increased by 380 h of cold exposure. Cold stress also induced a diminution in total membrane fatty acids, Zn2+, Fe3+, ATP, and ADP/O ratios; the content of cytochromes c + c1 and b oscillated. The contents of Ca2+, Na+, Pi, and cytochromes a + a3 were not affected, whereas matrix ADP, AMP, K+, and Mg2+ were markedly increased. Basal and oleic acid-stimulated respiration of mitochondria from cold-stressed rats was inhibited by GDP, carboxyatractyloside, or albumin. These agents did not affect basal respiration in control mitochondria. Western blot analysis showed enhanced expression of a protein of about 35 kDa, presumably the uncoupling protein 2, induced by long-term cold exposure. The overall data suggest that cold stress promoted decoupling of oxidative phosphorylation, and hence, changes in several matrix metabolites, by increasing free fatty acids and the UCP2 content.

The Membrane-bound L- and D-lactate dehydrogenase activities in mitochondria from Euglena gracilis.

The activity of the pyridine nucleotide-independent lactate dehydrogenase (iLDH) was characterized in mitochondria isolated from the protist Euglena gracilis. The dissociation constants for L- and D-lactate were similar, but the V(max) was higher with the d isomer. A ping-pong kinetic mechanism was displayed with 2,4-dichlorophenol-indolphenol (DCPIP), or coenzyme Q(1), reacting as the second substrate with the modified, reduced enzyme. Oxamate was a competitive inhibitor against both L- and D-lactate. Oxalate exerted a mixed-type inhibition regarding L- or D-lactate and also against DCPIP. The rate of L-lactate uptake was partially inhibited by mersalyl and lower than the rate of dehydrogenation, which was mersalyl-insensitive. These data suggested that the active site of L-iLDH was orientated toward the intermembrane space. The following observations indicated the existence of two stereo-specific iLDH enzymes in the inner membrane of Euglena mitochondria: a greater affinity of the D-iLDH for both inhibitors, D-iLDH thermo-stability at 70 degrees C and denaturation of L-iLDH, opposite signs in the enthalpy change for the association reaction of the isomers to the enzyme, differential solubilization of both activities with detergents, and different molecular mass.

Interactions of chromium with microorganisms and plants.

Chromium is a highly toxic non-essential metal for microorganisms and plants. Due to its widespread industrial use, chromium (Cr) has become a serious pollutant in diverse environmental settings. The hexavalent form of the metal, Cr(VI), is considered a more toxic species than the relatively innocuous and less mobile Cr(III) form. The presence of Cr in the environment has selected microbial and plant variants able to tolerate high levels of Cr compounds. The diverse Cr-resistance mechanisms displayed by microorganisms, and probably by plants, include biosorption, diminished accumulation, precipitation, reduction of Cr(VI) to Cr(III), and chromate efflux. Some of these systems have been proposed as potential biotechnological tools for the bioremediation of Cr pollution. In this review we summarize the interactions of bacteria, algae, fungi and plants with Cr and its compounds.

Multisite control of the Crabtree effect in ascites hepatoma cells.

AS-30D hepatoma cells, a highly oxidative and fast-growing tumor line, showed glucose-induced and fructose-induced inhibition of oxidative phosphorylation (the Crabtree effect) of 54% and 34%, respectively. To advance the understanding of the underlying mechanism of this process, the effect of 5 mM glucose or 10 mM fructose on the intracellular concentration of several metabolites was determined. The addition of glucose or fructose lowered intracellular Pi (40%), and ATP (53%) concentrations, and decreased cytosolic pH (from 7.2 to 6.8). Glucose and fructose increased the content of AMP (30%), glucose 6-phosphate, fructose 6-phosphate and fructose 1,6-bisphosphate (15, 13 and 50 times, respectively). The cytosolic concentrations of Ca2+ and Mg2+ were not modified. The addition of galactose or glycerol did not modify the concentrations of the metabolites. Mitochondria isolated from AS-30D cells, incubated in media with low Pi (0.6 mM) at pH 6.8, exhibited a 40% inhibition of oxidative phosphorylation. The data suggest that the Crabtree effect is the result of several small metabolic changes promoted by addition of exogenous glucose or fructose.

Mercury uptake and removal by Euglena gracilis.

