Inhibition Of Electron Flow Research Articles

Inhibition of electron transfer and uncoupling effects by emodin and emodinanthrone in Escherichia coli.

The anthraquinones emodin (1,3,delta-trihydroxy-6-methylanthraquinone) and emodinanthrone (1,3,8-trihydroxy-6-methylanthrone) inhibited respiration-driven solute transport at micromolar concentrations in membrane vesicles of Escherichia coli. This inhibition was enhanced by Ca ions. The inhibitory action on solute transport is caused by inhibition of electron flow in the respiratory chain, most likely at the level between ubiquinone and cytochrome b, and by dissipation of the proton motive force. The uncoupling action was confirmed by studies on the proton motive force in beef heart cytochrome oxidase proteoliposomes. These two effects on energy transduction in cytoplasmic membranes explain the antibiotic properties of emodin and emodinanthrone.

Evidence that energy conserving electron transport pathways to nitrate and cytochrome o branch at ubiquinone in Paracoccus denitrificans

The organisation and function of electron transport pathways in Paracoccus denitrificans has been studied with both inhibitors and electrode probes for O2 or N2O respiration and membrane potential. Myxothiazol completely inhibits electron flow through the cytochrome bc1 region of the electron transport chain, as judged by its effect on nitrous oxide respiration. Electron flow to oxygen via the cytochrome o oxidase was shown to be insensitive to myxothiazol in a mutant, HUUG 25, that lacks cytochrome c and in which the aa3 oxidase is therefore inactive. Myxothiazol did not inhibit nitrate reduction. It is concluded that myxothiazol is a specific inhibitor of electron flow through the cytochrome bc1 region and more potent than antimycin which does not give complete inhibition.

Binding of ioxynil to photosynthetic membranes

Two binding affinities for ioxynil to isolated thylakoids of the alga Bumilleriopsis filiformis are observed. Higher concentrations needed for the “low-affinity” binding site inhibit electron transport, while low ioxynil concentrations saturating the “high-affinity” binding site do not affect electron transport. For higher plant thylakoids ( Chenopodium, Spinacia) the high-affinity binding site apparently is also the site to inhibit electron flow. The time dependence known for electron-transport inhibition is also apparent for the binding of phenol-type inhibitors to thylakoids of higher plants. Long incubation times (40 min) with the herbicide are necessary to equilibrate the binding site. Time dependence can be abolished by decreased pH, short trypsin or lysyl-endoprotease digestion of the isolated membranes. These treatments only increase the accessibility of the binding site, but the amount of bound herbicide after equilibration does not change. As a second effect the number of binding sites is reduced by prolonged trypsin digestion. This is not observed by treatment with a lysyl-specific protease. In contrast to herbicides like atrazine or diuron, short trypsin treatment alters the accessibility only (presumably provided by a shielding protein) but not the properties of the binding site of phenol-type herbicides. In contrast, thylakoids from the alga Bumilleriopsis, either from wild-type or a diuron resistant mutant, do not exhibit a time-dependent binding.

A functional site of Ca 2+ in the oxygen-evolving photosystem II preparation from Synechococcus sp.

Treatment of the oxygen-evolving photosystem II preparation from the thermophilic cyanobacterium Synechococcus sp. with EDTA inhibited electron flow from Z to P680 and consequently induced a back electron flow from Q − a to P680 +. The inhibition was reversed fully by Ca 2+and partially by Mn 2+ and Mg 2+ when EDTA-treated preparations had been incubated with respective divalent metal cations for several minutes, whereas diphenylcarbazide had no effect on the recombination between q − a and P680 + in EDTA-treated preparations. It is concluded that Ca 2+ is essential for electron transport from Z to P680. Oxygen evolution Electron transport Photosystem II Ca 2+ Thermophilic cyanobacterium

Restoration by tetramethyl- p-phenylenediamine of photosynthesis in dibromothymoquinone-inhibited cells of the cyanobacterium Synechococcus sp.

