Alteration of plant mitochondrial proton conductance by free fatty acids. Uncoupling protein involvement

We characterized the uncoupling activity of the plant uncoupling protein from Solanum tuberosum (StUCP) using mitochondria from intact potato tubers or from yeast (Saccharomyces cerevisiae) expressing the StUCP gene. Compared with mitochondria from transfected yeast, StUCP is present at very low levels in intact potato mitochondrial membranes (at least thirty times lower) as shown by immunodetection with anti-UCP1 antibodies. Under conditions that ruled out undesirable effects of nucleotides and free fatty acids on uncoupling activity measurement in plant mitochondria, the linoleic acid-induced depolarization in potato mitochondria was insensitive to the nucleotides ATP, GTP, or GDP. In addition, sensitivity to linoleic acid was similar in potato and in control yeast mitochondria, suggesting that uncoupling occurring in potato mitochondria was because of a UCP-independent proton diffusion process. By contrast, yeast mitochondria expressing StUCP exhibited a higher sensitivity to free fatty acids than those from the control yeast and especially a marked proton conductance in the presence of low amounts of linoleic acid. However, this fatty acid-induced uncoupling was also insensitive to nucleotides. Altogether, these results suggest that uncoupling of oxidative phosphorylation and heat production cannot be the dominant feature of StUCP expressed in native potato tissues. However, it could play a role in preventing reactive oxygen species production as proposed for mammalian UCP2 and UCP3.     

Hydroxynonenal, a lipid peroxidation end product, stimulates uncoupling protein activity in Acanthamoeba castellanii mitochondria; the sensitivity of the inducible activity to purine nucleotides depends on the membranous ubiquinone redox state

We studied the influence of exogenously generated superoxide and exogenous 4-hydroxy-2-nonenal (HNE), a lipid peroxidation end product, on the activity of the Acanthamoeba castellanii uncoupling protein (AcUCP). The superoxide-generating xanthine/xanthine oxidase system was insufficient to induce mitochondrial uncoupling. In contrast, exogenously added HNE induced GTP-sensitive AcUCP-mediated mitochondrial uncoupling. In non-phosphorylating mitochondria, AcUCP activation by HNE was demonstrated by increased oxygen consumption accompanied by a decreased membrane potential and ubiquinone (Q) reduction level. The HNE-induced GTP-sensitive proton conductance was similar to that observed with linoleic acid. In phosphorylating mitochondria, the HNE-induced AcUCP-mediated uncoupling decreased the yield of oxidative phosphorylation. We demonstrated that the efficiency of GTP to inhibit HNE-induced AcUCP-mediated uncoupling was regulated by the endogenous Q redox state. A high Q reduction level activated AcUCP by relieving the inhibition caused by GTP while a low Q reduction level favoured the inhibition. We propose that the regulation of UCP activity involves a rapid response through the endogenous Q redox state that modulates the inhibition of UCP by purine nucleotides, followed by a late response through lipid peroxidation products resulting from an increase in the formation of reactive oxygen species that modulate the UCP activation.     

In Phosphorylating <Emphasis Type="Italic">Acanthamoeba castellanii</Emphasis> Mitochondria the Sensitivity of Uncoupling Protein Activity to GTP Depends on the Redox State of Quinone

In isolated Acanthamoeba castellanii mitochondria respiring in state 3 with external NADH or succinate, the linoleic acid-induced purine nucleotide-sensitive uncoupling protein activity is able to uncouple oxidative phosphorylation. The linoleic acid-induced uncoupling can be inhibited by a purine nucleotide (GTP) when quinone (Q) is sufficiently oxidized, indicating that in A. castellanii mitochondria respiring in state 3, the sensitivity of uncoupling protein activity to GTP depends on the redox state of the membranous Q. Namely, the inhibition of the linoleic acid-induced uncoupling by GTP is not observed in uninhibited state 3 respiration as well as in state 3 respiration progressively inhibited by complex III inhibitors, i.e., when the rate of quinol (QH₂)-oxidizing pathway is decreased. On the contrary, the progressive decrease of state 3 respiration by declining respiratory substrate availability (by succinate uptake limitation or by decreasing external NADH concentration), i.e., when the rate of Q-reducing pathways is decreased, progressively leads to a full inhibitory effect of GTP. Moreover, in A. castellanii mitochondria isolated from cold-treated cells, where a higher uncoupling protein activity is observed, the inhibition of the linoleic acid-induced proton leak by GTP is revealed for the same low values of the Q reduction level.     