The uptake and removal of mercury (added as HgCl2) from the culture medium by Euglena gracilis was studied. In cultures initiated in the light, cells accumulated a small fraction of the added heavy metal (5-13%). Mercury was both biologically and nonbiologically volatilized, and cell growth was partially inhibited; under these conditions the glutathione content was 3.2 nmol/10(6) cells. In contrast, in cultures initiated in the dark, mercury uptake by cells was two to three times higher, biological volatilization remained unchanged and nonbiological volatilization and growth were negligible; the glutathione content diminished to 1.4 nmol/10(6) cells. Biological mercury volatilization depended on cell density and metal concentration, but was light-independent. Thus, volatilization of mercury by Euglena appeared not to be an effective mechanism of resistance, whereas a high intracellular level of glutathione and a low mercury uptake seemed necessary for successful tolerance.

Modulation of 2-oxoglutarate dehydrogenase complex by inorganic phosphate, Mg(2+), and other effectors.

The interplay of inorganic phosphate (Pi) with other ligands such as Mg(2+), ADP, ATP, and Ca(2+) on the activation of 2-oxoglutarate dehydrogenase complex (2-OGDH) in both isolated enzyme complex and mitochondrial extracts was examined. Pi alone activated the enzyme, following biphasic kinetics with high (K(0.5) = 1.96+/-0.42 mM) and low (K(0.5) = 9.8+/-0.4 mM) affinity components for Pi. The activation by Pi was highly pH-dependent; it increased when the pH raised from 7.1 to 7.6, but it was negligible at pH values below 7.1. Mg-Pi and Mg-ADP, but not Mg-ATP, were more potent activators of 2-OGDH than free Pi and free ADP. ATP inhibited the 2-OGDH activity by chelating the free Mg(2+) and also as a Mg-ATP complex. With or without Mg(2+), ADP, and Pi activated the 2-OGDH by increasing the affinity for 2-OG and the V(m) of the reaction; ATP diminished the V(m), but it increased the affinity for 2-OG in the mitochondrial extract. Pi did not modify the 2-OGDH activation by Ca(2+). The results above mentioned were similar for both preparations, except for hyperbolic kinetics in the isolated enzyme and sigmoidal kinetics in the mitochondrial extracts when 2-oxoglutarate was varied. The data of this study indicated that physiological concentrations of Pi may exert a significant activation of 2-OGDH, which was potentiated by Mg(2+) and high pH, but surpassed by ADP.

Oxidative phosphorylation supported by an alternative respiratory pathway in mitochondria from Euglena.

The effect of antimycin, myxothiazol, 2-heptyl-4-hydroxyquinoline-N-oxide, stigmatellin and cyanide on respiration, ATP synthesis, cytochrome c reductase, and membrane potential in mitochondria isolated from dark-grown Euglena cells was determined. With L-lactate as substrate, ATP synthesis was partially inhibited by antimycin, but the other four inhibitors completely abolished the process. Cyanide also inhibited the antimycin-resistant ATP synthesis. Membrane potential was collapsed (<60 mV) by cyanide and stigmatellin. However, in the presence of antimycin, a H(+)60 mV) that sufficed to drive ATP synthesis remained. Cytochrome c reductase, with L-lactate as donor, was diminished by antimycin and myxothiazol. Cytochrome bc(1) complex activity was fully inhibited by antimycin, but it was resistant to myxothiazol. Stigmatellin inhibited both L-lactate-dependent cytochrome c reductase and cytochrome bc(1) complex activities. Respiration was partially inhibited by the five inhibitors. The cyanide-resistant respiration was strongly inhibited by diphenylamine, n-propyl-gallate, salicylhydroxamic acid and disulfiram. Based on these results, a model of the respiratory chain of Euglena mitochondria is proposed, in which a quinol-cytochrome c oxidoreductase resistant to antimycin, and a quinol oxidase resistant to antimycin and cyanide are included.

Sulfite and membrane energization induce two different active states of the Paracoccus denitrificans F0F1-ATPase.

