Thermodynamic parameters of free oxidation pathways in liver mitochondria

Two pathways of free oxidation in liver mitochondria were examined. One of these pathways is determined by the protonophoric action of free fatty acids, and the other pathway, by passive proton leakage in the absence of fatty acids. According to the model of the proton futile cycle of mitochondria, the protonophoric activity of fatty acids was defined as a quotient of the division of the acceleration of respiration by fatty acid by the coefficient of respiration control for the proton leakage. The temperature dependence of the palmitate protonophoric activity on the Arrhenius plot has a break at 22 degrees C and is characterized by the transition of activation energy from 120 to 60 kJ/mol. The dependence of the respiration rate in state 4 on the Arrhenius plot is linear and, the activation energy is 17 kJ/mol. It was concluded that the first pathway of free oxidation is determined by the cyclic transport of fatty acids with the participation of metabolic carriers, and this process depends on the membrane fluidity; the second pathway is determined by passive leakage of protons through membrane channels, without fatty acids and this process is independent on membrane fluidity.     

Effect of Temperature on Respiration of Mitochondria and Shoot Segments from Cold hardened and Nonhardened Wheat and Rye Seedlings

The effect of temperature on respiration of mitochondria and tissue segments from three wheat (Triticum aestivum L.) and one rye (Secale cereale L.) cultivar grown at 2 and 24 C has been examined. Discontinuities in Arrhenius plots of respiratory activity against temperature were observed for mitochondria and tissue segments from seedlings grown at both temperatures. The rates of respiration decreased abruptly below the transition temperatures, resulting in increased energy of activation values for respiration. Transition temperatures were observed from 6 to 10 C during tissue segment respiration, and from 10 to 14 C during respiration by isolated mitochondria. Respiratory control and efficiency of phosphorylation were not affected markedly by either reaction temperature or growth temperature of the seedlings. No correlation was observed between the cold hardiness of the cultivars and the temperature at which structural transitions occurred in the mitochondria. Dry matter content of the seedlings increased markedly during growth at 2 C, but no appreciable changes in the levels of mitochondrial protein were observed. The results support the view that changes other than fatty acid unsaturation are involved in the abrupt change in mitochondrial membrane properties at low temperature.     

Factors affecting the elicitation of sesquiterpenoid phytoalexin accumulation by eicosapentaenoic and arachidonic acids in potato

Eicosapentaenoic and arachidonic acids in extracts of Phytophthora infestans mycelium were identified as the most active elicitors of sesquiterpenoid phytoalexin accumulation in potato tuber slices. These fatty acids were found free or esterified in all fractions with elicitor activity including cell wall preparations. Yeast lipase released a major portion of eicosapentaenoic and arachidonic acids from lyophilized mycelium. Concentration response curves comparing the elicitor activity of the polyunsaturated fatty acids to a cell-free sonicate of P. infestans mycelium indicated that the elicitor activity of the sonicated mycelium exceeded that which would be obtained by the amount of eicosapentaenoic and arachidonic acids (free and esterified) present in the mycelium. Upon acid hydrolysis of lyophilized mycelium, elicitor activity was obtained only from the fatty acid fraction. However, the fatty acids accounted for only 21% of the activity of the unhydrolyzed mycelium and the residue did not enhance their activity. Centrifugation of the hydrolysate, obtained from lyophilized mycelium treated with 2n NaOH, 1 molarity NaBH₄ at 100°C, yielded a supernatant fraction with little or no elicitor activity. Addition of this material to the fatty acids restored the activity to that which was present in the unhydrolyzed mycelium. The results indicate that the elicitor activity of the unsaturated fatty acids is enhanced by heat and base-stable factors in the mycelium.     

Skeletal muscle mitochondrial respiration of malignant hyperthermia-susceptible patients. Ca2+-induced uncoupling and free fatty acids

1. Skeletal muscle mitochondria of malignant hyperthermia (MH)-susceptible patients showed normal oxidative phosphorylation but were more easily uncoupled than normal by exogenous Ca2+. 2. Fatty acids, in stimulating the mitochondrial ATPase activity, are responsible for the enhanced State 4 respiration in MH-susceptible patients. 3. These results imply that skeletal muscle mitochondria and free fatty acids are associated with the development of MH syndrome.     