Inhibition of electron flow from water to methyl viologen by 2,5-dibromo-3-methyl-6-isopropyl- p-benzoquinone (DBMIB) was reversed by the addition of N, N, N′, N′-tetramethyl- p-phenylenediamine (TMPD) to cells of a thermophilic cyanobacterium Synechococcus sp. The restored activity is not a simple Photosystem I reaction because it was suppressed by 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea (DCMU). TMPD was also found to restore photosynthetic oxygen evolution in the DBMIB-poisoned cells. That the restored oxygen evolution is coupled with CO 2 reduction was indicated by its sensitivity not only to electron-transport inhibitors such as DCMU and phenylmercuric acetate, but also to an inhibitor of CO 2 fixation (KCN), uncouplers (carbonyl cyanide m-chlorophenylhydrazone, methylamine and ammonium chloride) and an energy-transfer inhibitor ( N, N′-dicyclohexylcarbodiimide). In the DBMIB-poisoned cells, TMPD accelerated only P-700 reduction, while leaving the reduction kinetics of cytochrome c-553 and cytochrome f inhibited, indicating that P-700 is the site of electron entry from TMPD to Photosystem I. It is concluded that TMPD restores photosynthetic electron transport by accepting electrons in the plastoquinone region and in turn donating them directly to P-700, and thereby bypassing cytochrome c-553 and DBMIB-blocked cytochrome b 6- f complexes.

Activation of the human neutrophil NADPH oxidase results in coupling of electron carrier function between ubiquinone-10 and cytochrome b559.

The enzymatic activity underlying the respiratory burst in human neutrophils was examined in a subcellular fraction with high specific activity and shown to be a membrane-associated complex of a flavoprotein, ubiquinone-10, and cytochrome b559 in an approximate 1.3:1:2 molar ratio. Study of the redox poise of these electron carriers indicated that electron flow in the intact complex from unstimulated cells proceeded: NADPH----E-FAD----ubiquinone-10. Similar studies on the complex prepared from stimulated neutrophils indicated that electron flow proceeded: NADPH----E-FAD----ubiquinone-10----cytochrome b559----oxygen. The active enzyme complex was inhibited by p-chloromercuribenzoate. Inhibition persisted after removal of excess inhibitor, was reversed by dithiothreitol, and could be blocked by prior addition of substrate (NADPH). Inhibition of the active oxidase complex by p-chloromercuribenzoate also inhibited electron flow from NADPH to all purported electron carriers in the chain (i.e. E-FAD, ubiquinone-10, and cytochrome b559). We conclude that activation of the oxidase enzyme complex in the intact neutrophil resulted in linkage of electron carrier function between endogenous ubiquinone-10 and cytochrome b559 and was without demonstrable effect on proximal electron flow. The p-chloromercuribenzoate sensitive site(s) proximal to the initial electron acceptor (E-FAD) did not appear to be altered by the cellular activation process.

The high affinity of Paracoccus denitrificans cells for nitrate as an electron acceptor. Analysis of possible mechanisms of nitrate and nitrite movement across the plasma membrane and the basis for inhibition by added nitrite of oxidase activity in permeabilised cells