Production of endogenous matrix superoxide from mitochondrial complex I leads to activation of uncoupling protein 3

Superoxide generated using exogenous xanthine oxidase indirectly activates an uncoupling protein (UCP)-mediated proton conductance of the mitochondrial inner membrane. We investigated whether endogenous mitochondrial superoxide production could also activate proton conductance. When respiring on succinate, rat skeletal muscle mitochondria produced large amounts of matrix superoxide. Addition of GDP to inhibit UCP3 markedly inhibited proton conductance and increased superoxide production. Both superoxide production and the GDP-sensitive proton conductance were suppressed by rotenone plus an antioxidant. Thus, endogenous superoxide can activate the proton conductance of UCP3, which in turn limits mitochondrial superoxide production. These observations provide a departure point for studies under more physiological conditions.     

Activation and function of mitochondrial uncoupling protein in plants

Plant mitochondrial uncoupling protein (UCP) is activated by superoxide suggesting that it may function to minimize mitochondrial reactive oxygen species (ROS) formation. However, the precise mechanism of superoxide activation and the exact function of UCP in plants are not known. We demonstrate that 4-hydroxy-2-nonenal (HNE), a product of lipid peroxidation, and a structurally related compound, trans-retinal, stimulate a proton conductance in potato mitochondria that is inhibitable by GTP (a characteristic of UCP). Proof that the effects of HNE and trans-retinal are mediated by UCP is provided by examination of proton conductance in transgenic plants overexpressing UCP. These experiments demonstrate that the mechanism of activation of UCP is conserved between animals and plants and imply a conservation of function. Mitochondria from transgenic plants overexpressing UCP were further studied to provide insight into function. Experimental conditions were designed to mimic a bioenergetic state that might be found in vivo (mitochondria were supplied with pyruvate as well as tricarboxylic cycle acids at in vivo cytosolic concentrations and an exogenous ATP sink was established). Under such conditions, an increase in UCP protein content resulted in a modest but significant decrease in the rate of superoxide production. In addition, 13C-labeling experiments revealed an increase in the conversion of pyruvate to citrate as a result of increased UCP protein content. These results demonstrate that under simulated in vivo conditions, UCP is active and suggest that UCP may influence not only mitochondrial ROS production but also tricarboxylic acid cycle flux.     

The basal proton conductance of skeletal muscle mitochondria from transgenic mice overexpressing or lacking uncoupling protein-3.

The ability of native uncoupling protein-3 (UCP3) to uncouple mitochondrial oxidative phosphorylation is controversial. We measured the expression level of UCP3 and the proton conductance of skeletal muscle mitochondria isolated from transgenic mice overexpressing human UCP3 (UCP3-tg) and from UCP3 knockout (UCP3-KO) mice. The concentration of UCP3 in UCP3-tg mitochondria was approximately 3 microg/mg protein, approximately 20-fold higher than the wild type value. UCP3-tg mitochondria had increased nonphosphorylating respiration rates, decreased respiratory control, and approximately 4-fold increased proton conductance compared with the wild type. However, this increased uncoupling in UCP3-tg mitochondria was not caused by native function of UCP3 because it was not proportional to the increase in UCP3 concentration and was neither activated by superoxide nor inhibited by GDP. UCP3 was undetectable in mitochondria from UCP3-KO mice. Nevertheless, UCP3-KO mitochondria had unchanged respiration rates, respiratory control ratios, and proton conductance compared with the wild type under a variety of assay conditions. We conclude that uncoupling in UCP3-tg mice is an artifact of transgenic expression, and that UCP3 does not catalyze the basal proton conductance of skeletal muscle mitochondria in the absence of activators such as superoxide.     

Superoxide activates a GDP-sensitive proton conductance in skeletal muscle mitochondria from king penguin (Aptenodytes patagonicus)

We present the partial nucleotide sequence of the avian uncoupling protein (avUCP) gene from king penguin (Aptenodytes patagonicus), showing that the protein is 88-92% identical to chicken (Gallus gallus), turkey (Meleagris gallopavo), and hummingbird (Eupetomena macroura). We show that superoxide activates the proton conductance of mitochondria isolated from king penguin skeletal muscle. GDP abolishes the superoxide-activated proton conductance, indicating that it is mediated via avUCP. In the absence of superoxide there is no GDP-sensitive component of the proton conductance from penguin muscle mitochondria demonstrating that avUCP plays no role in the basal proton leak.     

