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.     

Complexes between uncouplers of oxidative phosphorylation

Summary We have examined the effects of two weak acid uncouplers of oxidative phosphorylation, 2,4-dinitrophenol and 5,6-dichloro-2-trifluoromethyl-benzimidazole, on the electrical properties of phospholipid bilayer membranes. All the effects they produce are consistent with the charged permeant species being a HA ₂ ^– complex formed between the neutral acid HA and its anion A^– and the current in the aqueous phases being carried by buffered hydrogen ions. When both uncouplers are present simultaneously, all the evidence we have obtained is consistent with the charged permeant species being a HAB^– complex formed between the neutral acid HA of one uncoupler and the anion B^– of the other. It was necessary, however, to take into account interfacial processes and the unstirred layers adjacent to the membrane, the adsorption of anions to the bilayer and the buffer level in the aqueous phases to explain the results in terms of this model. The degree to which these factors will complicate a comparison of results obtained on artificial systems and mitochondria is also discussed.     

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     

Effect of photosynthetic inhibitors and uncouplers of oxidative phosphorylation on nitrate and nitrite reduction in barley leaves

The effects of several photosynthetic inhibitors and uncouplers of oxidative phosphorylation on NO₃⁻ and NO₂⁻ assimilation were studied using detached barley (Hordeum vulgare L. cv Numar) leaves in which only endogenous NO₃⁻ or NO₂⁻ were available for reduction. Uncouplers of oxidative phosphorylation greatly increased NO₃⁻ reduction in both light and darkness, while photosynthetic inhibitors did not.

The NO₂⁻ concentration in the control leaves was very low in both light and darkness; 98% or more of the NO₂⁻ formed from NO₃⁻ was further assimilated in control leaves. More NO₂⁻ accumulated in the leaves in light and darkness in the presence of photosynthetic inhibitors. Of this NO₂⁻, 94% or more was further assimilated. It appears that metabolites, either external or internal to the chloroplast, capable of reducing NADP (which, in turn, could reduce ferredoxin via NADP reductase) might support NO₂⁻ reduction in darkness and light when photosynthetic electron flow is inhibited by photosynthetic inhibitors.

Nitrite assimilation was much more sensitive to uncouplers in darkness than in light: in darkness, 74% or more of NO₂⁻ formed from NO₃⁻ was further assimilated, whereas in light, 95% or more of the NO₂⁻ was further assimilated.

     

Inhibition of membrane transport in Streptococcus faecalis by uncouplers of oxidative phosphorylation and its relationship to proton conduction

We studied the effect of compounds that uncouple oxidative phosphorylation on membrane function in Streptoccocus faecalis, an organism which relies upon glycolysis for the generation of metabolic energy. At low concentrations (ranging from 10(-7) to 10(-4)m), tetrachlorosalicylanilide, tetramethyldipicrylamine, carbonylcyanide m-chlorophenylhydrazone, pentachlorophenol, and dicoumarol strongly inhibited energy-dependent transport of rubidium, phosphate, and certain amino acids. However, these compounds had little effect on the generation of adenosine triphosphate via glycolysis or on its utilization for the synthesis of macromolecules. They also did not seriously inhibit uptake of those monosaccharides and amino acids which do not require concurrent metabolism. It is proposed that the uncouplers interfere with the utilization of metabolic energy for membrane transport. The uncouplers accelerated the translocation of protons across the cytoplasmic membrane. It appears that a proton-impermeable membrane is required for transport, perhaps, because a proton gradient is involved in the coupling of metabolic energy to the translocation of substrates across the membrane.     

Inhibition of bacterial transport by uncouplers of oxidative phosphorylation. Effects of pentachlorophenol and analogues in Bacillus subtilis.

Analogues of the potent uncoupler of oxidative phosphorylation pentachlorophenol were tested as inhibitors of proline and glycine transport by Bacillus subtilis. These analogues included less highly substituted chlorophenols and pentachlorothiophenol. Like pentachlorophenol, they are non-competitive inhibitors of proline transport and uncompetitive inhibitors of glycine transport. However, the less highly substituted chlorophenols are weaker acids than pentachlorophenol and also weaker inhibitors. Analysis indicated that the anionic form of the uncouplers is the inhibiting species. Pentachlorothiophenol, a water-insoluble anion, is also a potent inhibitor. These results support previous studies that concluded that uncouplers of oxidative phosphorylation inhibit amino acid transport by binding at specific sites on proteins, the free energy of interaction stabilizing 'unproductive' conformations. Such specific interactions of uncoupler with protein are probably commonplace.     

