Prevention by uncouplers of lipophilic chelator inhibition at three sites of mitochondrial electron transport

Inhibition of coupled electron transport in mitochondrial cristae by hydrophobic chelators such as bathophenanthroline is partially prevented by uncoupling agents such as carbonylcyanide-m-chlorophenyl hydrazone. Reversal of inhibition is observed in all three sections of the electron transport chain where energy coupling occurs. Sites inhibited by hydrophilic bathophenanthroline sulfonate or orthophenanthroline do not show uncoupler relief of inhibition. We conclude that a buried non-heme iron or copper protein is closely associated with each energy coupling site.     

Lipophilic chelator inhibition of electron transport in Escherichia coli.

The lipophilic chelator bathophenanthroline inhibits electron transport in membranes from Escherichia coli. The less lipophilic 1,10-phenanthroline, bathophenanthroline sulfonate, and alpha,alpha-dipyridyl have little effect. Reduced nicotinamide adenine dinucleotide oxidase is more sensitive to bathophenanthroline inhibition than lactate oxidase activity. Evidence for two sites of inhibition comes from the fact that both reduced nicotinamide adenine dinucleotide menadione reductase and duroquinol oxidase activities are inhibited. Addition of uncouplers of phosphorylation before bathophenanthroline protects against inhibition.     

Coordinated, coenzyme Q reversible, 2,5-dibromothymoquionine inhibition of electron transport and ATPase in Escherichia coli.

2,6-dibromothymoquinone (DBMIB) and other coenzyme Q analogs partially inhibit electron transport and the membrane-bound Mg⁺⁺ stimulated ATPase of $\text{E. coli}$ membranes. The inhibitions by DBMIB are fully reversed by coenzyme Q₆, and other analogs show partial reversal by coenzyme Q₆. Electron transport reactions inhibited are NADH and lactate oxidase, NADH menadione reductase, lactate phenazinemethosulfate reductase and duroquinol oxidase. The concentrations of DBMIB required are similar for electron transport and ATPase inhibition and inhibitions are all increased by uncouplers. Electron transport and ATPase are not inhibited in a DBMIB insensitive mutant. Soluble ATPase extracted from the membranes does not show DBMIB inhibition under either high or low Mg⁺⁺ conditions. Lipophilic chelators show additional inhibition over DBMIB. It appears that coenzyme Q functions at three sites in $\text{E. coli}$ electron transport where ATPase activity is controlled. Coenzyme Q deficient mutants also show decreased electron transport and ATPase activity which is restored by coenzyme Q.     

Selective thiol inhibition of ferricyanide reduction in photosystem II of spinach chloroplasts

Selective inhibition of ferricyanide reduction in photosystem II by lipophilic thiols indicates a unique pathway of electron transport, which is not involved in reduction of class III acceptors or transfer of electrons to photosystem I. Both aromatic and aliphatic thiols induce the inhibition, but thiol binding reagents such as p-hydroxymercuribenzoate or N-ethylmaleimide do not inhibit. The inhibition can be observed using either dibromothymoquinone or bathophenanthroline to direct electrons away from photosystem I. No pretreatment of chloroplasts with thiols in the light was necessary to inhibit ferricyanide reduction by photosystem II or the O₂ evolution associated with ferricyanide reduction.     

Inhibition of mitochondrial electron transport by hydrophilic metal chelators. Determination of dehydrogenase topography

