Surface potential and the interaction of weakly acidic uncouplers of oxidative phosphorylation with liposomes and mitochondria

The pH dependence of the binding of weakly acidic uncouplers of oxidative phosphorylation to rat-liver mitochondria and liposomes is mainly determined by the pK_a of the uncoupler molecule.

The absorption and fluorescence excitation spectra of the anionic form of weakly acidic uncouplers of oxidative phosphorylation are red-shifted upon interaction with liposomal or mitochondrial membranes. The affinity for the liposomes, as deduced from the red shift, is independent of the degree of saturation of the fatty acid chains of different lecithins. The intensity of the spectra at one pH value is strongly dependent upon the surface charge of the liposomes. With positively charged liposomes the results obtained can be almost quantitatively explained with the Gouy-Chapman theory, but with negatively charged ones deviations are observed. At a particular pH, the divalent ion Ca²⁺ strongly influences the intensity of the spectra in the presence of negatively charged liposomes, but has no effect with neutral liposomes.

With mitochondrial membranes an effect of Ca²⁺ similar to that with negatively charged liposomes is observed. Depletion of the phospholipids of the mitochondria and subsequent restoration of the mitochondrial membrane with lecithin, strongly diminishes this effect, but restoration with negatively charged phospholipids does not influence it.

From these observations it is concluded that the anionic form of the uncoupler molecule when bound to mitochondria is located within the partly negatively charged phospholipid moiety of the membrane, with its anionic group pointing to the aqueous solution.     

The binding of uncouplers of oxidative phosphorylation to rat-liver mitochondria

The binding of different uncouplers of oxidative phosphorylation to rat-liver mitochondria was measured. At pH 7.2 and about 0.7 mg mitochondrial protein/ml the percentage bound of the uncoupler added was 84% for 2,3,4,5,6-pentachlorophenol (PCP), 40% for carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP), 35% for 4,5,6,7-tetrachloro-2-trifluoromethylbenzimidazole (TTFB), 4% for α′,α′-bis (hexafluoroacetonyl)acetone (1799), and less than 4% for 2,4-dinitrophenol. These percentages are constant up to amounts of uncoupler added several times the one needed for maximal uncoupling. The values found for FCCP and TTFB are in contradiction to the proposed stoichiometric interaction of uncouplers with the coupling sites of the mitochondrial membrane.From titration experiments of the rate of O₂ uptake by rat-liver mitochondria in State 4 as a function of the uncoupler concentration in the presence of albumin or of different types of liposomes the conclusion is drawn that the negative surface charge of the mitochondrial phospholipids may be an important parameter in determining the binding of anionic uncouplers to rat-liver mitochondria.     

Stimulation of ATP synthesis in Halobacterium halobium R1 by light-induced or artificially created proton electrochemical potential gradients across the cell membrane

The relationship between proton movement and phosphorylation in Halobacterium halobium R₁ has been investigated under anaerobic conditions. The light-induced changes in the bacteriorhodopsin are accompanied by proton movements across the cell membrane which result in pH changes in the suspending medium. The initial alkaline shift is shown to be closely paralleled by (and hence correlated with) ATP synthesis. Acidification of the medium in the presence of valinomycin, under conditions of low external potassium, brings about ATP synthesis in the dark.     

Regulation of dinitrogen fixation in intact Azotobacter vinelandii

1. In intact Azotobacter vinelandii the influence of oxygen on the levels of oxidized nicotinamide adenine dinucleotides and adenine nucleotides in relation to nitrogenase activity was investigated.

2. The hypothesis that a high (NADH + NADPH)/(NAD⁺ + NADP⁺) is the driving force for the transport of reducing equivalents to nitrogenase in intact A. vinelandii was found to be invalid. On the contrary, with a decreasing ratio of reduced to oxidized pyridine nucleotides, the nitrogenase activity of the whole cells increases.

3. By measuring oxidative phosphorylation and using 9-amino acridine as a fluorescent probe, it could be demonstrated that respiration-coupled transport of reducing equivalents to the nitrogenase requires a high energy level of the plasma membrane or possibly coupled to it, a high pH gradient over the cytoplasmic membrane. Furthermore nitrogen fixation is controlled by the presence of oxygen and the ATP/ADP ratio.     

Uncoupler-stimulated oxidation of choline by rat-liver mitochondria

The main product of uncoupler-stimulated oxidation of choline by rat-liver mitochondria is betaine, which is found almost exclusively extramitochondrially. The uncoupled oxidation of choline is stimulated by intramitochondrial phosphate. The effect of intramitochondrial phosphate is to induce adenine nucleotide efflux, which in its turn allows the efflux of betaine from the mitochondria.     

