Possible involvement of the 29 kDa protein in H-ATPase in the action of cationic uncoupler of oxidative phosphorylation. Effect of the (o-phenanthroline)2-Cu complex as a cationic uncoupler

The divalent cation (o-phenanthroline)₂-Cu²⁺ complex was found to uncouple oxidative phosphorylation in mitochondria. Its uncoupling activity depended on inorganic phosphate (P_i) in the incubation medium, and was inhibited by the SH-reagent N-ethylmaleimide, and retarded by ATP. The uncoupling by the (o-phenanthroline)₂-Cu²⁺ complex was suggested to be due to its modification of sulfhydryl groups in the 29 kDa protein in H⁺-ATPase.     

The spectrophotometric determination of micromolar concentrations of Co2+ using o-phenanthroline

Co2+ and o-phenanthroline formed a 1:3 complex with absorption maxima at 346, 332, 313, and 301 nm. The complex obeyed Beer's Law at the first three maxima. Standard curves constructed by monitoring the E346 at different concentrations of Co2+ had a maximum sensitivity of about 1 microM Co2+. At this concentration of Co2+ the delta E346 was 0.003 absorption units. This assay was not affected greatly by Ca2+, Mg2+, K+, Na+, or Tris. Erbium ions (Er3+) produced a small, nonspecific increase in absorbance at all wavelengths. Zn2+ also formed a complex with o-phenanthroline with maxima at 343, 328, and 313 nm. The absorbance of the Zn2+-o-phenanthroline complex was about 10% that of the Co2+-o-phenanthroline complex at 346 nm, but was still sufficient to cause interference at Zn2+ concentrations above 10 microM.     

(Na+ + K+)-ATPase : phosphorylation-dependent cross-linking of the alpha-subunits in the presence of Ca2+ and o-phenanthroline

In previous studies we had demonstrated that in the presence of 0.25 mM Cu2+ and 1.25 mM o-phenanthroline, cross-linking of the alpha-subunits of Na+ + K+)-dependent adenosine triphosphatase was induced by the addition of Na+ + ATP, and that the formation of the alpha,alpha-dimer was preceded by that of phosphoenzyme. The purpose of the present studies was the further evaluation of the role of phosphoenzyme in the process of cross-linking. Na+ + UTP did not induce cross-linking unless Mg2+ was also added. In contrast, Na+ + ATP-induced cross-linking did not require the addition of Mg2+. The different effects of ATP and UTP in the absence of added Mg2+ could be accounted for by the presence in the enzyme preparation of bound Mg2+ which supported enzyme phosphorylation by ATP but not by UTP. When the enzyme was phosphorylated by Pi, in the presence of Mg2 and ouabain, and the exposed to Cu2+ and o-phenanthroline, the alpha,alpha-dimer was obtained. Under these conditions, Na+ blocked both phosphorylation and cross-linking. These results indicate that it is the formation of phosphoenzyme per se that leads to conformational transitions favorable to cross-linking. They also suggest that Cu2+ and o-phenanthroline participate in the cross-linking reaction, but not in the phosphorylation reactions. In the digitonin-treated enzyme, Na+ and ATP induced the formation of phosphoenzyme, but not that of alpha,alpha-dimer. These findings indicate that in addition to phosphorylation, a proper orientation o alpha-subunits in an oligomer is also necessary for cross-linking.     

Role of the ADP/ATP-antiporter in fatty acid-induced uncoupling of Ca2+-loaded rat liver mitochondria

We show that Ca2+ loading of mitochondria substantially augments the myristate-induced decrease in the transmembrane electric potential difference (deltapsi). Such a Ca2+ action is without effect on the respiration rate and is not accompanied by the high-amplitude swelling when low concentrations of Ca2+ and myristate are used. The myristate-induced deltapsi decrease is prevented and reversed by cyclosporin A (CsA); the decrease is prevented and transiently reversed by nigericin. To explain these effects, we suggest that myristate induces opening of the mitochondrial permeability transition pore at a low-conductance state. Addition of carboxyatractylate (CAtr) after myristate induces the CsA-sensitive uncoupling, but when added after myristate and CsA, CAtr produces a decrease in deltapsi, if the interval between myristate and CsA addition is sufficiently long. The CAtr effect is completely reversed by EGTA and transiently reversed by nigericin. This suggests that the ADP/ATP-antiporter participates in the CsA-sensitive uncoupling when present as a pore complex constituent. ADP/ATP-antiporter that does not take part in the pore complex formation is involved in the CsA-insensitive uncoupling.     