Effect of bovine serum albumin on membrane potential in plant mitochondria

The membrane potential in highly coupled potato ( Solanum tuberosum L.) mitochondria, as measured by changes in safranine absorbance, was significantly increased by addition of bovine serum albumin. Purification of potato mitochondria on Percoll, in removing 50% of free unsaturated fatty acids, decreased the BSA-de-pendent membrane potential. The effect of added linoleic acid and of the natural accumulation of fatty acids during aging was studied. The response of membrane potential to addition of bovine serum albumin appeared to be directly correlated to the amount of free unsaturated fatty acids. Aging in vitro, in releasing free fatty acids, decreased respiratory control and ADP:O ratios and collapsed the membrane potential. During 2–3 h of incubation, addition of BSA completely restored membrane potential and oxidative phosphorylation.
It is concluded that both in fresh and in aged potato mitochondria the effect of bovine serum albumin on oxidative phosphorylation can be ascribed to an effect on membrane permeability to ions. BSA, in binding free unsaturated fatty acids, restored maximal membrane potential. The bovine serum albumin-dependent membrane potential appears to be a sensitive criterion of the functional integrity of the inner mitochondrial membrane.     

Influence of Severe Hypoglycemia on Mitochondrial and Plasma Membrane Function in Rat Brain

Abstract: Previous experiments have shown that severe hypoglycemia disrupts cerebral energy state in spite of a maintained cerebral oxygen consumption, suggesting uncoupling of oxidative phosphorylation. Other studies have demonstrated that hypoglycemia leads to loss of cerebral cortical phospholipids and phospholipid-bound fatty acids. The objective of the present study was, therefore, to study respiratory characteristics of brain mitochondria during severe hypoglycemia and to correlate respiratory activity to mitochondrial phospholipid composition. Mitochondria were isolated after 30 or 60 min of hypoglycemia with ceased EEG activity, and after a 90-min recovery period, and their resting (state 4) and ADP-stimulated (state 3) oxygen consumption rates and phospholipids and phospholipid-bound fatty acid content were measured. After 30 min of hypoglycemia, state 3 respiration decreased without any increase in state 4 respiration or change in ADP/O ratio. This decrease, which occurred with glutamate plus malate—but not with succinate—as substrates, was partly reversed by addition of bovine serum albumin and KCI. Chemical analyses of isolated mitochondria did not reveal changes in their phospholipid or fatty acid content. The results thus failed to account for the dissociation of cerebral energy state and oxygen consumption. It is emphasized, though, that uncoupling may well occur in vivo due to accumulation of free fatty acids and “futile cycling” of K⁺ and Ca²⁺. After 60 min of hypoglycemia, a moderate decrease in state 3 respiration was observed also with succinate as substrate, and there was some decrease in ADP/O ratios in KCI-containing media. However, the changes in ADP/O ratios were more conspicuous during recovery; in addition, state 4 respiration increased significantly. It is concluded that changes in mitochondrial function after 30 min of hypoglycemia are potentially reversible but that true mitochondrial failure develops in the recovery period following 60 min of hypoglycemia. This conclusion was corroborated by results demonstrating incomplete recovery of cerebral energy state. Since EEG and sensory evoked potentials return after 30 min but not after 60 min of hypoglycemia it seemed difficult to explain failure of return of electrophysiological function after 60 min of hypoglycemia solely by mitochondrial dysfunction; plasma membrane function was therefore assessed by measurements of extracellular potassium activity ([K⁺]_e). The results showed that whereas [K⁺]_e remained close to control in the recovery period following 30 min of hypoglycemia it rose progressively during recovery following 60 min of hypoglycemia. Possibly, inhibition of Na⁺ K⁺–activated ATPase could contribute to the permanent loss of spontaneous or evoked electrical activity.     

Involvement of Ca2+ release and activation of phospholipase A2 in mitochondrial dysfunction during anoxia