Redox state of quinone affects sensitivity of Acanthamoeba castellanii mitochondrial uncoupling protein to purine nucleotides

We studied FFA (free fatty acid)-induced uncoupling activity in Acanthamoeba castellanii mitochondria in the non-phosphorylating state. Either succinate or external NADH was used as a respiratory substrate to determine the proton conductance curves and the relationships between respiratory rate and the quinone reduction level. Our determinations of the membranous quinone reduction level in non-phosphorylating mitochondria show that activation of UCP (uncoupling protein) activity leads to a PN (purine nucleotide)-sensitive decrease in the quinone redox state. The gradual decrease in the rate of quinone-reducing pathways (using titration of dehydrogenase activities) progressively leads to a full inhibitory effect of GDP on LA (linoleic acid) induced proton conductance. This inhibition cannot be attributed to changes in the membrane potential. Indeed, the lack of GDP inhibitory effect observed when the decrease in respiratory rate is accompanied by an increase in the quinone reduction level (using titration of the quinol-oxidizing pathway) proves that the inhibition by nucleotides can be revealed only for a low quinone redox state. It must be underlined that, in A. castellanii non-phosphorylating mitochondria, the transition of the inhibitory effect of GDP on LA-induced UCP-mediated uncoupling is observed for the same range of quinone reduction levels (between 50% and 40%) as that observed previously for phosphorylating conditions. This observation, drawn from the two different metabolic states of mitochondria, indicates that quinone could affect UCP activity through sensitivity to PNs.     

No evidence for a basal,retinoic, or superoxide-induced uncoupling activity of the uncoupling protein 2 present in spleen or lung mitochondria

The phenotypes observed in mice whose uncoupling protein (Ucp2) gene had been invalidated by homologous recombination (Ucp2(-/-) mice) are consistent with an increase in mitochondrial membrane potential in macrophages and pancreatic beta cells. This could support an uncoupling (proton transport) activity of UCP2 in the inner mitochondrial membrane in vivo. We used mitochondria from lung or spleen, the two organs expressing the highest level of UCP2, to compare the proton leak of the mitochondrial inner membrane of wild-type and Ucp2(-/-) mice. No difference was observed under basal conditions. Previous reports have concluded that retinoic acid and superoxide activate proton transport by UCP2. Spleen mitochondria showed a higher sensitivity to retinoic acid than liver mitochondria, but this was not caused by UCP2. In contrast with a previous report, superoxide failed to increase the proton leak rate in kidney mitochondria, where no UCP2 expression was detected, and also in spleen mitochondria, which does not support stimulation of UCP2 uncoupling activity by superoxide. Finally, no increase in the ATP/ADP ratio was observed in spleen or lung of Ucp2(-/-) mice. Therefore, no evidence could be gathered for the uncoupling activity of the UCP2 present in spleen or lung mitochondria. Although this may be explained by difficulties with isolated mitochondria, it may also indicate that UCP2 has another physiological significance in spleen and lung.     

Proton leak induced by reactive oxygen species produced during in vitro anoxia/reoxygenation in rat skeletal muscle mitochondria

Superoxide anion generation and the impairment of oxidative phosphorylation yield were studied in rat skeletal muscle mitochondria submitted to anoxia/reoxygenationk in vitro. Production of superoxide anion was detected after several cycles of anoxia/reoxygenationk. Concomitantly, a decrease of state 3 respiration and phosphorylation yield (ADP/O) were observed. The latter resulted from a proton leak. The presence of palmitic acid during anoxia/reoxygenationk cycles led to a dose-dependent inhibition of superoxide anion production together with a partial protection of the ADP/O ratio measured after anoxia/reoxygenationk. The ADP/O decrease was shown to be due to a permeability transition pore-sustained proton leak, as it was suppressed by cyclosporine A. The permeability transition pore activation was induced during anoxia/reoxygenationk by superoxide anion, as it was cancelled by the spin trap (POBN), which scavenges superoxide anion and by palmitic acid, which induces mitochondrial uncoupling. It can be proposed that the palmitic acid-induced proton leak cancels the production of superoxide anion by mitochondria during anoxia/reoxygenationk and therefore prevents the occurrence of the superoxide anion-induced permeability transition pore-mediated proton leak after anoxia/reoxygenationk.     