(1) The apparent K m for nitrate of the electron-transport system in intact cells of Paracoccus denitrificans was less than 5 μM. In contrast the apparent K m for nitrate of inverted membrane vesicles oxidising NADH was greater than 50 μM. When azide, a competitive inhibitor, was present, the apparent K m observed with the vesicles was raised to 0.64 mM, consistent with values previously reported for purified preparations of the reductase. In membrane vesicles the nitrate reductase is probably not rate-limiting for NADH-nitrate oxido-reductase activity, and thus a lower limit for K m (NO 3 −) is obtained. It is suggested that the very low K m (NO 3 −) in intact cells must arise from either a transport process or a nitrate-specific pore that allows access of nitrate directly to the active site of its reductase from the periplasm. (2) The swelling of spheroplasts has been studied under both aerobic and anaerobic conditions to probe possible mechanisms of nitrate and nitrite transport across the plasma membrane of P. denitrificans. Nitrate reductase was inhibited by azide to prevent reduction of internal nitrate. No evidence for operation of either nitrate-nitrite antiport or proton-nitrate symport was obtained. (3) Measurements from the fluorescence intensity of 8-anilino-naphthalene-1-sulphonate of the rates of decay of diffusion potentials generated by addition of potassium salts to valinomycin-treated plasma membrane vesicles from P. denitrificans showed that the permeability of the membrane to anions is SCN − > NO 3 −, NO 2 −, pyruvate, acetate > CI − > SO 4 2−. In the presence of a protonophore the rate of decay of the diffusion potential was considerably enhanced with potassium acetate or potassium nitrite, but not with potassium salts of nitrate, chloride or pyruvate. This result indicates that HNO 2 and CH 3COOH can rapidly and passively diffuse across the cell membrane. This finding suggests that transport systems for nitrite are in general probably not required in bacteria. The failure of a protonophore to enhance the dissipation of the diffusion potential generated by potassium nitrate is evidence against the operation of a proton-nitrate symporter. (4) Low concentrations of added nitrite very strongly inhibit electron flow to oxygen in anaerobically grown cells, provided that they have been treated with Triton X-100 or an uncoupler. This inhibition is not observed with aerobically grown cells. It is concluded that the inhibitory species is a reaction product or an intermediate of the nitrite reductase reaction. The requirement for collapse of protonomotive force by uncoupler or permeabilising the plasma membrane suggests that any such species could be negatively charged. Nitroxyl anion (NO −) can be considered, as its conjugate acid is a postulated intermediate between nitrite and nitrous oxide; nitroxyl anion can bind to heme centres to give nitrosyl derivatives. (5) The basis for the ability of permeabilised, but not intact, cells of P. denitrificans to reduce oxygen and nitrate simultaneously is discussed.

Effect of papain digestion on redox-linked proton translocation in b- c1 complex from beef heart reconstituted into liposomes

Papain treatment of the cytochrome b- c 1 complex from beef heart results in partial proteolysis of core protein II, the iron-sulphur protein and the 15-kDa subunit. Under these conditions a significant inhibition of electron flow and complete suppression of proton translocation in the complex reconstituted into liposomes are observed. Kinetic experiments indicate a correlation between the digestion of core protein II and 15-kDa subunit and the suppression of proton translocation. The results suggest an active involvement of polypeptides of the complex in stabilizing the semiquinone species and/or providing pathways to exchange protons between bound quinone systems and aqueous phases.

Calmodulin is not involved in the regulation of exogenous NADH oxidation by plant mitochondria

The oxidation of exogenous NADH by mitochondria from potato (Solanum tuberosum L., cv. Bintje) tubers, measured with different electron acceptors, oxygen, cytochrome c, duroquinone and ubiquinone 1, was greatly enhanced under high salt conditions compared to low salt conditions, confirming the stimulatory effect of electrostatic screeening of negative membrane charges by cations. In addition to this nonspecific stimulation, the oxidation of exogenous NADH showed a specific dependence on Ca2+. Results presented here suggest that calmodulin was not directly involved in the regulation of exogenous NADH oxidation by potato mitochondria: (1) Calmodulin antagonists were found to inhibit electron flow at several sites in a nonspecific manner. (2) Using a phenothiazine‐Affi Gel column, it was not possible to demonstrate the presence of calmodulin in Triton X‐100 solubilized mitochondria. (3) Fractions eluted from a calmodulin‐Sepharose column with EGTA [ethyleneglycolbis (β‐aminoethylether)‐N, N, N′, N′‐tetraacetic acid] did not display any activity related to mitochondrial electron transport, suggesting that NADH dehydrogenase had no specific affinity for calmodulin. The possible indirect involvement of calmodulin in the regulation of exogenous NADH oxidation by Ca2+ is discussed.