During anoxic incubation, depletion of mitochondrial ATP was followed by release of Ca2+ with concomitant increase in the rate of state 4 respiration due to disruption of the diffusion barrier against protons. The external addition of ATP and its non-metabolizable analog, beta,gamma-methylene adenosine 5'-triphosphate, prevented both the release of Ca2+ and increase in the rate of state 4 respiration. Addition of EGTA, which did not prevent release of the ion, resulted in little increase in the respiration rate. Addition of an inhibitor of mitochondrial phospholipase A2, such as quinacrine, dibucaine, or chlorpromazine, also prevented increase in the respiration rate without affecting Ca2+ release from mitochondria during anoxic incubation. Non-esterified polyunsaturated fatty acids were also found to be liberated from anoxic mitochondria. External addition of the ATP-analog, EGTA, and inhibitors of phospholipase A2 suppressed the liberation of non-esterified polyunsaturated fatty acids. Melittin and Ca2+, which activate phospholipase A2, increased the rate of state 4 respiration and the liberation of fatty acids. These findings support the hypothesis proposed previously that the following sequence changes occurs in mitochondria during anoxia; depletion of ATP, liberation of free calcium from mitochondria, and disruption of the diffusion barrier against H+ of the inner membrane. The results also indicate another event; activation of phospholipase A2 by release Ca2+ which results in H+ leakiness of the inner membrane.     

Metabolism and compartmentation of endogenous fatty acids in aged mouse liver mitochondria

Isolated mouse liver mitochondria were loaded with endogenous free fatty acids by aging in vitro. The oxidation and compartmentation of these fatty acids was studied. ATP-supported carnitine-dependent and carnitine-independent oxidation pathways of about equal activity were identified. The carnitine-dependent activity was abolished by nagarse and tetrathionate. It was also absent in mitoplasts. Hence the endogenous pool of free fatty acids which served as substrate for this pathway was located in the outer membrane. The carnitine-independent pathway was strongly inhibited by low concentrations of atractyloside suggesting that a pool of fatty acids located in the inner membrane was utilized. The occurrence of free fatty acids in the outer and inner membranes was confirmed by direct assay. The endogenous respiratory activity was also stimulated by oligomycin which was insensitive to nagarse, atractyloside, carnitine, and ATP suggesting that the stimulation was due to utilization of endogenous ATP and fatty acids localized within the inner membrane. Bovine serum albumin preferentially reduced the carnitine-independent activity presumably by binding the endogenous fatty acids suggesting that albumin has a higher affinity for free fatty acids of the inner than of the outer membrane.     

Interactions of lipoidal materials and a pyridazinone inhibitor of chloroplast development

Formation of chloroplast pigments was inhibited, and free fatty acids accumulated in mustard (Brassica juncea [L.] Coss.) cotyledons and in barley (Hordeum vulgare L.) first leaves developed after treatment with 4-chloro-5- (dimethylamino)-2- (α, α, α-trifluoro-m-tolyl) -3 (2H) -pyridazinone. The inhibitor reduced the amount of fatty acids found in polar lipids (galactolipids) of barley chloroplasts and increased the amount in nonpolar lipids while having little effect on total content of bound fatty acids. The inhibition of chlorophyll formation was circumvented by D-α-tocopherol acetate, phytol, farnesol, and squalene, and by unsaturated fatty acids and their methyl esters. The protective action can be explained partially by an interaction external to the plant whereby 4-chloro-5- (dimethylamino) -2- (α, α, α-trifluoro-m-tolyl) -3 (2H) -pyridazinone partitioned out of the aqueous phase and into the lipid phase, thus limiting availability of the inhibitor to plants. However, the amount of inhibitor reaching the cotyledons of tocopherol-protected mustard seedlngs was still in excess of the amount necessary to cause white foliage, but it failed to produce the effect. Tocopherol treatment did not prevent the 4-chloro-5- (dimethylamino) -2- (α, α, α-trifluoro-m-tolyl) -3 (2H) -pyridazinone-induced buildup of fatty acids in mustard cotyledons but did partially circumvent the effect in barley leaves. The amount of linolenic acid relative to linoleic acid was reduced in barley leaves and chloroplasts by 4-chloro-5- (dimethylamino) -2- (α, α, α-trifluoro-m-tolyl) -3 (2H) -pyridazinone action and this effect was circumvented by tocopherol.     