The topography of the inner mitochondrial membrane was investigated using inhibitors of electron transport on preparations of beef heart mitochondria and electron transport particles of opposite orientation. Reductions of juglone, ferricyanide, indophenol, coenzyme Q, duroquinone, and cytochrome c by NADH are inhibited to different extents on both sides of the membrane by the impermeant hydrophilic chelators bathophenanthroline sulfonate and orthophenanthroline. The extent of inhibition for each acceptor increased in the order given. At least two chelator-sensitive sites are present on each membrane face between the flavoprotein and coenzyme Q and a chelator-sensitive site is present on the matrix face between the sites of coenzyme Q and duroquinone interaction. Duroquinol oxidation in mitochondria only is stimulated by bathophenanthroline sulfonate. Juglone reduction is stimulated in electron transport particles (only) by p-hydroxymercuribenzenesulfonate, but after mercurial treatment, juglone reduction in both particles and mitochondria is more sensitive to bathophenanthroline sulfonate.Succinate dehydrogenase components are inhibited by hydrophilic orthophenanthroline or bathophenanthroline sulfonate in mitochondria only. Electron flow between the dehydrogenases of succinate and NADH occurs via a chelator-sensitive site located on the matrix face of the membrane. Inter-complex electron flow is prevented by rotenone or thenoyltrifluoroacetone. The lack of succinate-indophenol reductase inhibition by bathophenanthroline sulfonate in the presence of rotenone or thenoyltrifluoroacetone indicates that the rotenone-sensitive site may be located on the matrix face and demonstrates that electrons flow between the NADH and succinate dehydrogenases via a hydrophilic chelator and rotenone-thenoyltrifluoroacetone-sensitive site on the matrix face of the membrane. Inhibition by hydrophilic chelators only in mitochondria indicates that succinate dehydrogenase as well as NADH dehydrogenase has a transmembranous orientation.     

ATP synthesis and hydrolysis in chloroplast membranes. Differential inhibition by antibodies to chloroplast coupling factor 1.

1. Divalent antibodies against chloroplast coupling factor 1 inhibited the factor ATPase, ATP synthesis, hydrolysis and Pi-ATP exchange in chloroplasts. These antibodies also inhibited coupled electron flow rates but not the basal or uncoupled rates. 2. Several types of non-precipitating, modified antibodies prepared from the original antibody preparation strongly inhibited the ATPase and Pi-ATP exchange reaction but had little effect on ATP formation. 3. It is suggested that the inhibition of ATP synthesis by the divalent antibodies is probably due to an indirect blocking of the active site, while the inhibition of ATP-utilizing reactions by the modified antibodies is related to their effect on the transfer of ATP from a non-catalytic to a catalytic site on coupling factor 1, via an energy-dependent conformational change.     

Inhibition of purified mitochondiral ATPase (F1) by bathophenanthroline and relief of the inhibition by uncouplers.

Low concentrations of bathophenanthroline inhibit the ATPase activity of purified beef-heart F₁. The inhibition is antagonized by ATP in a fashion consistent with the involvement of a regulatory site on the enzyme. Various uncouplers, including FCCP, S-13, TTFB, dicoumarol and 2,4-dinitrophenol, relieve the bathophenanthroline inhibition, in concentrations similar to those known to uncouple mitochondrial oxidative phosphorylation.     

Inhibition of transplasma membrane electron transport by transferrin-adriamycin conjugates.

Transplasma membrane electron transport from HeLa cells, measured by reduction of ferricyanide or diferric transferrin in the presence of bathophenanthroline disulfonate, is inhibited by low concentrations of adriamycin and adriamycin conjugated to diferric transferrin. Inhibition with the conjugate is observed at one-tenth the concentration required for adriamycin inhibition. The inhibitory action of the conjugate appears to be at the plasma membrane since (a) the conjugate does not transfer adriamycin to the nucleus, (b) the inhibition is observed within three minutes of addition to cells, and (c) the inhibition is observed with NADH dehydrogenase and oxidase activities of isolated plasma membranes. Cytostatic effects of the compounds on HeLa cells show the same concentration dependence as for enzyme inhibition. The adriamycin-ferric transferrin conjugate provides a more effective tool for inhibition of the plasma membrane electron transport than is given by the free drug.     

Inhibition of energy-transducing functions of chloroplast membranes by lipophilic iron chelators.