Bypasses of the antimycin A block of mitochondrial electron transport in relation to ubisemiquinone function

Two different bypasses around the antimycin block of electron transport from succinate to cytochrome c via the ubiquinol-cytochrome c oxidoreductase of intact rat liver mitochondria were analyzed, one promoted by N, N, N′, N′-tetramethyl- p-phenylenediamine (TMPD) and the other by 2,6-dichlorophenolindophenol (DCIP). Both bypasses are inhibited by myxothiazol, which blocks electron flow from ubiquinol to the Rieske iron-sulfur center, and by 2-hydroxy-3-undecyl-1,4-naphthoquinone, which inhibits electron flow from the iron-sulfur center to cytochrome c 1. In the bypass promoted by TMPD its oxidized form (Wurster's blue) acts as an electron acceptor from some reduced component prior to the antimycin block, which by exclusion of other possibilities is ubisemiquinone. In the DCIP bypass its reduced form acts as an electron donor, by reducing ubisemiquinone to ubiquinol; reduced DCIP is regenerated again at the expense of either succinate or ascorbate. The observations described are consistent with and support current models of the Q cycle. Bypasses promoted by artificial electron carriers provide an independent approach to analysis of electron flow through ubiquinol-cytochrome c oxidoreductase.

A Nitroxide Diuron Analog as a Probe for the Mode of Action of Herbicides

A new spin label nitroxide which is also a herbicide of the urea family has been synthesized. The nitroxide spin label derivative of diuron (ditempuron, DTPU) inhibits electron flow at the site of diuron inhibition of photosystem II (PSII) activity, and it is also reduced by electron flow from one of the first acceptors of PSII. The reduction of the nitroxide group of DTPU is inhibited by diuron or by trypsin treatment of the thylakoids but not by DNP-INT or DBMIB. It is suggested that DTPU binds specifically to and accepts electrons at the Qв site.

Reevaluation of the Role of Bicarbonate and Formate in the Regulation of Photosynthetic Electron Flow in Broken Chloroplasts

The stimulation of the Hill reaction in CO(2)-depleted broken chloroplasts (Pisum sativum L. cv Rondo) by the total amount of dissolved CO(2) and HCO(3) (-) (bicarbonate(*)) was measured at several formate concentrations. Formate appears to be a competitive inhibitor of the bicarbonate(*) stimulation of electron flow. From these experiments we have obtained a reactivation constant (K(r)) of 78 +/- 31 micromolar NaHCO(3) and an inhibition constant (K(i)) of 2.0 +/- 0.7 millimolar HCOONa at pH 6.5. In the absence of formate, significant electron flow was measured at a bicarbonate(*) concentration well below K(r), suggesting that electron flow from Q, the primary electron acceptor of photosystem II, to plastoquinone can proceed when no bicarbonate(*) is bound to the regulatory site at the Q(B)-protein. If so, bicarbonate(*) stimulation of electron flow is mainly a diminution of the inhibition of electron flow by formate. In view of the results, it is proposed that regulation of linear electron flow by bicarbonate(*) and formate is a mechanism that could link cell metabolism to photosynthetic electron flow.

In vitro studies on pathways and regulation of electron transport to nitrogenase with a cell-free extract from heterocysts of Anabaena variabilis

A cell-free preparation of heterocysts from Anabaena variabilis showed high nitrogenase activities with several physiological electron donors, dependent on addition of an ATP-generating system. Light-induced acetylene reduction with the artificial electron donor to photosystem I, diaminodurol, exhibited the same light saturation as with hydrogen as donor. Inhibitors of electron flow through plastoquinone affected light-induced, hydrogen- or NADH-dependent nitrogenase activity in a similar way. Several uncoupling agents were without effect, indicating that energized membranes are not a prerequisite for nitrogen fixation. We conclude that NADH or hydrogen deliver electrons to nitrogenase via photosystem I and ferredoxin, feeding in at the plastoquinone site.