The influence of diffusion potentials across liposomal membranes on the fluorescence intensity of 1-anilinonaphthalene-8-sulphonate

The influence of diffusion potentials across different phospholipid membranes on the fluorescence intensity of 1-anilinonaphthalene-8-sulphonate (ANS) was studied. With liposomes or chloroform spheres covered with a monolayer of egg lecithin, no specific effects were found. With liposomes of soy-bean phospholipids, generation of a diffusion potential leads to an enhancement or decrease, depending on the direction of the potential, of the intensity of ANS fluorescence. This effect is mainly due to a change in quantum yield of the bound ANS. These data support a mechanism according to which ANS molecules are pushed into or pulled out of the membrane by a potential, but not an electrophoretic one in which the potential causes movement of ANS across the membrane.     

The mechanism of energy conservation and transduction by mitochondrial cytochrome c oxidase

Oxidation of ferrocytochrome c by molecular oxygen catalysed by cytochrome c oxidase (cytochrome aa₃) is coupled to translocation of H⁺ ions across the mitochondrial membrane. The proton pump is an intrinsic property of the cytochrome c oxidase complex as revealed by studies with phospholipid vesicles inlayed with the purified enzyme. As the conformation of cytochrome aa₃ is specifically sensitive to the electrochemical proton gradient across the mitochondrial membrane, it is likely that redox energy is primarily conserved as a conformational “strain” in the cytochrome aa₃ complex, followed by relaxation linked to proton translocation. Similar principles of energy conservation and transduction may apply on other respiratory chain complexes and on mitochondrial ATP synthase.     

A second mechanism for sodium extrusion in Halobacterium halobium: a light-driven sodium pump.

Membrane vesicles from a red mutant of $\text{Halobacterium}\text{halobium}$ R₁ accumulate protons when illuminated causing the pH of the suspension to rise. Sodium is extruded from the vesicles and a membrane potential is formed. This potential and the proton uptake are abolished by valinomycin if K⁺ is present. In contrast, Na⁺-efflux is uninhibited by valinomycin even though no membrane potential is detectable and H⁺ influx does not occur. $\text{Bis}$ (hexafluoracetonyl)acetone (1799) stimulates proton uptake but does not abolish membrane potential. We propose that a light-dependent sodium pump is present. Passive proton uptake occurs in response to the electrical gradient created by this light-driven Na⁺ pump in contrast to the $\text{active}$ proton, and passive Na⁺ flux that occurs in response to the light-driven proton pump described in vesicles of the parent strain of $\text{H.}\text{halobium}$ R₁.     

Delayed light studies on photosynthetic energy conversion. VII. Effect of the high energy state, coupled to 2,3,5,6-tetramethyl p-phenylenediamine-catalyzed cyclic electron flow, on millisecond emission from chloroplasts and digitonin subchloroplast particles

The effect of 2,3,5,6-tetramethyl p-phenylenediamine-catalyzed cyclic electron flow on millisecond delayed light emission from chloroplasts has been compared to the effect on subchloroplast particles. Non-cyclic electron flow of both chloroplasts and subchloroplast particles was blocked with 3-(3,4-dichlorophenyl)-1,1-dimethylurea. 2,3,5,6-tetramethyl p-phenylenediamine-catalyzed cyclic electron flow increased the millisecond delayed emission by 2–4 times in both chloroplasts and subchloroplast particles. Uncoupling conditions which collapse only the pH gradient component of the proton motive force reduced the 2,3,5,6-tetramethyl p-phenylenediamine stimulation of delayed light in chloroplasts but not in particles. The 2,3,5,6-tetramethyl p-phenylenediamine stimulation of delayed light in particles was sensitive to uncoupling conditions which are presumed to destroy the transmembrane potential. Energy transfer inhibitors were without effect on the 2,3,5,6-tetramethyl p-phenylenediamine stimulation in both chloroplasts and particles.

The 2,3,5,6-tetramethyl p-phenylenediamine stimulation of millisecond delayed emission appears to reflect the particular form of the proton motive force; in chloroplasts it seems to be correlated with the proton concentration gradient, whereas in particles it is more closely correlated with the transmembrane potential.     