Lipophilic metal chelators inhibit various energy-transducing functions of chloroplasts. The following observations were made 1. Photophosphorylation coupled to any known mode of electron transfer, i.e. whole-chain noncyclic, the partial noncyclic Photosystem I or Photosystem II reactions, or cyclic, is inhibited by several lipophilic chelators, but not by hydrophilic chelators. 2. The light- and dithioerythritol-dependent Mg2+-ATPase was also inhibited by the lipophilic chelators. 3. Electron transport through either partial reaction. Photosystem I or Photosystem II was not inhibited by lipophilic chelators. Whole-chain coupled electron transport was inhibited by bathophenanthroline, and the inhibition was not reversed by uncouplers. The diketone chelators diphenyl propanedione and nonanedione inhibited the coupled, whole-chain electron transport and the inhibition was reversed by uncouplers, a pattern typical of energy transfer inhibitors. The electron transport inhibition site is localized in the region of platoquinone leads to cytochrome f. This inhibition site is consistent with other recent work (Prince et al. (1975) FEBS Lett. 51, 108 and Malkin and Aparicio (1975) Biochem. Biophys. Res. Commun. 63, 1157) showing that a non-heme iron protein is present in chloroplasts having a redox potential near + 290 mV. A likely position for such a component to function in electron transport would be between plastoquinone and cytochrome f. just where our data suggests there to be a functional metalloprotein. 4. Some of the lipophilic chelators induce H+ leakiness in the chloroplast membrane, making interpretation of their phosphorylation inhibition difficult. However, 1-3 mM nonanedione does not induce significant H+ leakiness, while inhibiting ATP formation and the Mg2+-ATPase. Nonanedione, at those concentrations, causes a two- to four-fold increase in the extent of H+ uptake. 5. These results are consistent with, but do not prove, the involvement of a non-heme iron or a metalloprotein in chloroplast energy transduction.     

Oligomycin effects on ATPase and photophosphorylation of pea chloroplast thylakoid membranes

Oligomycin inhibited the membrane-bound, Ca²⁺-dependent ATPase of pea (Pisum sativum var. Progress No. 9) chloroplasts up to 50%, but only after treating the membranes with trypsin, whether or not the trypsin step was needed for full activity. The energy-linked Mg²⁺-dependent (light- and dithiothreitol (DTT)-activated) ATPase of pea thylakoids could be inhibited up to 100% under specified conditions. The data indicate that oligomycin does not interfere with activation processes, and it failed to inhibit the ATPase of solubilized chloroplast coupling factor 1 under any circumstances. Photophosphorylation, previously thought insensitive to oligomycin, was inhibited 30% in the case of pea chloroplasts, and this increased to 50% inhibition after pretreating the chloroplasts with either trypsin or DTT. The nature of inhibition of phosphorylation was complex, with apparent small components of electron transport inhibition and uncoupling, as well as energy transfer inhibition.     

K+-independent effects of valinomycin in photosynthetic systems

With chromatophores ofRhodospirillum rubrum, valinomycin inhibited electron transport in the presence or absence of K⁺. NH₄Cl had no effect on photophosphorylation but uncoupled with valinomycin present. ATPase activity was stimulated by NH₄Cl plus valinomycin but not by either alone. K⁺ partially reversed the inhibition of phosphorylation and the stimulation of ATPase by valinomycin plus NH₄Cl.With chloroplasts, valinomycin inhibited coupled but not basal electron transport. The inhibition was only partially reversed by uncouplers. Valinomycin stimulated the light-activated Mg²⁺-dependent ATPase similar to several uncouplers such as quinacrine, methylamine, and S-13. In addition, valinomycin inhibited delayed light emission and stimulated the H⁺/e^– ratio. These contrasting activities in chloroplasts are not easily explained.Contribution number 389 of the Charles F. Kettering Research Laboratory.     

Involvement of transferrin in the reduction of iron by the transplasma membrane electron transport system

Nonpermeable electron acceptors can be reduced by a transplasma membrane electron transport system in suspensions of intact cells. Here we report that diferric transferrin is reduced by HeLa S3 cells. The reduction is recorded spectrophotometrically as the formation of the ferrous complex of bathophenanthroline disulfonate. Ferric ammonium citrate can also be used as an electron acceptor, and the presence of low concentrations of diferric transferrin greatly stimulates the reduction of trivalent iron under these conditions. Likewise very low concentrations of ferricyanide, which does not give rise to a ferrous bathophenanthroline disulfonate complex formation, have a strong stimulatory effect on the complex formation when ferric ammonium citrate is the source of ferric iron. Apotransferrin is a potent inhibitor of the reaction. The inhibition occurs at the concentration necessary for complete occupancy of the transferrin receptors. The inhibition can be demonstrated also when high concentrations of ferricyanide are used as electron acceptor. The possible mechanism behind the reported phenomena is discussed, and it is concluded that the transplasma membrane electron transport system can be involved in the process of cellular iron uptake.     