Substrate-specific changes in mitochondrial respiration in skeletal and cardiac muscle of hibernating thirteen-lined ground squirrels

During torpor, the metabolic rate (MR) of thirteen-lined ground squirrels (Ictidomys tridecemlineatus) is considerably lower relative to euthermia, resulting in part from temperature-independent mitochondrial metabolic suppression in liver and skeletal muscle, which together account for ~40 % of basal MR. Although heart accounts for very little (<0.5 %) of basal MR, in the present study, we showed that respiration rates were decreased up to 60 % during torpor in both subsarcolemmal (SS) and intermyofibrillar (IM) mitochondria from cardiac muscle. We further demonstrated pronounced seasonal (summer vs. winter [i.e., interbout] euthermia) changes in respiration rates in both mitochondrial subpopulations in this tissue, consistent with a shift in fuel use away from carbohydrates and proteins and towards fatty acids and ketones. By contrast, these seasonal changes in respiration rates were not observed in either SS or IM mitochondria isolated from hind limb skeletal muscle. Both populations of skeletal muscle mitochondria, however, did exhibit metabolic suppression during torpor, and this suppression was 2- to 3-fold greater in IM mitochondria, which provide ATP for Ca²⁺- and myosin ATPases, the activities of which are likely quite low in skeletal muscle during torpor because animals are immobile. Finally, these changes in mitochondrial respiration rates were still evident when standardized to citrate synthase activity rather than to total mitochondrial protein.     

The lipid composition of flight muscle mitochondria isolated from the blowfly, Phormia regina.

The role of lipids in membrane structure and function was studied by measuring the major lipid classes in mitochondria isolated from flight muscle of the blowfly, Phormia regina. Approximately 98% of the total lipid is phospholipid. Neutral lipid constitutes the remaining 2% of the total. Phosphatidylethanolamine accounts for 55–60% of the phospholipid. A molecular ratio of 4:1:1 is found for phosphatidylethanolamine, phosphatidylcholine, and cardiolipin (diphosphatidylglycerol). The neutral lipids include cholesterol, about 20%, and quinone, 40–45% of the total. The free fatty acid content of the neutral lipid fraction is variable, apparently being generated by endogenous phospholipase activity. The fatty acids of the neutral and phospholipid classes are predominantly 14–18 carbon acids; long-chain fatty acids of 20 and 22 carbons are essentially absent. The neutral lipid fraction contains 43% saturated and 51% monoenoic fatty acids. More than 65% of the phospholipid fatty acids are unsaturated. The principal fatty acids are palmitic, palmitoleic, oleic, linoleic, and linolenic. No trace of α- or β-tocopherol is detected. As vitamin E is considered an important naturally occuring antioxidant that prevents lipid peroxidation, the apparent absence of α- and β-tocopherol in these mitochondria coupled with intense oxidative activity of the mitochondria leads to the suggestion that blowfly flight muscle mitochondria may be particularly susceptible to peroxidative damage.     

Studies on essential fatty acid deficiency. Effect of the deficiency on the lipids in liver mitochondria and oxidative phosphorylation

1. Dietary deficiency of essential fatty acids results in a twofold increase in the neutral lipid content of liver mitochondria as compared with the corresponding value for stock-fed rats. 2. Deficiency produces changes in the pattern of the constituent fatty acids of the main phospholipid fractions of liver mitochondria which are similar to those previously reported for the lipids of whole liver. There is a fall in the content of C_18:2 acid and to a smaller extent of C_20:4 acid associated with a rise of C_16:1, C_18:1 and C_20:3 acids. 3. Deficiency results in small decreases in the phosphorylation quotients of liver mitochondria during oxidation of succinate and pyruvate, but the values lie within the range reported for normal mitochondria. Mitochondrial respiration with succinate is decreased as a result of deficiency but no change was observed with pyruvate as substrate.     

Biogenesis of mitochondria: The effects of altered steady-state membrane lipid composition on mitochondrial-energy metabolism in Saccharomyces cerevisiae

A chemostat culture technique has been developed for the growth of an unsaturated fatty acid auxotroph of Saccharomyces cerevisiae. Any chosen steady-state cellular unsaturated fatty acid level between 75 and 15% of the total fatty acids could be established and maintained. In all cultures the steady-state glucose concentrations were maintained at levels below that which induces catabolite repression.The efficiency of oxidative phosphorylation as determined from the molar growth yield decreased as the cellular unsaturated fatty acid composition was lowered. The number of moles of ATP produced by oxidative phosphorylation per mole of glucose utilized was 7.2, 4.8, 0.7, and 0.4 for cells in which 75, 50, 44, and 34%, respectively, of the total fatty acids were unsaturated.The lesion in oxidative phosphorylation was a direct result of lowering the membrane unsaturated fatty acid composition as the respiratory activities and cytochrome content of cells and mitochondria were unaffected by a decrease in the cellular unsaturated fatty acid level from the wild-type value of about 75% down to about 34%.In cells which contained lipids with 22–28% unsaturated fatty acids, cyanide-sensitive respiration was absent, and the levels of all mitochondrial cytochromes were less than 10% of normal. The reduction in the levels of cytochromes aa₃ and b appeared to be a consequence of a loss of mitochondrial protein synthetic activity in such cells. The level of cytochrome c was also greatly decreased, indicating that the cellular unsaturated fatty acid composition was affecting either the synthesis in the cytoplasm of mitochondrial proteins or the assembly of these proteins in the mitochondria.     

