The site of inhibition of photosynthetic electron transfer by amphotericin B.

The inhibitory effect of the polyene antibiotic, amphotericin B, on photosynthetic electron transfer has been investigated. Treatment of chloroplasts with the inhibitor results in the release of plastocyanin from its site in the chloroplast membrane. This release is accompanied by a shift in the pH curve for ferricyanide photoreduction from water, which is similar to that observed when chloroplasts are treated by sonication or passage through a French press. Delayed light emission from photosystem 2 is not destroyed by amphotericin B treatment, indicating that photosystem 2 is not damaged. Amphotericin B does not inhibit photoreduction of ferricyanide from water by chloroplast preparations which are deficient in plastocyanin, such as maize bundle-sheath chloroplast fragments, Euglena chloroplasts, or maize mesophyll chloroplasts passed through a French press. Chloroplasts treated with amphotericin B are not able to photooxidize plastocyanin. This result demonstrates that little structural damage occurs to the membrane during treatment with the antibiotic as a capacity to photooxidize plastocyanin is observed only in damaged chloroplast membranes.     

Multiple sites of DCIP reduction by sonicated Oat chloroplasts: Role of plastocyanin

1. Oat chloroplasts, in the presence of 0.02 M methylamine, reduce 2,6 dichlorophenolindophenol (DCIP) at a rate of 350–500 μmoles/mg chl per h, in saturating light. Brief sonication for approx. 1 min lowers the rate to approx. 50 μmoles/mg chl per h; longer sonication does not reduce activity further. During brief sonication, plastocyanin is lost from the chloroplasts. When plastocyanin is added back to sonicated fragments, DCIP reduction is approximately doubled to 100 μmoles/mg chl per h.

2. When oxidized plastocyanin is added, a transient is observed when light is first turned on: this is due to a reduction of the plastocyanin before DCIP reduction begins. When reduced plastocyanin is added, a different transient occurs: this is due to a fast photoreduction of DCIP by the plastocyanin and is followed by the slower steady state reduction of DCIP by water. When light is turned off before complete reduction of DCIP, a transient reduction of oxidized plastocyanin by reduced DCIP is seen. Insensitivity of these transients to 3(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) and the greater effectiveness of 710 nm light, along with the known capacity of plastocyanin to mediate electron transfer to System I, prove that an intrinsically fast reduction of DCIP occurs at a site close to the primary photoreduced product of System I.

3. After brief sonication and washing, no residual plastocyanin was detected in chloroplast fragments, and the rate of the slow DCIP reduction (about 50μmoles/mg chl per h) sustained by such fragments was essentially identical to that maintained by fragments of mutants lacking System I activity. Following et al.⁹, the simplest explanation for this slow DCIP reduction is that is occurs at a site close to System II and the system I is not involved.

4. A very slow transient reduction of DCIP occurs after extinguishing light; this presumably involves another reduction site close to System II, as suggested by ⁹.     

Inhibition of photosynthetic electron transport by amphotericin B

By assaying partial reactions of the photosynthetic electron transport system using thylakoids from spinach as well as from the algae Bumilleriopsis, Dunaliella , and Anabaena , it was demonstrated that the polyene antibiotic amphotericin B has no specific effect on plastocyanin. Pretreating spinach and algal thylakoids with this antibiotic decreased photosystem-II as well as photosystem-I activity regardless of whether the membranes contained plastocyanin or cytochrome c-553. Different sensitivity of cell-free electron transport activity against this antibiotic was observed due to the species used. With Dunaliella , the photosystem-II region was inhibited more strongly than photosystem-I, while Bumilleriopsis chloroplasts – although not containing plastocyanin – exhibited a stronger inhibition of the photosystem-I region. Apparently, amphotericin B mainly solubilizes redox compounds that form connecting pools in the photosynthetic electron transport chain.     