On the functional role of coupling factor B in the mitochondrial H+ -ATPase

Evidence for the presence of a functionally important vicinal dithiol in mitochondrial coupling factor B (FB) has been presented earlier (Sanadi, D. R. (1982) Biochim. Biophys. Acta 683, 39-56). FB was completely inactivated by 38 micron of copper o-phenanthroline or 0.63 mM iodosobenzoate, and the kinetics were consistent with intramolecular disulfide formation as were polyacrylamide gel patterns which showed that FB which had been treated with copper o-phenanthroline had a different mobility from that of untreated FB. ATP-Pi exchange activity and ATP-induced binding of bis[3-propyl-5-oxoisoxazol-4-yl]pentamethine oxonol (oxonol VI) to H+ -ATPase were also inhibited by the thiol oxidizing reagents, although oligomycin-sensitive ATPase activity was unaffected. F0 isolated from H+ -ATPase rebinds purified F1 with the restoration of ATP-induced oxonol-binding activity. Prior treatment of F0 (but not of F1) with copper o-phenanthroline abolished the oxonol-binding activity of reconstituted F0-F1. 115Cd binds tightly to H+ -ATPase and the bound protein can be recovered by gel electrophoresis in phosphate buffer in the presence of sodium dodecyl sulfate at a position corresponding to FB. Prior treatment of the H+ -ATPase with copper o-phenanthroline abolished 115Cd binding. The results indicate that the major effect of these inhibitors is on FB dithiol and leave little doubt that Cd2+ is indeed bound to a vicinal dithiol group.     

Some modification in the assay of Fe2+ in 1–10, o-phenanthroline extracts of fresh plant tissues

Summary The over-estimation of Fe²⁺ by a spectrophotometer due to simultaneous extraction of certain colouring matter in o-phenanthroline extracts can be avoided by making determinations on an atomic absorption spectrophotometer. It is shown that estimates of iron by atomic absorption spectrophotometer did not include measurable amount of Fe³⁺ due to a high specificity of o-phenanthroline for plant Fe²⁺.     

A cyanine dye tri-S-C7(5). Phosphate-dependent cationic uncoupler of oxidative phosphorylation in mitochondria

The trinuclear cyanine dye, tri-S-C7(5), at about 10 microM stimulated State 4 respiration of rat liver mitochondria more than 6-fold and released oligomycin-inhibited respiration completely. Thus, the dye is concluded to be a very effective cationic uncoupler of oxidative phosphorylation in mitochondria. However, for exhibition of its uncoupling action, the presence of Pi (or arsenate) was necessary, and a phosphate-transport inhibitor, N-ethylmaleimide or mersalyl, inhibited its action. The stimulation of phosphate transport via the Pi carrier by the dye is suggested to be directly related to the uncoupling action.     

Why is inorganic phosphate necessary for uncoupling of oxidative phosphorylation by Cd2+ in rat liver mitochondria?

The phosphate (Pi)-dependent uncoupling action of Cd2+ in oxidative phosphorylation in rat liver mitochondria was studied mainly in terms of Pi transport. Cd2+ at 2 microM caused full uncoupling in the presence of 10 mM Pi, but no uncoupling in the absence of Pi. Cd2+ released state 4 respiration after a certain lag-time, and then the respiration increased progressively with time. After its addition, Cd2+ was taken up by mitochondria in a similar period to the lag time before respiratory release. KIH-201, a potent and specific inhibitor of Pi transport via the Pi/H+ symporter, abolished the uncoupling completely. Cd2+ caused dissipation of the electric transmembrane potential (delta psi) and swelling of mitochondria in a Pi-dependent manner. Uncoupling by Cd2+ was found to take place in parallel with the uptake of Pi into mitochondria via the Pi/H+ symporter, suggesting that the uncoupling was due to acceleration of H+ influx through the Pi/H+ symporter activated by Cd2+.     

2-Phenylcarbamoylisatogen,a novel uncoupler of mitochondrial oxidative phosphorylation

2-Phenylcarbamoylisatogen has been shown to stimulate succinate oxidation by rat liver mitochondria, but to inhibit the phosphorylation of ADP. The hydrolysis of ATP was also stimulated and an additive action with 2,4-dinitrophenol was demonstrated. The possible mechanism of the uncoupling action of 2-phenylcarbamoylisatogen is discussed.     

