Inhibition of Ca++-stimulated respiration by uncouplers.

In a phosphate medium at pH 6.6 low concentrations of uncouplers such as p-trifluoromethoxyphenylhydrazone carbonylcyanide and 2,4-dinitrophenol inhibit the oxidation of beta-hydroxybutyrate and succinate, when added during Ca++-accumulation. The inhibition depends on the amount of accumulated Ca++, and is released by N,N,N',N'-tetramethyl-p-phenylendiamine plus ascorbate as substrate. Under identical conditions the uncouplers have no inhibitory effect when added to mitochondria during state 3 respiration or during accumulation of Sr++. Inhibition of respiration by the decrease of transmembranal succinate transport or by accumulation of oxaloacetate can be excluded. It is suggested that accumulation of Ca++ in the presence of phosphate induces structural alteration of the mitochondrial membrane, which on the one hand changes the accessibility or sensitivity of dehydrogenases to uncouplers and causes leakage of accumulated Ca++ on the other.     

Isothiocyanates. A new class of uncouplers.

This paper describes the uncoupling effect of three isothiocyanates: p-bromophenylisothiocyanate, 4,4'-diisothiocyanatebiphenyl and beta-naphtylemthylisothiocyanate on the respiration of Ehrlich-Lettré cells and isolated mitochondria. The isothiocyanates are similar to other uncouplers (such as 2,4-dinitrophenol and carbonyl cyanide p-trifluoromethoxyphenylhydrazone) in that they: 1. stimulate respiration of state 4 mitochondria; 2. stimulate mitochondrial ATPase activity; 3. release the inhibition of mitochondrial respiration by oligomycin and 4. inhibit both mitochondrial respiration and mitochondrial ATPase activity at higher molar concentrations. The incoupling activity of these isothiocyanates correlates well with their biological activity. Maximal activation of a latent mitochondrial ATPase activity of rat liver mitochondria in the presence of p-bromophenylisothiocyanate was found at a concentration of 15 muM. The investigated isothiocyanates differ significantly in their solubility in organic solvents and their chemical reactivity. We assume that the greater the partition coefficient in a series of isothiocyanates grouped according to the increasing value of log P (partition coefficient for the system octanol/water, 25 degrees C), the greater will be their uncoupling activity, but only up to a certain degree. Any further increase of log P will be marked by a decrease of this activity.     

Isothiocyanates. A new class of uncouplers

This paper describes the uncoupling effect of three isothiocyanates: p-bromophenylisothiocyanate, 4,4′-diisothiocyanatebiphenyl and β-naphtylmethylisothiocyanate on the respiration of Ehrlich-Lettré cells and isolated mitochondria. The isothiocyanates are similar to other uncouplers (such as 2,4-dinitrophenol and carbonyl cyanide p-trifluoromethoxyphenylhydrazone) in that they: 1. stimulate respiration of state 4 mitochondria; 2. stimulate mitochondrial ATPase activity; 3. release the inhibition of mitochondrial respiration by oligomycin and 4. inhibit both mitochondrial respiration and mitochondrial ATPase activity at higher molar concentrations. The uncoupling activity of these isothiocyanates correlates well with their biological activity. Maximal activation of a latent mitochondrial ATPase activity of rat liver mitochondria in the presence of p-bromophenylisothiocyanate was found at a concentration of 15 μM. The investigated isothiocyanates differ significantly in their solubility in organic solvents and their chemical reactivity. We assume that the greater the partition coefficient in a series of isothiocyanates grouped according to the increasing value of log P (partition coefficient for the system octanol/water, 25 °C), the greater will be their uncoupling activity, but only up to a certain degree. Any further increase of log P will be marked by a decrease of this activity.     

Inhibition of nitrite assimilation by uncouplers of phosphorylation

Summary Carbonyl cyanide m-chlorophenylhydrazone (CCCP) and pentachlorophenol (PCP), two powerful uncouplers of phosphorylation, specifically inhibit the assimilation of nitrite in the course of nitrate reduction. These results support our former conclusion that high-energy phosphate is involved in the metabolism of nitrite.     