Electron flow to dimethylsulphoxide or trimethylamine-N-oxide generates a membrane potential in Rhodopseudomonas capsulata.

Under dark and essentially anaerobic conditions electron flow to either dimethylsulphoxide or trimethylamine-N-oxide in cells of Rhodopseudomonas capsulata has been shown to generate a membrane potential. This conclusion is based on the observation of a red shift in the carotenoid absorption band which is a well characterised indicator of membrane potential in this bacterium. The magnitude of the dimethylsulphoxide- or trimethylamine-N-oxide-dependent membrane potential was reduced either by a protonophore uncoupler of oxidative phosphorylation or synergistically by a combination of a protonophore plus rotenone, an inhibitor of electron flow from NADH dehydrogenase. These findings, together with the observation that venturicidin, an inhibitor of the proton translocating ATPase, did not reduce the membrane potential, show that electron flow to dimethylsulphoxide or trimethylamine-N-oxide is coupled to proton translocation. Thus contrary to some previous proposals dark and anaerobic growth of Rps. capsulata in the presence of dimethylsulphoxide or trimethylamine-N-oxide cannot be regarded as purely fermentative.

Cations stimulate electron transport associated with Photosystem II and inhibit electron flow linked with Photosystem I in spheroplasts of the cyanobacterium Synechococcus cedrorum

Cations stimulate electron transport associated with Photosystem II and inhibit electron flow linked with Photosystem I in spheroplasts of the cyanobacterium Synechococcus cedrorum

Action of the antitumor and antispermatogenic agent lonidamine on electron transport in ehrlich ascites tumor mitochondria

The effect of lonidamine, an antispermatogenic and antitumor drug, on the oxygen consumption, ATPase activity, and redox state of the electron carriers of Ehrlich ascites tumor mitochondria has been studied. Lonidamine inhibits ADP- and uncoupler-stimulated respiration on various NAD- and FAD-linked substrates, but does not affect state 4 respiration. Experiments to determine its site of action showed that lonidamine does not significantly inhibit electron flow through cytochrome oxidase. Electron flow through site 2, the ubiquinone-cytochrome b-cytochrome c 1 complex, also was unaffected by lonidamine, which failed to inhibit the oxidation of duroquinol. Moreover, inhibition of electron flow through site 2 was also excluded because of the inability of the N,N,N′,N′-tetramethyl- p-phenylenediamine bypass to relieve the lonidamine inhibition of the oxidation of pyruvate + malate. The F 0F 1ATPase activity and vectorial H + ejection are also unaffected by lonidamine. The inhibition of succinate oxidation by lonidamine was found to take place at a point between succinate and iron-sulfur center S3. Spectroscopic experiments demonstrated that lonidamine inhibits the reduction of mitochondrial NAD + by pyruvate + malate and other NAD-linked substrates in the transition from state 1 to state 4. However, lonidamine does not inhibit reduction of added NAD + by submitochondrial vesicles or by soluble purified NAD-linked dehydrogenases. These observations, together with other evidence, suggest that electron transport in tumor mitochondria is inhibited by lonidamine at the dehydrogenase-coenzyme level, particularly when the electron carriers are in a relatively oxidized state and/or when the inner membrane-matrix compartment is in the condensed state. The action of lonidamine in several respects resembles the selective inhibition of electron transport in tumor cells produced by cytotoxic macrophages.

On the inhibition of photosyhthetic electron transfer in the plastoquinone-cytochrome ƒ region by hydroxyquinoline- N-oxides

By examining the effect of high concentrations of 2- n-heptyl-4-hydroxyquinoline- N-oxide (HQNO) on electron transport in both thylakoids and isolated cytochrome b 6/ƒ complexes, we have determined that HQNO ( I 50 ≊ 50 μ M ) inhibits electron flow between plastoquinone and cytochrome ƒ. Based on the similarity in the inhibition pattern of reactions that involve quinol oxidation by HQNO compared to dibromothymoquinone and the dinitrophenyl ether of iodonitrothymol, we propose that cytochrome b 6 is the target for alkyl hydroxyquinoline- N-oxides in the plastoquinone-cytochrome ƒ region.