Reactions of plastocyanin and cytochrome 553 with Photosystem I of Scenedesmus

Chloroplast material active in photosynthetic electron transport has been isolated from Scenedesmus acutus (strain 270/3a). During homogenization, part of cytochrome 553 was solubilized, and part of it remained firmly bound to the membrane. A direct correlation between membrane cytochrome 553 and electron transport rates could not be found. Sonification removes plastocyanin, but leaves bound cytochrome 553 in the membrane. Photooxidation of the latter is dependent on added plastocyanin. In contrast to higher plant chloroplasts, added soluble cytochrome 553 was photooxidized by 707 nm light without plastocyanin present. Reduced plastocyanin or cytochrome 553 stimulated electron transport by Photosystem I when supplied together or separately. These reactions and cytochrome 553 photooxidation were not sensitive to preincubation of chloroplasts with KCN, indicating that both redox proteins can donate their electrons directly to the Photosystem I reaction center. Scenedesmus cytochrome 553 was about as active as plastocyanin from the same alga, whereas the corresponding protein from the alga Bumilleriopsis was without effect on electron transport rates.

It is suggested that besides the reaction sequence cytochrome 553 → plastocyanin → Photosystem I reaction center, a second pathway cytochrome 553 → Photosystem I reaction center may operate additionally.     

The Effect of Polyene Antibiotics on Photosynthetic Electron Transfer

The effect of four polyene antibiotics and digitonin on photosyntheticelectron transfer by maize mesophyll chloroplasts has been investigated.All five compounds, at concentrations between 0.1 mM and 20mM, inhibited photosystem 2 activity as measured by the photo-reductionof ferricyanide and 2,6-dichlorophenolindophenol from water.Etruscomycin, amphotericin B, and digitonin were more inhibitorythan filipin and nystatin. Photosystem 1 activity was inhibitedby 1 mM concentrations of etruscomycin, amphotericin B, anddigitonin but not by filipin and nystatin. In all cases whereinhibition occurred, it was temperature dependent. The inhibition of photosystem 1 activity could be relieved byplastocyanin. Etruscomycin and digitonin, at concentrationsof 0.5 mM and above, caused disintegration of the chloroplasts,and this disintegration was accompanied by a two- to three-foldincrease in photosystem 1 activity in the presence of plastocyanin.It is concluded that the action of polyene antibiotics resultsin the release of plastocyanin from its site in the photosyntheticelectron transfer chain. The results are discussed in termsof the abilities of polyene antibiotics and digitonin to formcomplexes with sterols.     

The reduction of plastocyanin by plastoquinol-1 in the presence of chloroplasts. A dark electron transfer reaction involving components between the two photosystems.

The reduction of plastocyanin by plastoquinol-1 was efficiently catalysed by disrupted chloroplasts or etioplasts in the dark. The reaction was inhibited by 2,5-dibromomethylisopropyl-p-benzo-quinone which inhibits photosynthetic electron transport between plastoquinone and cytochrome f. Evidence is presented that the reduction took place via cytochrome f, and that plastoquinone-9 was not involved. Triton X-100 and organic solvents were inhibitory, but partial fractionation was achieved without loss of activity by density gradient centrifugation in the presence of high digitonin concentrations. All active material contained cytochromes b-559LP and b-563 in addition to cytochrome f, but these b-type cytochromes were not directly involved. Other 1-electron acceptors could be used in place of plastocyanin, for instance ferricyanide and Pseudomonas cytochrome c-551. The reaction can be applied to give a sensitive dark assay for active cytochrome f. It is suggested that cytochrome f possesses two sites for interaction with redox reagents: a hydrophilic site with which plastocyanin reacts by electron transfer and a hydrophobic site with which plastoquinol reacts by hydrogen atom transfer.     