Proton leak modulation in testicular mitochondria affects reactive oxygen species production and lipid peroxidation

Mitochondrial proton leak can account for almost 20% of oxygen consumption and it is generally accepted that this process contributes to basal metabolism. In order to clarify the role of basal proton leak in testicular mitochondria, we performed a comparative study with kidney and liver mitochondrial fractions. Proton leak stimulated by linoleic acid and inhibited by guanosine diphosphate (GDP) was detected, in a manner that was correlated with protein levels for uncoupling protein 2 (UCP2) in the three fractions. Modulation of proton leak had an effect on reactive oxygen species production as well as on lipid peroxidation, and this effect was also tissue‐dependent. However, a possible role for the adenine nucleotide transporter (ANT) in testicular mitochondria proton leak could not be excluded. The modulation of proton leak appears as a possible and attractive target to control oxidative stress with implications for male gametogenesis. Copyright © 2010 John Wiley & Sons, Ltd.     

Functional characterisation of UCP1 in the common carp: uncoupling activity in liver mitochondria and cold-induced expression in the brain

Mammalian uncoupling protein 1 (UCP1) mediates nonshivering thermogenesis in brown adipose tissue. We previously reported on the presence of a UCP1 orthologue in ectothermic fish and observed downregulation of UCP1 gene expression in the liver of the common carp. Neither the function of UCP1, nor the mode of UCP1 activation is known in carp liver mitochondria. Here, we compared the proton conductance at 25°C of liver mitochondria isolated from carp either maintained at 20°C (warm-acclimated, WA) or exposed to 8°C (cold-acclimated, CA) water temperature for 7–10 days. Liver mitochondria from WA carp had higher state four rates of oxygen consumption and greater proton conductance at high membrane potential. Liver mitochondria from WA, but not from CA, carp showed a strong increase in proton conductance when palmitate (or 4-hydroxy-trans-2-nonenal, HNE) was added, and this inducible proton conductance was prevented by addition of GDP. This fatty acid sensitive proton leak is likely due to the expression of UCP1 in the liver of WA carp. The observed biochemical properties of proton leak strongly suggest that carp UCP1 is a functional uncoupling protein with broadly the same activatory and inhibitory characteristics as mammalian UCP1. Significant UCP1 expression was also detected in our previous study in whole brain of the carp. We here observed a twofold increase of UCP1 mRNA in carp brain following cold exposure, suggesting a role of UCP1 in the thermal adaptation of brain metabolism. In situ hybridization located the UCP1 gene expression to the optic tectum responsible for visual system control, the descending trigeminal tract and the solitary tract. Taken together, this study characterises uncoupling protein activity in an ectotherm for the first time. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

High membrane potential promotes alkenal-induced mitochondrial uncoupling and influences adenine nucleotide translocase conformation

Mitochondria generate reactive oxygen species, whose downstream lipid peroxidation products, such as 4-hydroxynonenal, induce uncoupling of oxidative phosphorylation by increasing proton leak through mitochondrial inner membrane proteins such as the uncoupling proteins and adenine nucleotide translocase. Using mitochondria from rat liver, which lack uncoupling proteins, in the present study we show that energization (specifically, high membrane potential) is required for 4-hydroxynonenal to activate proton conductance mediated by adenine nucleotide translocase. Prolonging the time at high membrane potential promotes greater uncoupling. 4-Hydroxynonenal-induced uncoupling via adenine nucleotide translocase is prevented but not readily reversed by addition of carboxyatractylate, suggesting a permanent change (such as adduct formation) that renders the translocase leaky to protons. In contrast with the irreversibility of proton conductance, carboxyatractylate added after 4-hydroxynonenal still inhibits nucleotide translocation, implying that the proton conductance and nucleotide translocation pathways are different. We propose a model to relate adenine nucleotide translocase conformation to proton conductance in the presence or absence of 4-hydroxynonenal and/or carboxyatractylate.     