Comparative study of effects of the tyrosine-copper(II) complex on xenobiotic hydroxylation and lipid peroxidation]

It has been found that NADPH-dependent hydroxylation of dimethylaniline, aniline, p- and o-nitroanisol and lipid peroxidation is inhibited by the tyrosine-copper (II) complex (low molecular weight analog of superoxide dismutase), which is indicative of a possibility of superoxide radicals formation in these reactions. The inhibition of the above-mentioned reactions with Tyr2-Cu2+ is less pronounced or absent, if cumole hydroperoxide is used as cosubstrate instead of NADPH. Differences in the Tyr2-Cu2+ complex effects on the cumule hydroperoxide-dependent xenobiotics hydroxylation and lipid peroxidation catalyzed by various forms of cytochrome P-450, e. g. microsomal, soluble and incorporated into liposomes, have been found. The data obtained suggest that the efficiency of the inhibitory effect of the Tyr2-Cu2+ complex depends on the type of cosubstrates (NADPH, cumole hydroperoxide) and substrates used as well as on the form of cytochrome P-450.     

Regulation of the chloroplast H+-ATPase by light. The involvement of Mg2+ ions

The involvement of Mg2+ ions in the light-dependent regulation of the chloroplast H+-ATPase was studied in both type C and osmotically shocked type A chloroplasts. The following results were obtained. ATPase activity measured under dark, partially uncoupling conditions, following light activation with dithiothreitol and pyocyanine, was markedly enhanced by the presence of Mg2+ in the activation stage. This Mg2+ effect required concentrations in the millimolar range, was rather slow (time range of minutes), reversible, rather unspecific and did not involve changes in the affinity to dithiothreitol. Dark deactivation of the ATPase in the absence of substrate was accelerated by Mg2+. The dark effect of Mg2+ also required millimolar concentrations, but was fast (time range of seconds), highly specific for Mg2+, and did not involve thiol oxidation. The major effect of the absence of Mg2+ from the light-activation stage or of its presence in the dark interval between activation and assay was the induction of an 'abnormal' sensitivity to uncouplers: after these treatments ATP hydrolysis was not stimulated but rather inhibited by NH4Cl or other uncouplers. The pretreatments in the light without Mg2+ or dark with Mg2+ did not affect the membrane proton permeability, nor the proton pumping coupled to ATPase activity. The results are discussed in terms of Mg2+-dependent regulation of the enzyme complex at the level of subunit interaction and its effect on the affinity to protons.     

The effect of cardiotoxin on (Ca2+ + Mg2+)-ATPase of the erythrocyte and sarcoplasmic reticulum

The effects of cardiotoxin on the ATPase activity and Ca2+-transport of guinea pig erythrocyte and rabbit muscle sarcoplasmic reticulum (Ca2+ + Mg2+)-ATPase (E.C.3.6.1.3) were investigated. Erythrocyte (Ca2+ + Mg2+)-ATPase was inhibited by cardiotoxin in a time- and dose-dependent fashion and inhibition appears to be irreversible. Micromolar calcium prevented this inhibitory effect. Specificity for (Ca2+ + Mg2+)-ATPase inhibition by cardiotoxin was indicated since a homologous neurotoxin had no effect. Cardiotoxin did not affect (Ca2+ + Mg2+)-ATPase activity from sarcoplasmic reticulum, but Ca2+-transport was 50% inhibited. This inhibition was not due to an increased Ca2+-efflux and could be the result of an intramolecular uncoupling of ATPase activity from Ca2+-transport. Inhibition of Ca2+-transport by cardiotoxin could not be prevented by millimolar concentrations of Ca2+. It is suggested that the biological effects of cardiotoxin could be a consequence of inhibition of plasma membrane (Ca2+ + Mg2+)-ATPases.     

A cyanine dye tri-S-C7(5). Phosphate-dependent cationic uncoupler of oxidative phosphorylation in mitochondria

The trinuclear cyanine dye, tri-S-C₇(5), at about 10 μM stimulated State 4 respiration of rat liver mitochondria more than 6-fold and released oligomycin-inhibited respiration completely. Thus, the dye is concluded to be a very effective cationic uncoupler of oxidative phosphorylation in mitochondria. However, for exhibition of its uncoupling action, the presence of P_i (or arsenate) was necessary, and a phosphate-transport inhibitor, N-ethylmaleimide or mersalyl, inhibited its action. The stimulation of phosphate transport via the P_i carrier by the dye is suggested to be directly related to the uncoupling action.     

The electronic structure of Fe2+ in reaction centers from Rhodopseudomonas sphaeroides. II. Extended x-ray fine structure studies.