The function of cytoplasmic membrane of Paracoccus denitrificans in controlling the rate of reduction of terminal acceptors.

The rate of reduction of terminal acceptors (nitrate, nitrite, and oxygen) in anaerobically grown cells of Paracoccus denitrificans increased on permeabilization of cytoplasmic membrane. It was proved that under aerobic conditions the increase of the rate of nitrate reduction was caused by: (i) the abolishment of the permeability barrier for nitrate, (ii) the enhancement of the influx of redox equivalents to the respiratory chain due to the stimulation of succinate dehydrogenase reaction, and (iii) the inhibition of electron flow to oxygen by endogenously formed nitrite. Nitrite inhibits oxygen reduction by its interaction with the terminal part of the respiratory chain (I50 = 15 microM) localized at the inner aspect of the cytoplasmic membrane. The distribution of nitrite between intact cells and the suspension medium follows the Nernst equation for monovalent anion. The possible physiological consequences of the low intracellular nitrite concentration are discussed.

Interaction of Chloroplasts with Inhibitors

Several effects on pea (Pisum sativum L. var Onwards) chloroplasts of a new diphenylether herbicide, fomesafen (5-[2-chloro-4-trifluoromethyl-phenoxy]-N-methanesulfonyl-2 -nitrobenzamide) have been compared with those of a herbicide of related structure, nitrofluorfen (2-chloro-1-[4-nitrophenoxy]-4-[trifluoromethyl]benzene). Although both compounds produce the same light-dependent symptoms of desiccation and chlorosis indicative of a common primary mechanism of action, this study is concerned with a more broadly based investigation of different effects on the electron transport system. Comparisons have also been made with other compounds interacting with the chloroplast. Unlike nitrofluorfen, fomesafen has little effect as an inhibitor of electron flow or energy transfer. Both compounds have the ability to stimulate superoxide production through a functional electron transport system, and this involves specifically the p-nitro substituent. The stimulation, which is not likely to be an essential part of the primary herbicidal effect, is diminished under conditions that remove the coupling factor. Evidence suggests that both diphenylethers may be able to bind to the coupling factor, and kinetic studies reveal this for dibromothymoquinone as well. Such a binding site might be an important feature in allowing the primary effect of the diphenylether herbicides to be expressed.

Interactions of herbicides and azidoquinones at a Photosystem II binding site in the thylakoid membrane

6-Azido-5-decyl-2,3-dimethoxy- p-benzoquinone (6-azido-Q 0C 10) was found to replace the native plastoquinone at B (the second stable electron acceptor to Photosystem II (PS II)). The 6-azido-Q 10C 10 would accept electrons from the primary electron-accepting quinone, Q, thus allowing electron transport through PS II to the plastoquinone pool in thylakoids. The synthetic azidoquinone also competes with the PS II herbicides ioxynil and atrazine for binding. This observation strongly favors the hypothesis that PS II herbicides block electron transport by replacing the native quinone which acts as the second electron carrier on the reducing side of PS II (termed B). Covalent linkage of 6-azido-Q 0C 10 to its binding environment by ultraviolet irradiation greatly reduces herbicide-binding affinity but does not lead to a loss in herbicide-binding sites. We take this as evidence that covalent attachment of 6-azido-Q 0C 10 allows some freedom of quinone head-group movement such that the herbicides can enter the binding site. This indicates that the protein determinants which regulate quinone and herbicide binding are very closely related, but not identical. A compound somewhat related to 6-azido-Q 0C 10 is 2-azido-3-methoxy-5-geranyl-6-methyl- p-benzoquinone (2-azido-Q 2). This compound was found to be an ineffective competitor with respect to herbicide binding. Thus, interactions with protein-binding determinants are highly dependent on the molecular structure of quinones. The 2-azido-Q 2 was an inhibitor of electron flow in the intersystem portion of the chain.