Inhibition of photosynthetic oxygen evolution by protonophoric uncouplers

The protonophoric uncouplers carbonyl cyanide m-chlorophenylhydrazone (CCCP), 2,3,4,5,6-pentachlorophenol (PCP) and 4,5,6,7-tetrachloro-2-trifluoromethylbenzimidazole (TTFB) inhibited the Hill reaction with K₃[Fe(CN)₆] (but not with SiMo) in chloroplast and cyanobacterial membranes (the I₅₀ values were approx. 1–2, 4–6 and 0.04–0.10 M, respectively). The inhibition is due to oxidation of the uncouplers on the Photosystem II donor side (ADRY effect) and their subsequent reduction on the acceptor side, ie. to the formation of a cyclic electron transfer chain around Photosystem II involving the uncouplers as redox carriers. The relative amplitude of nanosecond chlorophyll fluorescence in chloroplasts was increased by DCMU or HQNO and did not change upon addition of uncouplers, DBMIB or DNP-INT; the HQNO effect was not removed by the uncouplers. The uncouplers did not inhibit the electron transfer from reduced TMPD or duroquinol to methylviologen which is driven by Photosystem I. These data show that CCCP, PCP and TTFB oxidized on the Photosystem II donor side are reduced by the membrane pool of plastoquinone (Q_p) which is also the electron donor for K₃ [Fe(CN)₆] in the Hill reaction as deduced from the data obtained in the presence of inhibitors. Inhibition of the Hill reaction by the uncouplers was maximum at the pH values corresponding to the pK of these compounds. It is suggested that the tested uncouplers serve as proton donors, and not merely as electron donors on the oxidizing side of Photosystem II.Abbreviations ADRY- acceleration of the deactivation reactions of the water-splitting enzyme system Y - ANT2p- 2-(3-chloro-4-trifluoromethyl) anilino-3,5-dinitrothiophene - CCCP- carbonyl cyanide m-chlorophenylhydrazone - DBMIB- 2,5-dibromo-3-methyl 6-isopropyl-p-benzoquinone - DCMU- 3-(3,4-dichlorophenyl)-1,1-dimethylurea - DNP-INT- 2-iodo-6-isopropyl-3-methyl 2,4,4-trinitrodiphenyl ether - DPC- 1,5-diphenylcarbazide - DPIP- 2,6-dichlorophenolindophenol - FCCP- carbonyl cyanide p-trifuoromethoxyphenylhydrazone - FeCy- potassium ferricyanide - HQNO- 2-n-heptyl-4-hydroxyquinoline N-oxide - (MN)₄- the tetranuclear Mn cluster of water oxidizing complex - P680- photoactive Chl of the reaction center of Photosystem II - PCP- 2,3,4,5,6-pentachlorophenol - PS- photosystem - Q_A and Q_B- primary and secondary plastoquinones of PS II - Q_C and Q_Z- plastoquinone binding sites in the cytochrome blf complex - Q_p- membrane pool of plastoquinone - SiMo- sodium silicomolybdate - TMPD- N,N,N-tetramethyl-p-phenylenediamine - TTFB- 4,5,6,7-tetrachloro-2-trifluoromethylbenzimidazole - WOC- water oxidixing complex - Y_Z- tyrosine-161 of the Photosystem II D1 polypeptide     

Inhibition of DNA replication in Escherichia coli by dibromophenol and other uncouplers.

DNA replication in Escherichia coli is inhibited by uncouplers such as 2,4-dibromophenol and 3,3'4',5-tetrachlorosalicylanilide. Inhibition occurs in either aerobically or anaerobically growing cells or in cells made permeable by toluene. With anaerobically growing cells, inhibition by dibromophenol is reversible and occurs under conditions in which there is no change in pools of ATP or deoxynucleoside triphosphates. With toluenized cells, inhibition is not due to breakdown of deoxynucleoside triphosphates. The rates of protein and RNA synthesis are not inhibited either in vivo or in toluenized cells by concentrations of dibromophenol or tetrachlorosalicylanilide which inhibit replication. It is generally believed that uncouplers inhibit many other cellular processes by collapsing a proton gradient across a membrane. However, the relative effectiveness of eight uncouplers and related compounds to inhibit replication did not parallel their ability to transport protons into E. coli cells. Therefore, the inhibition by uncouplers does not suggest that replication depends on a chemiosmotic process. A possible explanation for the uncoupler sensitivity is provided by the finding that many of the purified enzymes tested, including DNA polymerases II and III, are inhibited by dibromophenol and tetrachlorosalicylanilide.     