Extended x-ray absorption fine structure (EXAFS) studies were performed on reaction centers (RC) of the photosynthetic bacterium Rhodopseudomonas sphaeroides R-26. RC containing two, one, and no quinones (2Q, 1Q, 0Q) samples were studied. The average ligand distance of the first coordination shell was determined to be 2.10 +/- 0.02 A with a more distant shell at 4.14 +/- 0.05 A. The Fe2+ site in RC was found to have a very large structural disorder parameter, from which a spread in ligand distance per iron site of approximately +/- 0.1 A was deduced. The most likely coordination number of the first shell is six, with a mixture of oxygens and nitrogens as ligands. The edge absorption results are consistent with the Fe2+ being in distorted octahedral environment. The EXAFS spectra of the 2Q and 1Q samples with and without O-phenanthroline were found to be the same. This indicates that either the secondary quinone and o-phenanthroline do not bind to Fe2+ or that they replace an equivalent ligand. The 0Q sample showed a 12% decrease in the EXAFS amplitude, which was restored upon addition of o-phenanthroline. These results can be explained by either a loss of a ligand or a severe conformational change when the primary quinone was removed.     

Chlortetracycline-mediated continuous Ca2+ oscillations in mitochondria of digitonin-treated Tetrahymena pyriformis

Ca2+ transport in mitochondria was studied in situ using digitonin-permeabilized cells of the ciliate protozoan Tetrahymena pyriformis GL. In the presence of oxidizable substrates and inorganic phosphate, mitochondria were able to accumulate a large amount of the added Ca2+ without subsequent uncoupling and mitochondrial damage. However, the maximal Ca2+ uptake dramatically decreased in the presence of micromolar concentrations of the fluorescent calcium indicator, chlortetracycline, which in aerobic conditions caused an uncoupling of the respiration in Ca2+-loaded mitochondria. Moreover, on reaching hypoxia, when the rate of oxygen diffusion from the air to the stirred incubation medium became a limiting factor, continuous Ca2+ oscillations were observed. Ca2+ fluxes were synchronous with the cyclic changes of the membrane potential and were followed with a significant delay by the changes of the membrane-associated fluorescence of Ca-chlortetracycline complexes. Both the chlortetracycline-induced uncoupling of the respiration and the oscillations were prevented by either EGTA or ruthenium red. It is suggested that in conditions of the limited rate of respiration the oscillations are generated as a result of the functioning of the two Ca2+-transport pathways: a Ca2+ uniport and a chlortetracycline-mediated electroneutral Ca2+ efflux.     

Functional effect of hydrogen peroxide on the sarcoplasmic reticulum membrane: uncoupling and irreversible inhibition of the Ca2+-ATPase protein

The chemical treatment of sarcoplasmic reticulum vesicles with H2O2 affects both Ca2+ transport and the hydrolytic activity supported by the Ca2+-ATPase protein. Ca2+ transport was much more sensitive to inhibition than ATPase activity and the decrease in Ca2+ transport was not the result of an increase in membrane permeability. The Ca2+/Pi uncoupling can be attributed to the own catalytic mechanism of the enzyme. Under conditions of high uncoupling, Ca2+ binding to the transport sites was barely affected and accumulation of phosphorylated species during the enzyme cycling gave almost maximal levels. These are features defining intramolecular uncoupling mediated by a phosphorylated form of the enzyme. Severe inhibition of the hydrolytic activity was observed when higher peroxide concentrations and leaky vesicles were used. These experimental conditions diminished maximal Ca2+ binding and the steady-state phosphoenzyme level. The low hydrolytic activity can be ascribed to a decrease in the rate of enzyme dephosphorylation.     

Transesterification of Moz-Asn-Leu-Gly-OEt in methanol. Confirmation of Ca2+ mediated catalysis

During the recrystallization of the crude protected tripeptide ester Moz-Asn-Leu-Gly-OEt (obtained by an enzymatic coupling reaction) in methanol/water, the transesterification of this compound to methyl ester was observed. The involvement of Ca2+ in this process was indicated by the results obtained in the following experiments: 1) incubation of crude Moz-Asn-Leu-Gly-OEt in methanol solutions of o-phenanthroline and EDTA; 2) recrystallization of the crude Moz-Asn-Leu-Gly-OEt in ethanol/water followed by incubation in methanol; 3) determination of the Ca2+ content of the crude Moz-Asn-Leu-Gly-OEt. After recrystallization Moz-Asn-Leu-Gly-OEt lost the ability to be transesterified in methanol. However, in the presence of crude Moz-Asn-Leu-Gly-OEt, or calcium acetate, or a mixture of calcium chloride/sodium acetate, the compound was transesterified, suggesting that the transesterification of crude and recrystallized compounds occurs through different mechanisms. On the basis of these results, and of the conformational data obtained for these peptides by 1H-NMR, we suggest models for the transesterification reactions.     