Activation of the latent ATPase activity of inside-out vesicles from plasma membranes of Paracoccus denitrificans was studied. Several factors were found to induce activation: heat, membrane energization by succinate oxidation, methanol, oxyanions (sulfite, phosphate, arsenate, bicarbonate) and limited proteolysis with trypsin. Among the oxyanions, sulfite induced the higher increase in ATPase activity. Sulfite functioned as a nonessential activator that slightly modified the affinity for ATP and increased notoriously the Vmax. There was a competitive effect between sulfite, bicarbonate and phosphate for ATPase activation; their similar chemical geometry suggests that these oxyanions have a common binding site on the enzyme. Dithiothreitol did not affect the ATPase activity. ATPase activation by sulfite was decreased by uncoupler, enhanced by trypsin and inhibited by ADP, oligomycin and venturicidin. In contrast, activation induced by succinate was less sensitive to ADP, oligomycin, venturicidin and trypsin. It is proposed that the active states induced by sulfite and succinate reflect two conformations of the enzyme, in which the inhibitory subunit epsilon is differently exposed to trypsin.

Substrate oxidation and ATP supply in AS-30D hepatoma cells.

The oxidation of several metabolites in AS-30D tumor cells was determined. Glucose and glycogen consumption and lactic acid production showed high rates, indicating a high glycolytic activity. The utilization of ketone bodies, oxidation of endogenous glutamate, and oxidative phosphorylation were also very active: tumor cells showed a high respiration rate (100 ng atoms oxygen (min x 10(7) cells)(-1)), which was 90% oligomycin-sensitive. AS-30D tumor cells underwent significant intracellular volume changes, which preserved high concentrations of several metabolites. A high O(2) concentration, but a low glucose concentration were found in the cell-free ascites liquid. Glutamine was the oxidizable substrate found at the highest concentration in the ascites liquid. We estimated that cellular ATP was mainly provided by oxidative phosphorylation. These data indicated that AS-30D hepatoma cells had a predominantly oxidative and not a glycolytic type of metabolism. The NADH-ubiquinol oxido reductase and the enzyme block for ATP utilization were the sites that exerted most of the control of oxidative phosphorylation (flux control coefficient = 0.3-0.42).

Chromate efflux by means of the ChrA chromate resistance protein from Pseudomonas aeruginosa.

Everted membrane vesicles of Pseudomonas aeruginosa PAO1 harboring plasmid pCRO616, expressing the ChrA chromate resistance protein, accumulated four times more (51)CrO(4)(2-) than vesicles from plasmidless cells, indicating that a chromate efflux system functions in the resistant strain. Chromate uptake showed saturation kinetics with an apparent K(m) of 0.12 mM chromate and a V(max) of 0. 5 nmol of chromate/min per mg of protein. Uptake of chromate by vesicles was dependent on NADH oxidation and was abolished by energy inhibitors and by the chromate analog sulfate. The mechanism of resistance to chromate determined by ChrA appears to be based on the active efflux of chromate driven by the membrane potential.

Determining and understanding the control of flux. An illustration in submitochondrial particles of how to validate schemes of metabolic control.

Two complementary methods were used to determine how the rate of respiration and that of ATP hydrolysis were controlled in rat liver submitochondrial particles. In the first, 'direct control analysis' method, respiration was titrated with malonate, antimycin or cyanide at 20, 30 and 37 degrees C, to determine the flux control exerted by succinate dehydrogenase, cytochrome bc1 complex and cytochrome c oxidase, respectively. Together, the three respiratory complexes only controlled the flux by about 50%, leaving the other 50% of flux control to the H+ leak. In the second, 'elasticity based' method, the elasticity coefficients of the respiratory chain or the H+-ATPase and the H+ leak towards the H+ gradient were determined. Then, the flux control coefficients were calculated using the connectivity and summation laws of metabolic control theory. The correspondence between the flux control coefficients determined in the two ways validated the two methods. This allowed us to use the second method to analyse what was the kinetic origin of the observed distribution of control. Control of ATP hydrolysis by the ATPase decreased with increasing ATPase activity; hence, the control exerted by the H+ leak increased with increasing ATPase activity, due to a diminishing elasticity towards the H+ gradient. Reverse electron transport was mainly controlled by the ATPase; the sum of flux control coefficients of succinate dehydrogenase, NADH-CoQ oxidoreductase, and H+-ATPase yielded a value greater than one, indicating that the H+ leak exerted a significant negative control on this pathway.