Fasting reveals largely intact systemic lipid mobilization mechanisms in respiratory chain complex III deficient mice

Mice homozygous for the human GRACILE syndrome mutation (Bcs1l^c.A232G) display decreased respiratory chain complex III activity, liver dysfunction, hypoglycemia, rapid loss of white adipose tissue and early death. To assess the underlying mechanism of the lipodystrophy in homozygous mice (Bcs1l^p.S78G), these and wild-type control mice were subjected to a short 4-hour fast. The homozygotes had low baseline blood glucose values, but a similar decrease in response to fasting as in wild-type mice, resulting in hypoglycemia in the majority. Despite the already depleted glycogen and increased triacylglycerol content in the mutant livers, the mice responded to fasting by further depletion and increase, respectively. Increased plasma free fatty acids (FAs) upon fasting suggested normal capacity for mobilization of lipids from white adipose tissue into circulation. Strikingly, however, serum glycerol concentration was not increased concomitantly with free FAs, suggesting its rapid uptake into the liver and utilization for fuel or gluconeogenesis in the mutants. The mutant hepatocyte mitochondria were capable of responding to fasting by appropriate morphological changes, as analyzed by electron microscopy, and by increasing respiration. Mutants showed increased hepatic gene expression of major metabolic controllers typically associated with fasting response (Ppargc1a, Fgf21, Cd36) already in the fed state, suggesting a chronic starvation-like metabolic condition. Despite this, the mutant mice responded largely normally to fasting by increasing hepatic respiration and switching to FA utilization, indicating that the mechanisms driving these adaptations are not compromised by the CIII dysfunction.Summary statementBcs1l mutant mice with severe CIII deficiency, energy deprivation and post-weaning lipolysis respond to fasting similarly to wild-type mice, suggesting largely normal systemic lipid mobilization and utilization mechanisms.     

Effect of phospholipids from Saccharomyces cerevisiae at different stages of development on restoration of succinooxidase activity in lipid-depleted mitochondria

Phospholipids extracted fromSaccharomyces cerevisiae at different stages of development after glucose repression contain three major fatty acids: palmitic, palmitoleic and oleic. The ratio palmitic: palmitoleic strongly decreases beginning at the 6th hour of growth.To test the effect of fatty acid composition and in particular of unsaturation on succinoxidase activity, all these phospholipids, phospholipids from commercial yeast, and Asolectin were incubated with lipid-depleted yeast mitochondria. The amount of P bound was not much different for the various phospholipids; succinoxidase activity was restored best by Asolectin; the least effective reactivation was given by phospholipids from yeast at the middle stages of growth. There are not great differences between the various phospholipids and there is no correlation with unsaturation. If we compare the pattern of appearance of respiration during morphogenesis of yeast mitochondria with the pattern of the capability of the phospholipids from cells at different stages of mitochondrial morphogenesis to restore activity of lipid-depleted yeast mitochondria, we find no correlation. The results of this investigation are consistent with the idea that changes in phospholipids and changes in enzyme activities are not linked by a causal relation.     

Percentage recovery of free fatty acids from bovine muscle lipids by nonchromatographic techniques

The inability of silicic acid to completely separate the neutral lipids from phospholipids has been reported by several investigators (1,2). Hornstein et al. (3) increased the polarity of the solvent system and reported a clean separation of the phospholipid fraction by adsorption on activated silicic acid. Studies on bovine lipids by Hood and Allen (2) utilized acid-washed Florisil to separate the lipid fractions claiming that silicic acid incompletely separates the free fatty acids from the phospholipids. Work performed in this laboratory (4) on bovine lipids confirmed that phospholipids could be effectively separated from free fatty acids by adsorption on silicic acid by incorporating the solvent system described by Hornstein et al. (3). The liquid-liquid partition procedure of Hamilton and McDonald (5) was also found to be sensitive enough to partition the extremely small amount of free fatty acids from the esterified fatty acids. This paper provides evidence for the effectiveness of these methods in separating the frec fatty acids by incorporating an internal standard [1-¹⁴C]palmitic acid.     