The role of superoxide in xanthine oxidase-induced autooxidation of linoleic acid

The effect of hydroxyperoxyoctadecadienoic acid, e.g. 13-hydroperoxy-cis,9,trans-11-octadecadienoic acid, on the autooxidation of linoleic acid induced by superoxide radical was examined in a system containing xanthine oxidase, acetaldehyde, and diethylenetriaminepentaacetic acid dissolved in an aqueous phosphate buffer containing 10% ethanol. The superoxide radical is required for autooxidation, as shown by essentially complete inhibition on the addition of superoxide dismutase. Pure linoleic acid was not readily oxidized, but the addition of lipid hydroperoxide markedly stimulated the autooxidation. Addition of 2.8 microM FeCl3 did not produce an increase in the rate of xanthine oxidase-induced autooxidation. Spontaneous autooxidation, a process slower than xanthine oxidase-induced autooxidation, was detectable on the time scale of these observations but was slower than the xanthine oxidase-induced autooxidation. Initiation of linoleic acid autooxidation is postulated to result from a reaction between superoxide and lipid hydroperoxide. The nature of this reaction is uncertain, but it does not appear to depend on iron catalysis.     

Cloning of mouse mitochondrial thymidine kinase 2 cDNA

In this communication, we show that the plant uncoupling mitochondrial protein (PUMP) present in potato tuber mitochondria is induced by aging at 28 degrees C and that this induction is strongly stimulated when the potato tubers are stored at low temperature (4 degrees C). PUMP activity was detected by the degree of linoleic acid (LA)-induced ATP-sensitive mitochondrial uncoupling measured as a function of the decrease in membrane potential (delta psi). The PUMP content was evaluated by immunoblot analysis using polyclonal antibodies raised against potato PUMP that specifically detected a 32 kDa band. In agreement with the effect of LA on delta psi, the content of the 32 kDa band increased during storage and was stimulated by low temperature. These results support the proposed role of PUMP in plant thermogenesis and possibly in fruit ripening and senescence.     

Fatty acid activation of the reconstituted brown adipose tissue mitochondria uncoupling protein

The effect of fatty acids, palmitoyl-CoA, and N',N-dicyclohexylcarbodiimide on the ion conductance of the reconstituted brown adipose tissue mitochondria uncoupling protein was investigated. 1, 5, and 10 microM palmitic acid induced a specific, GDP inhibited, increase in proton conductance in proteoliposomes containing the uncoupling protein but not in proteoliposomes prepared with purified protein extracts of liver mitochondria. 10 microM oleic acid, like palmitic acid, increased proton conductance in proteoliposomes prepared with the uncoupling protein. Palmitoyl-CoA and caprylic acid had no effect on increasing proton conductance. Similar to the observation in mitochondria, there was no effect of palmitic acid on Cl-conductance, but unlike mitochondria its activation by palmitoyl-CoA or inhibition by N',N-dicyclohexylcarbodiimide was lost. The results, obtained in an isolated system, provide support for the contention that long chain fatty acids act as an acute physiological activator of the uncoupling protein.     

Xanthine dehydrogenase AtXDH1 from <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> is a potent producer of superoxide anions via its NADH oxidase activity

Xanthine dehydrogenase AtXDH1 from Arabidopsis thaliana is a key enzyme in purine degradation where it oxidizes hypoxanthine to xanthine and xanthine to uric acid. Electrons released from these substrates are either transferred to NAD⁺ or to molecular oxygen, thereby yielding NADH or superoxide, respectively. By an alternative activity, AtXDH1 is capable of oxidizing NADH with concomitant formation of NAD⁺ and superoxide. Here we demonstrate that in comparison to the specific activity with xanthine as substrate, the specific activity of recombinant AtXDH1 with NADH as substrate is about 15-times higher accompanied by a doubling in superoxide production. The observation that NAD⁺ inhibits NADH oxidase activity of AtXDH1 while NADH suppresses NAD⁺-dependent xanthine oxidation indicates that both NAD⁺ and NADH compete for the same binding-site and that both sub-activities are not expressed at the same time. Rather, each sub-activity is determined by specific conditions such as the availability of substrates and co-substrates, which allows regulation of superoxide production by AtXDH1. Since AtXDH1 exhibits the most pronounced NADH oxidase activity among all xanthine dehydrogenase proteins studied thus far, our results imply that in particular by its NADH oxidase activity AtXDH1 is an efficient producer of superoxide also in vivo.     