Inhibition of amino acid transport in Bacillus subtilis by uncouplers of oxidative phosphorylation.

The effect of three uncouplers of oxidative phosphorylation, trifluoromethoxycarbon-ylcyanidephenylhydrazone (FCCP), 3,3′,4′,5-tetrachlorosalicylanilide (TCSA), and pentachlorophenol (PCP), on transport of glycine and proline by Bacillus subtilis were examined. FCCP inhibited proline uptake uncompetitively, but glycine uptake competitively. TCSA inhibited proline uptake noncompetitively, but glycine uptake competitively. PCP inhibited proline uptake noncompetitively, but glycine uptake uncompetitively. The results indicate that these uncouplers inhibit amino acid transport by interacting at specific sites rather than by reducing any central supply of energy used to fuel metabolic processes.     

Modulation of the chloroplast ATPase by tight ADP binding. Effect of uncouplers and ATP

Inactivation of the membrane-bound ATPase by tight ADP binding was studied under nonenergized conditions. The energy state of the system was controlled either by omitting MgCl₂, preventing ATP hydrolysis, or by addition of an uncoupler which dissipates the . In the absence of Mg²⁺, ATP prevents the inactivation of the enzyme by ADP, in a competitive manner. This effect of ATP resembles that of GDP with Mg²⁺ present. In the presence of nigericin, Mg²⁺, and ATP, inactivation occurs after a 10–15-sec interval, during which the enzyme is able to hydrolyze ATP at a relatively rapid rate. The degree of inactivation is proportional to the level of bound ADP detected. This behavior is different from that of the coupled ATPase (no uncoupler added), where inactivation is attained only upon exhaustion of the ATP by its hydrolysis, despite the finding that ADP binds tightly to the active ATPase at all stages of the reaction. Higher levels of tightly bound ADP were detected in the presence of an uncoupler. We suggest that the interval during which the enzyme becomes inactive is that required for the enzyme to generate and bind ADP, and to change from the active to the inactive conformation. These results support the mechanism suggested previously for the modulation of the ATPase by tight nucleotide binding.     

Stimulation of microsecond-delayed fluorescence from spinach chloroplasts by uncouplers and by phosphorylation

Delayed fluorescence, as measured with a laser phosphoroscope, is stimulated not inhibited by uncouplers during the first 100 μs after the light is turned off. This is true only wen uncouplers cause an increase in the rate of electron transport. When ADP and P_i cause an increase in the electron transport rate, microsecond-delayed fluorescence is also increased. Indeed, there is a complex quantitative relationship between the rate of electron transport and the initial intensity of delayed fluorescence under a wide range of conditions.

Uncouplers or ADP and P_i also increase the rate of decay of delayed fluorescence so that after about 150 μs they become inhibitory, as already reported by many authors.

Microsecond-delayed fluorescence continues to rise with rising light intensities long after the rate of reduction of exogenous acceptor is light-saturated.

These observations suggest a correlation of the rate of electron transport both with the intensity of the 5–100 μs-delayed fluorescence and with the rate of decay in the intensity of delayed fluorescence. The data imply that the decrease in intensity of millisecond-delayed fluorescence which has often been noted with uncouplers is probably not due to the elimination of a membrane potential. It seems more likely that the decrease in millisecond-delayed fluorescence is a reflection of the rate of disappearance of some other electron transport-generated condition, a condition which is uncoupler-insensitive. Certainly stimulations of microsecond-delayed fluorescence by electron transport which has been uncoupled by gramicidin suggest that ion gradients are not an essential component of the conditions responsible for delayed fluorescence.     