Effects of Hydroxamic Acids Isolated from Gramineae on Adenosine 5'-triphosphate Synthesis in Chloroplasts

Two hydroxamic acids isolated from maize extracts, 2,4-dihydroxy-7-methoxy-1,4-(2H)-benzoxazin-3(4H)-one (DIMBOA) and the 2-O-beta-d-glucopyranoside of DIMBOA, inhibit photophosphorylation by spinach chloroplasts. Both cyclic and noncyclic photophosphorylations were inhibited to the same extent. The concentrations producing 50% inhibition for DIMBOA and its glucoside were about 1 and 4 millimolar, respectively. These compounds inhibit coupled electron transport but do not affect basal or uncoupled electron transport. Both acids inhibit the ATPase activities of membrane-bound coupling factor 1 (CF(1)) and of purified CF(1). On the basis of these results, it is concluded that DIMBOA and its glucoside act as energy transfer inhibitors of photophosphorylation.     

Inhibition of energy-transducing functions of chloroplasts by spegazzinine.

The effects of spegazzinine, a dihydroindole alkaloid, on various energy-transducing functions of chloroplasts were studied. The following observations were made, (i) Spegazzinine inhibited both cyclic and noncyclic photophosphorylation in isolated spinach chloroplast. The I₅₀ value was about 80 μm. Over a concentration range which gave marked inhibition of ÀTP synthesis, there was no effect on basal or uncoupled electron flow or light-induced proton accumulation by isolated thylakoids, while the fraction of electron transport stimulated by coupled phosphorylation was reduced to the basal level by spegazzinine. (ii) The regulatory effect of low concentrations of ATP on proton movements and electron transport was diminished by the alkaloid, (iii) Spegazzinine also inhibited with similar efficiency the ATPase activities of membrane-bound coupling factor 1 (CF₁) and of purified CF₁. One mole of spegazzinine per mole of CF₁ seemed to be required to inhibit the ATPase activity, (iv) The allosteric effect of ADP on ATPase activity was not affected by spegazzinine. (v) On the basis of these results it is concluded that spegazzinine acts as an energy transfer inhibitor of hotophosphorylation and that its site of action may be at or near the catalytic site of ATPase.     

On the functional organization of electron transport from plastoquinone to Photosystem I

Signal I, the EPR signal of P-700, induced by long flashes as well as the rate of linear electron transport are investigated at partial inhibition of electron transport in chloroplasts. Inhibition of plastoquinol oxidation by dibromothymoquinone and bathophenanthroline, inhibition of plastocyanin by KCN and HgCl₂, and inhibition by 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide are used to study a possible electron exchange between electron-transport chains after plastoquinone. (1) At partial inhibition of plastocyanin the reduction kinetics of P-700⁺ show a fast component comparable to that in control chloroplasts and a new slow component. The slow component indicates P-700⁺ which is not accessible to residual active plastocyanin under these conditions. We conclude that P-700 is reduced via complexed plastocyanin. (2) The rate of linear electron transport at continuous illumination decreases immediately when increasing amounts of plastocyanin are inhibited by KCN incubation. This is not consistent with an oxidation of cytochrome f by a mobile pool of plastocyanin with respect to the reaction rates of plastocyanin being more than an order of magnitude faster than the rate-limiting step of linear electron transport. It is evidence for a complex between the cytochrome b₆ - f complex and plastocyanin. The number of these complexes with active plastocyanin is concluded to control the rate-limiting plastoquinol oxidation. (3) Partial inhibition of the electron transfer between plastoquinone and cytochrome f by dibromothymoquinone and bathophenanthroline causes decelerated monophasic reduction of total P-700⁺. The P-700 kinetics indicate an electron transfer from the cytochrome b₆ - f complex to more than ten Photosystem I reaction center complexes. This cooperation is concluded to occur by lateral diffusion of both complexes in the membrane. (4) The proposed functional organization of electron transport from plastoquinone to P-700 in situ is supported by further kinetic details and is discussed in terms of the spatial distribution of the electron carriers in the thylakoid membrane.     