Redox State of Endogenous Coenzyme Q Modulates the Inhibition of Linoleic Acid-Induced Uncoupling by Guanosine Triphosphate in Isolated Skeletal Muscle Mitochondria

The skeletal muscle mitochondria contain two isoforms of uncoupling protein, UCP2 and mainly UCP3, which had been shown to be activated by free fatty acids and inhibited by purine nucleotides in reconstituted systems. On the contrary in isolated mitochondria, the protonophoretic action of muscle UCPs had failed to be demonstrated in the absence of superoxide production. We showed here for the first time that muscle UCPs were activated in state 3 respiration by linoleic acid and dissipated energy from oxidative phosphorylation by decreasing the ADP/O ratio. The efficiency of UCPs in mitochondrial uncoupling increased when the state 3 respiratory rate decreased. The inhibition of the linoleic acid-induced uncoupling by a purine nucleotide (GTP), was not observed in state 4 respiration, in uninhibited state 3 respiration, as well as in state 3 respiration inhibited by complex III inhibitors. On the contrary, the progressive inhibition of state 3 respiration by n -butyl malonate, which inhibits the uptake of succinate, led to a full inhibitory effect of GTP. Therefore, as the inhibitory effect of GTP was observed only when the reduced state of coenzyme Q was decreased, we propose that the coenzyme Q redox state could be a metabolic sensor that modulates the purine nucleotide inhibition of FFA-activated UCPs in muscle mitochondria.     

Regulation of uncoupling protein activity in phosphorylating potato tuber mitochondria

In isolated potato tuber mitochondria, palmitic acid (PA) can induce a H+ leak inhibited by GTP in the phosphorylating (state 3) respiration but not in the resting (state 4) respiration. The PA-induced H+ leak is constant when state 3 respiration is decreased by an inhibition of the succinate uptake with n-butyl malonate (nBM). We show that the efficiency of inhibition by GTP is decreased when state 3 respiration is progressively inhibited by antimycin A (AA) and is restored following subsequent addition of nBM. We propose that in phosphorylating potato tuber mitochondria, the redox state of ubiquinone, which can antagonistically be varied with AA and nBM, modulates inhibition of the PA-activated UCP-sustained H+ leak by GTP.     

Characterisation of the control of respiration in potato tuber mitochondria using the top-down approach of metabolic control analysis.

Control over oxidative phosphorylation by purified potato mitochondria was determined using the top-down approach of metabolic control analysis. The control over the respiration rate, phosphorylation rate, proton-leak rate and proton motive force exerted by the respiratory chain, phosphorylation reactions and the proton leak were measured over a range of phosphorylation rates from resting (state 4) to maximal (state 3). These rates were obtained by adding different amounts of hexokinase in the presence of glucose, or different amounts of oligomycin in the presence of ADP. The respiratory substrate was NADH or succinate, both of which feed electrons directly to ubiquinone. The rate of oxygen consumption by the alternative oxidase pathway was negligible with NADH as substrate but was measurable with succinate and was subtracted. Control over the respiration rate in potato mitochondria was predominantly exerted by the respiratory chain at all rates except close to state 4, where control by the proton leak was equally or more important. For oxidation of NADH, the flux control coefficient over the respiration rate exerted by the respiratory chain in state 3 was between 0.8 and 1.0, while in state 4, control over the respiration rate was shared about equally between the chain and the proton leak. The control over the phosphorylation rate was predominantly exerted by the respiratory chain, although at low rates control by the phosphorylation system was also important. For oxidation of NADH, the flux control coefficient over the phosphorylation rate exerted by the respiratory chain in state 3 was 0.8-1.0, while near state 4 the flux control coefficients over the phosphorylation rate were about 0.8 for the phosphorylation system and 0.25 for the chain. Control over the proton leak rate was shared between the respiratory chain and the proton leak; the phosphorylation system had negative control. For oxidation of NADH, the flux control coefficients over the leak rate in state 3 were 1.0 for the leak, 0.4 for the chain and -0.4 for the phosphorylation system, while in state 4 the flux control coefficients over leak rate were about 0.5 for the leak and 0.5 for the chain. Control over the magnitude of the protonmotive force was small, between -0.2 and +0.2, reflecting the way the system operates to keep the protonmotive force fairly constant; the respiratory chain and the phosphorylation system had equal and opposite control and there was very little control by the proton leak except near state 4.