Effect of cyanide on mitochondrial membrane depolarization induced by uncouplers

In this work, it was found that the ability of common uncouplers – carbonyl cyanide p-trifluoromethoxyphenyl-hydrazone (FCCP) and 2,4-dinitrophenol (DNP) – to reduce membrane potential of isolated rat liver mitochondria was diminished in the presence of millimolar concentrations of the known cytochrome c oxidase inhibitor – cyanide. In the experiments, mitochondria were energized by addition of ATP in the presence of rotenone, inhibiting oxidation of endogenous substrates via respiratory complex I. Cyanide also reduced the uncoupling effect of FCCP and DNP on mitochondria energized by succinate in the presence of ferricyanide. Importantly, cyanide did not alter the protonophoric activity of FCCP and DNP in artificial bilayer lipid membranes. The causes of the effect of cyanide on the efficiency of protonophoric uncouplers in mitochondria are considered in the framework of the suggestion that conformational changes of membrane proteins could affect the state of lipids in their vicinity. In particular, changes in local microviscosity and vacuum permittivity could change the efficiency of protonophore-mediated translocation.     

Bacterial resistance to uncouplers

Uncoupler resistance presents a potential challenge to the conventional chemiosmotic coupling mechanism. InE. coli, an adaptive response to uncouplers was found in cell growing under conditions requiring oxidative phosphorylation. It is suggested that uncoupler-resistant mutants described in the earlier literature might represent a constitutive state of expression of this low energy shock adaptive response. In the environment, bacteria are confronted by nonclassical uncoupling factors such as organic solvents, heat, and extremes of pH. It is suggested that the low energy shock response will aid the cell in coping with the effects of natural uncoupling factors. The genetic analysis of uncoupler resistance has only recently began, and is yielding interesting and largely unexpected results. InBacillus subtilis, a mutation in fatty acid desaturase causes an increased content of saturated fatty acids in the membrane and increased uncoupler resistance. The protonophoric efficiency of uncouplers remains unchanged in the mutants, inviting nonorthodox interpretations of the mechanism of resistance. InE. coli, two loci conferring resistance to CCCP and TSA were cloned and were found to encode multidrug resistance pumps. Resistance to one of the uncouplers, TTFB, remained unchanged in strains mutated for the MDRs, suggesting a resistance mechanism different from uncoupler extrusion.     

Release of respiratory control by uncouplers: the question of stoichiometry

An alternative kinetic model of uncoupler action is envisaged in which the observed stoichiometry of uncoupler and titratable factor do not reflect chemical binding reactions. This model assumes (a) that highly effective uncouplers dissolve in the membrane, whereas less effective uncouplers remain largely in aqueous solution; (b) that a high affinity exists between respiratory chains and “low energy states”; (c) that maximal respiration requires the presence of only a small number of low-energy states and forms high-energy from low-energy states; (d) that uncouplers act by a bimolecular “collision” process without binding. In this model, respiratory chains must be capable of driving energy equivalents into a storage mode, with no effect on respiration rate until the store is almost filled.     

Regulation of the Hill reaction by cations and its abolishment by uncouplers

Concurrent measurements of P-700 turnover and the reduction of K₃Fe(CN)₆ revealed an identical relative quantum yield for both reactions in isolated pea chloroplasts as well as chloroplast particles from wild type Scenedesmus. On the other hand, chloroplast particles of wild type Scenedesmus showed the same relative quantum yield for the Hill reaction as those of the P-700-free mutant No. 8, indicating that P-700 is not required for ferricyanide reduction.Several metal ions, such as Mg²⁺, Ca²⁺, Na⁺ and K⁺ stimulated the reduction of K₃Fe(CN)₆. In short wavelength light, the stimulation was a function of light intensity, varying in magnitude from an approximate doubling of the yield in low intensities to only a slight increase at light saturation. P-700 was totally unaffected by the cations.The effect of the metal salts was abolished in the presence of uncouplers of photophosphorylation.The data reconcile several divergent results concerning the effect of divalent cations on the reduction of ferricyanide which have been reported in the recent literature. On the whole the experiments suggest that the Hill acceptor can be reduced at two sites. The stimulation of the Hill reaction by metal ions is proposed to be due to an activation of Photosystem II and a more efficient utilization of quanta at the expense of radiationless de-excitation.