The equilibrium between the mitochondrial ATPase (F1) and its natural inhibitor in submitochondrial particles

1. The reversible equilibrium between the mitochondrial ATPase (F₁) and its naturally occurring inhibitor in Mg-ATP submitochondrial particles has been studied under different conditions.2. High ionic strength favours dissociation of the ATPase inhibitor as tested by ATPase and ATP-driven transhydrogenase activities.3. Dissociation of the ATPase inhibitor results in an increased maximal velocity of the ATPase activity measured in the presence of uncoupler and an increased affinity for adenine nucleotides, in particular for ATP.4. Association of the ATPase inhibitor with inhibitor-depleted Mg-ATP particles causes a slowing of the initial rate of succinate oxidation.5. The antibiotic aurovertin stimulates the ATPase activity of Mg-ATP particles preinculbated in the presence of a supply of oxidative energy. Bound aurovertin impedes the association of inhibitor-deficient particles with ATPase inhibitor.6. The fluorescence of aurovertin bound to inhibitor-containing particles is much less than that of aurovertin bound to inhibitor-depleted particles.7. The oligomycin-sensitivity-conferring protein, added either alone or in the presence or absence of membranous components of the ATPase complex, has little or no effect on the fluorescence of the F₁-aurovertin complex.8. It is suggested that the ATPase inhibitor brings F₁ in a conformation denoted 1F₁ that binds aurovertin with a low quantum yield, a decreased affinity and an increased binding capacity.     

Isolation and characterization of a rubredoxin and two ferredoxins from Desulfovibrio africanus

Rubredoxin and two distinct ferredoxins have been purified from Desulfovibrio africanus. The rubredoxin has a molecular weight of 6000 while the ferredoxins appear to be dimers of identical subunits of approximately 6000 to 7000 molecular weight. Rubredoxin contains one iron atom, no acid-labile sulfide and four cysteine residues per molecule. Its absorbance ratio A₂₇₈/A₄₉₀ is 2.23 and its amino acid composition is characterized by the absence of leucine and a preponderance of acidic amino acids.

The two ferredoxins, designated I and II, are readily separated on DEAE-cellulose. The amino acid compositions of ferredoxins I and II show them to be different protein species; the greater number of acidic amino acid residues in ferredoxin I than in ferredoxin II appears to account for separation based on electronic charge. Both ferredoxins contain four iron atoms, four acid-labile sulfur groups and either four (ferredoxin II) or six (ferredoxin I) cysteine residues per molecule. Spectra of the two ferredoxins differ from those of ferredoxins of other Desulfovibrio species by exhibiting a pronounced absorption peak at 283 nm consistent with an unusual high content of aromatic residues. The A₃₈₅/A₂₈₃ absorbance ratio of ferredoxins I and II are 0.56 and 0.62, respectively.

The N-terminal sequencing data of the two ferredoxins clearly indicate that ferredoxins I and II are different protein species. However, the two proteins exhibit a high degree of homology.

The physiological activity of ferredoxins I and II appears to be similar as far as the electron transfer in the phosphoroclastic reaction is concerned.     

Structural requirements of alkyl acyldithiocarbazates for the uncoupling of oxidative phosphorylation in mitochondria

A structure-activity relationship study on the uncoupling of alkyl acyldithiocarbazates was carried out. Greater activity was observed with increasing alkyl chain length, the optimum being C₉. A further increase in alkyl chain length caused a decrease in the activity. Thione-thiol tautomeric forms with a dissociable proton were found to be of primary importance for the uncoupling and the role of the acyl group was auxiliary.The reactivity of the SH group of alkyl acyldithiocarbazates with an SH-reagent was very low. These compounds facilitated the valinomycin-induced swelling of non-respiring mitochondria and non-sonicated lecithin liposomes in isotonic potassium acetate solution.     

Formation of ATP by the adenosine triphosphatase complex from spinach chloroplasts reconstituted together with bacteriorhodopsin

The energy-linked ATPase complex has been isolated from spinach chloroplasts. This protein complex contained all the subunits of the chloroplast coupling factor (CF₁) as well as several hydrophobic components. When the activated complex was reconstituted with added soybean phospholipids, it catalyzed the exchange of radioactive inorganic phosphate with ATP. Sonication of the complex into proteoliposomes together with bacteriorhodopsin yielded vesicles that catalyzed light-dependent ATP formation. Both the ³²P_i-ATP exchange reactions and ATP formation were sensitive to uncouplers such as 3-tert-butyl-5,2′-dichloro-4′-nitrosalicylanilide, bis-(hexafluoroacetonyl)acetone and carbonyl cyanide-p-trifluoromethoxyphenyl-hydrazone, that act to dissipate a proton gradient. The energy transfer inhibitors dicyclohexylcarbodiimide, triphenyltin chloride and 2-β-d-glucopyranosyl-4,6′-dihydroxydihydrochalcone were also effective inhibitors of both reactions.     