Inhibition of energy-transducing functions of chloroplast membranes by lipophilic iron chelators

Lipophilic metal chelators inhibit various energy-transducing functions of chloroplasts. The following observations were made.1. Photophosphorylation coupled to any known mode of electron transfer, i.e. whole-chain noncyclic, the partial noncyclic Photosystem I or Photosystem II reactions, or cyclic, is inhibited by several lipophilic chelators, but not by hydrophilic chelators.2. The light- and dithioerythritol-dependent Mg²⁺-ATPase was also inhibited by the lipophilic chelators.3. Electron transport through either partial reaction, Photosystem I or Photosystem II was not inhibited by lipophilic chelators. Whole-chain coupled electron transport was inhibited by bathophenanthroline, and the inhibition was not reversed by uncouplers. The diketone chelators diphenyl propanedione and nonanedione inhibited the coupled, whole-chain electron transport and the inhibition was reversed by uncouplers, a pattern typical of energy transfer inhibitors.The electron transport inhibition site is localized in the region of plastoquinone → cytochrome f. This inhibition site is consistent with other recent work (Prince et al. (1975) FEBS Lett. 51, 108 and Malkin and Aparicio (1975) Biochem. Biophys. Res. Commun. 63, 1157) showing that a non-heme iron protein is present in chloroplasts having a redox potential near +290 mV. A likely position for such a component to function in electron transport would be between plastoquinone and cytochrome f, just where our data suggests there to be a functional metalloprotein.4. Some of the lipophilic chelators induce H⁺ leakiness in the chloroplast membrane, making interpretation of their phosphorylation inhibition difficult. However, 1–3 mM nonanedione does not induce significant H⁺ leakiness, while inhibiting ATP formation and the Mg²⁺-ATPase. Nonanedione, at those concentrations, causes a two- to four-fold increase in the extent of H⁺ uptake.5. These results are consistent with, but do not prove, the involvement of a non-heme iron or a metalloprotein in chloroplast energy transduction.     

Stimulation of photosynthesis by carbonyl compounds and chelators

A number of carbonyl compounds including bicarbonate, ethylene carbonate, dimethylcarbonate, propylene carbonate, bis-pentamethylene urea, and glycidol, and several chelators were tested for their effect on photosynthetic reactions in isolated spinach chloroplasts. It was found that carbonyl compounds inhibited the DCMU-insensitive silicomolybdate reduction by photosystem II but stimulated the O₂ evolution associated with ferricyanide reduction in presence of DBMIB and the H₂O→methylviologen reaction. Many chelators behaved in the same manner except 1,10-phenanthroline which shows the opposite effect. The carbonyl compounds did not uncouple because they stimulated the proton gradients associated with noncyclic photophosphorylation, whereas some chelators, such as bathocuproine or bathophenanthroline inhibited the proton gradients 100%. Electron transport in presence of ADP and inorganic phosphate showed a stimulation of rates beyond that obtained in presence of an uncoupler. The data are discussed in terms of inhibition of cyclic electron flow around PS II which leads to increased electron transport rates toward PS I.     