Complex I-Associated Hydrogen Peroxide Production Is Decreased and Electron Transport Chain Enzyme Activities Are Altered in n-3 Enriched fat-1 Mice

The polyunsaturated nature of n-3 fatty acids makes them prone to oxidative damage. However, it is not clear if n-3 fatty acids are simply a passive site for oxidative attack or if they also modulate mitochondrial reactive oxygen species (ROS) production. The present study used fat-1 transgenic mice, that are capable of synthesizing n-3 fatty acids, to investigate the influence of increases in n-3 fatty acids and resultant decreases in the n-6∶n-3 ratio on liver mitochondrial H₂O₂ production and electron transport chain (ETC) activity. There was an increase in n-3 fatty acids and a decrease in the n-6∶n-3 ratio in liver mitochondria from the fat-1 compared to control mice. This change was largely due to alterations in the fatty acid composition of phosphatidylcholine and phosphatidylethanolamine, with only a small percentage of fatty acids in cardiolipin being altered in the fat-1 animals. The lipid changes in the fat-1 mice were associated with a decrease (p<0.05) in the activity of ETC complex I and increases (p<0.05) in the activities of complexes III and IV. Mitochondrial H₂O₂ production with either succinate or succinate/glutamate/malate substrates was also decreased (p<0.05) in the fat-1 mice. This change in H₂O₂ production was due to a decrease in ROS production from ETC complex I in the fat-1 animals. These results indicate that the fatty acid changes in fat-1 liver mitochondria may at least partially oppose oxidative stress by limiting ROS production from ETC complex I.     

Long-chain free fatty acids from Momordica cochinchinensis leaves as attractants to its insect pest,Aulacophora foveicollis Lucas (Coleoptera: Chrysomelidae)

Extraction, thin layer chromatography, and gas chromatography–mass spectrometry of young, mature, and senescent leaves of Momordica cochinchinensis Spreng revealed 13 free fatty acids, representing a total of 82.29, 91.30, and 68.52% of fatty acids in young, mature, and senescent leaves, respectively. Palmitic acid was the predominant fatty acid followed by stearic acid in three types of leaves. The free fatty acids from young, mature, and senescent leaves attracted female Aulacophora foveicollis Lucas (Coleoptera: Chrysomelidae) at the minimal concentrations of 4, 2, and 8 μg, respectively; whereas the mixtures of synthetic fatty acids mimicking free fatty acids of young, mature, and senescent leaves showed attraction at the minimal concentrations of 4, 2, and 10 μg, respectively, in Y-shaped glass tube olfactometer bioassay under laboratory condition. The results indicate that A. foveicollis may employ long-chain free fatty acids as an olfactory cue for host location. The individual synthetic fatty acids mimicking the proportions detected in three types of leaves were also evaluated through olfactometer bioassay. Only synthetic palmitic acid at the minimal amount of 2.17 μg attracted the insect. A synthetic blend of fatty acids mimicking 8 μg free fatty acid concentration of mature leaves or an amount of 5.42 μg palmitic acid produced the highest attraction of the insect. Hence 5.42 μg palmitic acid might be used for insect pest management program such as baited traps.     

Uncoupling proteins in mitochondria of plants and some microorganisms.

Uncoupling proteins, members of the mitochondrial carrier family, are present in mitochondrial inner membrane and mediate free fatty acid-activated, purine-nucleotide-inhibited H+ re-uptake. Since 1995, it has been shown that the uncoupling protein is present in many higher plants and some microorganisms like non-photosynthetic amoeboid protozoon, Acanthamoeba castellanii and non-fermentative yeast Candida parapsilosis. In mitochondria of these organisms, uncoupling protein activity is revealed not only by stimulation of state 4 respiration by free fatty acids accompanied by decrease in membrane potential (these effects being partially released by ATP and GTP) but mainly by lowering ADP/O ratio during state 3 respiration. Plant and microorganism uncoupling proteins are able to divert very efficiently energy from oxidative phosphorylation, competing for deltamicroH+ with ATP synthase. Functional connection and physiological role of uncoupling protein and alternative oxidase, two main energy-dissipating systems in plant-type mitochondria, are discussed.