ATP-dependent spectral response of oxonol VI in an ATP-Pi exchange complex

(1) Energy transduction in an ATPase complex (complex V) has been studied in two reactions catalyzed by this system, i.e., ATP-dependent spectral shift of oxonol VI, and ATP-P_i exchange activity. (2) Aurovertin alone inhibits 50% of the oxonol shift at 2 μM, and no further inhibition occurs at up to 12 μM. In combination with even weakly effective uncouplers, 4 μM aurovertin fully abolishes the oxonol response. No such effects are observed in the presence of oligomycin and uncouplers. (3) No pH gradient is detectable by quenching of 9-amino-6-chloro-2-methoxyacridine; and nigericin is without effect on the oxonol response. Valinomycin is inhibitory even in the absence of added potassium, due to ammonium ions introduced during the purification steps. Thiocyanate inhibits the dye response by only 10–27%, depending on the preparation. The extent of the oxonol response depends on the ATP / ADP ratio rather than the phosphorylation potential. (4) The dye response in the ATPase complex is 4–7-times less sensitive to bile salts than in submitochondrial particles. The inhibition by cardiolipin can be reversed by the addition of phospholipids. (5) The possibility is discussed that the oxonol response in the ATPase complex reflects, at least in part, a more local, ATP-dependent and energy-related process.     

Effect of hemin and isonicotinic acid hydrazide on the uptake of iron from transferrin by isolated rat liver mitochonodria

Isolated rat liver mitochondria accumulate iron from the suspending medium when [⁵⁹Fe] transferrin is used as a model compound. The accumulation proceeds by two different mechanisms, i.e. by an energy-dependent and an energy-independent mechanism. The energy-dependent uptake of iron from transferrin is inhibited by hemin and stimulated by isonicotinic acid hydrazide. The energy-independent uptake of [⁵⁹Fe] transferrin is influenced neither by hemin nor by isonicotinic acid hydrazide.     

An active proton pump of intact vacuoles isolated from Tulipa petals

Anthocyanin pigments within Tulipa petal vacuoles provide the means for real-time spectrophotometric monitoring of vacuolar sap pH and for studying ATP-dependent proton transport in isolated, intact vacuoles. Spectra of petal extracts were used to select empirically those wavelengths giving an approximately linear variation in anthocyanin absorbance with pH over a pH range of interest. A sensitive single-beam spectrophotometer with vertical optics was used to minitor absorbance changes of intact, settled vacuoles. Substrates and inhibitors of vacuolar ATPase (Lin, W., Wagner, G.J., Siegelman, H.W. and Hind, Q. (1977) Biochim. Biophys. Acta 465, 110–117) were added to probe proton transport. Acidification of the vacuole sap occurred following addition of MgATP, but not CaATP. Proton accumulation was inhibited by 10 μM Dio 9, an inhibitor of tonoplast ATPase in vitro, and the proton gradient established by addition of MgATP was dissipated after addition of 10 μM CCCP. No pumping response was observed with intact protoplasts. Potential differences across the tonoplast were directly measured by impaling vacuoles with glass microelectrodes. Potential differences of 10–20 mV (inside positive) were recorded when vacuoles were suspended in 0.7 M mannitol/10 mM Hepes buffer (adjusted to pH 8.0 with KOH), and 0.5 mM dithiothreitol. Addition of MgATP increased the potential difference by 2–5 mV.     

Purification,characterization and biological activity of three forms of ferredoxin from the sulfate-reducing bacterium Desulfovibrio gigas

Three forms of ferredoxin FdI, FdI′, and FdII have been isolated from Desulfovibrio gigas, a sulfate reducer. They are separated by a combination of DEAE-cellulose and gel filtration chromatographic procedures. FdI and FdI′ present a slight difference in isoelectric point which enables the separation of the two forms over DEAE-cellulose, while FdII is easily separated from the two other forms by gel filtration. The three forms have the same amino acid composition and are isolated in different aggregation states. Molecular weight determinations by gel filtration gave values of 18 000 for FdI and FdI′ and 24 000 for FdII, whereas a value of 6000 is determined when dissociation is accomplished with sodium dodecyl sulfate. The electronic spectra are different and their ultraviolet-visible absorbance rations are 0.77, 0.87 and 0.68 respectively for FdI, FdI′ and FdII. Despite these differences, the physiological activities of the three forms are similar as far as the reduction of sulfite by molecular hydrogen is concerned.