Trifluoperazine inhibition of electron transport and adenosine triphosphatase in plant mitochondria

Trifluoperazine inhibits ADP-stimulated respiration in mung bean (Phaseolus aureus) mitochondria when either NADH, malate, or succinate serve as substrates (IC50 values of 56, 59, and 55 microM, respectively). Succinate:ferricyanide oxidoreductase activity of these mitochondria was inhibited to a similar extent. The oxidation of ascorbate/TMPD was also sensitive to the phenothiazine (IC50 = 65 microM). Oxidation of exogenous NADH was inhibited by trifluoperazine even in the presence of excess EGTA [ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid] (IC50 = 60 microM), indicating an interaction with the electron transport chain rather than with the dehydrogenase itself. In contrast, substrate oxidation in Voodoo lily (Sauromatum guttatum) mitochondria was relatively insensitive to the phenothiazine. The results suggest the bc1 complex to be a major site of inhibition. The membrane potential of energized mung bean mitochondria was depressed by micromolar concentrations of trifluoperazine, suggesting an effect on the proton-pumping capability of these mitochondria. Membrane-bound and soluble ATPases were equally sensitive to trifluoperazine (IC50 of 28 microM for both), implying the site of inhibition to be on the F1. Inhibition of the soluble ATPase was not affected by EGTA, CaCl2, or exogenous calmodulin. Trifluoperazine inhibition of electron transport and phosphorylation in plant mitochondria appears to be due to an interaction with a protein of the organelle that is not calmodulin.     

The relation of the alteration of photosystem. II. Activity to the inhibition of energy transfer and the proton pump in tris-washed chloroplasts

Washing of spinach chloroplasts with high concentrations of Tris³ induces pH-dependent changes in chloroplast reactions. At high pH (8.4) Tris washing causes the inhibition of Photosystem 2 activity which can be prevented by the maintenance of reducing conditions during washing. Washing at low pH (7.2) causes an enhancement of oxygen evolution and increased rate of ferricyanide photoreduction which is not influenced by the presence of reducing conditions. The increased rate of electron flow is accompanied by the inhibition of light mediated phosphorylating activity, acid-induced ATP synthesis, light-induced proton uptake and light triggered Mg²⁺ ATPase activity. Tris treatment at low pH also causes a sensitization of Photosystem 2 activity such that oxygen evolution is inhibited by low concentrations of tris at high pH. This inhibition of the stimulated electron flow is not accompanied by a reconstitution of the photophosphorylation activity. A detailed analysis of the effect of tris treatment on Photosystem II activity and membrane dependent energy conversion shows that the treatment of chloroplasts causes an inhibition of the energy conversion process which is independent of the effect on oxygen evolution. Determination of the presence of coupling factor (as determined by ATPase activity) and membrane osmotic properties reveal normal levels of enzyme activity and osmotic response in treated chloroplasts. The inhibition of the energy conversion process is accompanied by reduced capacity to maintain a proton gradient. Kinetic analysis of the proton uptake reaction reveals that Tris treatment renders the grana membranes more permeable to protons.     

Sulphydryl groups in photosynthetic energy conservation. I. Light-dependent inhibition of photophosphorylation by the sulphydryl reagent 2-2'dithio bis-(5-nitropyridine).

1. The sulphydryl reagent 2-2'dithio bis-(5-nitropyridine) (DTNP) inhibited photophosphorylation when the chloroplasts were preincubated with the reagent in the light. A maximum inhibition of about 50% was obtained in the presence of pyocyanine and MgCl 2 at 0.3 mumol DTNP per mg chlorophyll and was completed in about 40 s of preillumination. 2. Dithioerythritol, ADP plus Pi (or arsenate) and uncouplers prevented the inhibition when present during the preillumination while phloridzin, Dio-9 and discarine B were ineffective. Low concentrations of ADP or ATP afforded partial protection but other nucleotides had no effect. 3. DTNP inhibited the coupled electron transport rate to the basal level and had no effect on the uncoupled electron transport. The stimulation of proton uptake and inhibition of electron transport by ATP was prevented by DTNP. 4. The trypsin-activated but not the light- and dithioerythritol-triggered ATPase was inhibited by light preincubation of chloroplasts with DTNP. 5. Reversal of DTNP inhibition of photophosphorylation was obtained by a second preillumination in the presence of thiol groups. 6. More DTNP reacted with chloroplasts in the light than in the dark. Two mol of thione were formed in the light per mol of DTNP disappeared. 7. The results suggested that DTNP inhibition is related to the oxidation by DTNP of chloroplast vicinal dithiols probably exposed by a light-induced conformational change.