The cyanine dye triS-C4(5) as a cationic uncoupler of oxidative phosphorylation: interaction with mitochondria detected by derivative spectrophotometry

Derivative spectrophotometry was used to study the interaction of the cationic uncoupler triS-C4(5) with mitochondria. The uncoupling action of this dye is dependent on the presence of Pi in the incubation medium. The second derivative spectrum of the dye changed with the incubation period, becoming similar to the spectrum in chloroform; but, after a time, the spectral pattern reverted to the original spectrum. The change in the spectrum in the presence of Pi was much more rapid than in its absence. The degree of spectral change agreed with the relative amount of bound dye determined directly. Thus, the spectral change reflects the binding of dye to the mitochondria, dependent on their energy state. The greater binding without Pi does not cause uncoupling but does cause shrinkage. In contrast, the lesser binding in the presence of Pi causes uncoupling and the swelling of mitochondria. These facts indicate that the dye does not penetrate the mitochondrial membrane. This refutes the idea that uncoupling by lipophilic cations is caused by the electrophoretic transfer of the uncoupler to the mitochondrial matrix space.     

Effect of ebselen on Ca2+ transport in mitochondria

The seleno-organic compound ebselen mimics the glutathione-dependent, hydroperoxide reducing activity of glutathione peroxidase. The activity of glutathione peroxidase determines the rate of hydroperoxide-induced Ca2+ release from mitochondria. Ebselen stimulates Ca2+ release from mitochondria, accelerates mitochondrial respiration and uncoupling, and induces mitochondrial swelling, indicating a deterioration of mitochondrial function. These manifestations are abolished by cyclosporine A, a potent inhibitor of the mitochondrial permeability transition. However, when ebselen-induced Ca2+ cycling is prevented with ruthenium red, an inhibitor of the Ca2+ uniporter, or by chelation of extramitochondrial Ca2+ by EGTA, no detectable elevation of swelling or uncoupling is observed. The release of Ca2+ from mitochondria is delayed in the absence of rotenone, i.e. when pyridine nucleotides are maintained in the reduced state due to succinate-driven reversed electron flow. We suggest that ebselen induces Ca2+ release from intact mitochondria via an NAD+ hydrolysis-dependent mechanism.     

Temperature dependence of charge recombination from the P+QA- and P+QB- states in photosynthetic reaction centers isolated from the thermophilic bacterium Chloroflexus aurantiacus.

The temperature dependence of charge recombination from the P+QA- and from the P+QB- states produced by a flash was studied in reaction centers isolated from the photosynthetic thermophilic bacterium Chloroflexus aurantiacus. P designates the primary electron donor; QA and QB the primary and secondary quinone electron acceptors respectively. In QB-depleted reaction centers the rate constant (kAP) for P+QA- recombination was temperature independent between 0-50 degrees C (17.6 +/- 0.7 s-1 at pH 8 and pH 10). The same value was obtained in intact membranes in the presence of o-phenanthroline. Upon lowering the temperature from 250 K to 160 K, kAP increased by a factor of two and remained constant down to 80 K. The overall temperature dependence of kAP was consistent with an activationless process. Ubiquinone (UQ-3) and different types of menaquinone were used for QB reconstitution. In UQ-3 reconstituted reaction centers charge recombination was monoexponential (rate constant k = 0.18 +/- 0.03 s-1) and temperature independent between 5-40 degrees C. In contrast, in menaquinone-3- and menaquinone-4-reconstituted reaction centers P+ rereduction following a flash was markedly biphasic and temperature dependent. In menaquinone-6-reconstituted reaction centers a minor contribution from a third kinetic phase corresponding to P+QA- charge recombination was detected. Analysis of these kinetics and of the effects of the inhibitor o-phenanthroline at high temperature suggest that in detergent suspensions of menaquinone-reconstituted reaction centers a redox reaction removing electrons from the quinone acceptor complex competes with charge recombination. Instability of the semiquinone anions is more pronounced when QB is a short-chain menaquinone. From the temperature dependence of P+ decay the activation parameters for the P+QB- recombination and for the competing side oxidation of the reduced menaquinone acceptor have been derived. For both reactions the activation enthalpies and entropies change markedly with menaquinone chain length but counterbalance each other, resulting in activation free energies at ambient temperature independent of the menaquinone tail. When reaction centers are incorporated into phospholipid vesicles containing menaquinone-8 a temperature-dependent, monophasic, o-phenanthroline-sensitive recombination from the P+QB- state is observed, which is consistent with the formation of stable semiquinone anions. This result seems to indicate a proper QB functioning in the two-subunit reaction center isolated from Chlorflexus aurantiacus when the complex is inserted into a lipid bilayer.