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.     

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.     

Fatty acids as uncouplers of oxidative phosphorylation

This review summarizes data on the uncoupling effect of fatty acids on oxidative phosphorylation in mitochondria of various animal and plant tissues.     

Inhibition of erythrocyte plasma membrane NADH dehydrogenase by nucleotides and uncouplers

Erythrocyte ghost NADH dehydrogenase is inhibited in a competitive fashion by ATP and ADP whereas other nucleoside di- and triphosphates, cyclic nucleosides, as well as non-phosphorylating ATP analogs are relatively ineffective. In addition, this enzyme, measured with ferricyanide as electron acceptor, is inhibited by uncouplers of oxidative phosphorylation (proton-conducting reagents), the inhibition being competitive in character (i.e., the uncouplers were without influence upon maximum velocity). The effectiveness of the uncouplers was in the order of their hydrophobic character with the presence of the alkyl side chain rendering nonyl-dinitrophenol much more active than 2,6-dinitrophenol itself. Hydrophobic compounds that are not protonophores (e.g., eosin, proflavin or valinomycin) were not inhibitory. Whereas adenine nucleotides probably inhibit NADH oxidation competitively through structural similarity with the substrate, it appears unlikely that uncouplers compete at the NADH site directly. Rather, the apparently-competitive inhibition in the latter case may reflect competition for proton transfer to an acceptor residing in a hydrophobic region of the enzyme complex.     

Inhibition of erythrocyte plasma membrane NADH dehydrogenase by nucleotides and uncouplers

Erythrocyte ghost NADH dehydrogenase is inhibited in a competitive fashion by ATP and ADP whereas other nucleoside di- and triphosphates, cyclic nucleosides, as well as non-phosphorylating ATP analogs are relatively ineffective. In addition, this enzyme, measured with ferricyanide as electron acceptor, is inhibited by uncouplers of oxidative phosphorylation (proton-conducting reagents), the inhibition being competitive in character (i.e., the uncouplers were without influence upon maximum velocity). The effectiveness of the uncouplers was in the order of their hydrophobic character with the presence of the alkyl side chain rendering nonyl-dinitrophenol much more active than 2,6-dinitrophenol itself. Hydrophobic compounds that are not protonophores (e.g., eosin, proflavin or valinomycin) were not inhibitory. Whereas adenine nucleotides probably inhibit NADH oxidation competitively through structural similarity with the substrate, it appears unlikely that uncouplers compete at the NADH site directly. Rather, the apparently-competitive inhibition in the latter case may reflect competition for proton transfer to an acceptor residing in a hydrophobic region of the enzyme complex.     

Escherichia coli mutants resistant to uncouplers of oxidative phosphorylation

Two mutant strains of Escherichia coli K 12 Doc-S resistant to the uncoupling agents 4,5,6,7-tetrachloro-2-trifluoromethyl benzimidazole and carbonyl cyanide m-chlorophenylhydrazone were isolated. These strains, designated TUV and CUV, were capable of (a) growth, (b) the transport of succinate and L-proline and (c) electron-transport-linked oxidative synthesis of ATP in the presence of titres of uncoupler which inhibited these processes in strain Doc-S. The inhibition of transport of L-proline by a fixed titre of uncoupler was sharply pH dependent in strain Doc-S: uptake was unaffected at pH 7.6 but completely inhibited at pH 5.6. This pH dependence was not shown by the resistant strains. We believe that uncouplers were equally accessible to their site(s) of action in the energy-conserving membrane of the sensitive and resistant strains. We conclude that uncoupler resistance in these strains of E. coli has arisen as a consequence of mutations which directly affect a specific site of uncoupler action within the cytoplasmic membrane, rather than as a consequence of a decrease in the permeability of cells to uncoupler.