The site of KCN inhibition in the photosynthetic electron transport pathway

Treatment of chloroplasts with high concentrations of KCN inhibits reactions which involve Photosystem I (e.g. electron transport from water or diaminodurene to methylviologen), but not those assumed to by-pass Photosystem I (e.g. electron transport from water to quinonediimides). The spectrophotometric experiments described in this paper showed that KCN inhibits the oxidation of cytochrome f by far-red light without blocking its reduction by red light. Both optical and EPR experiments indicated that KCN does not inhibit the photooxidation of P700 but markedly slows down the subsequent dark decay (reduction). Reduction of P700 by Photosystem II is prevented by KCN. It is concluded that KCN blocks electron transfer between cytochrome f and P700, i.e. the reaction step which is believed to be mediated by plastocyanin. In KCN-poisoned chloroplasts the slow dark reduction of P700 following photooxidation is greatly accelerated by reduced 2,6-dichlorophenolindophenol or by reduced N-methylphenazonium methosulfate (PMS), but not by diaminodurene. It appears that the reduced indophenol dye and reduced PMS are capable of donating electrons directly to P700, at least partially by-passing the KCN block.     

Distinct trans-plasma membrane redox pathways reduce cell-impermeable dyes in HeLa cells

Trans-plasma membrane electron transport (tPMET) in mammalian cells has been demonstrated using artificial cell-impermeable dyes, but the extent to which reduction of these dyes involves distinct pathways remains unclear. Here we compare the properties of three commonly used dyes, WST-1, FeCN and DCIP. The presence of an intermediate electron carrier (mPMS or CoQ(1)) was obligatory for WST-1 reduction, whereas FeCN and DCIP were reduced directly. FeCN reduction was, however, greatly enhanced by CoQ(1), whereas DCIP was unaffected. Superoxide dismutase (SOD) and aminooxyacetate (AOA), a malate/aspartate shuttle inhibitor, strongly inhibited WST-1 reduction and reduced DCIP reduction by 40-60%, but failed to affect FeCN reduction, indicating involvement of mitochondrial TCA cycle-derived NADH and a possible role for superoxide in WST-1 but not FeCN reduction. Reduction of all three substrates was similarly inhibited by dicoumarol, diphenyleneiodonium and capsaicin. These results demonstrate that WST-1 FeCN and DCIP are reduced by distinct tPMET pathways.     

EGTA, a calcium chelator, inhibits electron transport in photosystem II of spinach chloroplasts at two different sites

A fifteen minute incubation of spinach chloroplasts with the divalent Ca²⁺ chelator, EGTA, in concentrations 50–250 μM, inhibits electron transport through both photosystems. All photosystem II partial reactions, including indophenol, ferricyanide and the DCMU-insensitive silicomolybdate reduction are inhibited from 70–100%. The photosystem II donor reaction, diphenyl carbazide → indophenol, is also inhibited, indicating that the inhibition site comes after the Mn²⁺ site, and that the first Ca²⁺ effect noted (site II) is not on the water oxidation enzyme, as is commonly assumed, but between the Mn²⁺ site and plastoquinone A pool. The other photosystem II effect of EGTA (Ca²⁺ site I), occurs in the region between plastoquinone A and P700 in the electron transport chain of chloroplasts. About 50% inhibition of the reaction ascorbate + TMPD → methyl viologen is given by incubation with 200 μM EGTA for 15 min. Ca²⁺ site II activity can be restored with 20 mM CaCl₂. Ca²⁺ site I responds to Ca²⁺ and plastocyanin added jointly. More than 90% activity in the ascorbate + TMPD → methylviologen reaction can be restored. Various ways in which Ca²⁺ ions could affect chloroplast structure and function are discussed. Since EGTA is more likely to penetrate chloroplast membranes than EDTA, which is known to remove CF₁, the coupling factor, from chloroplast membranes, and since Mg²⁺ ions are ineffective in restoring activity, it is concluded that Ca²⁺ may function in the electron transport chain of chloroplasts in a hitherto unsuspected manner.     

Localization of the reaction side of plastocyanin from immunological and kinetic studies with inside-out thylakoid vesicles

(1) The effect of four active antisera against plastocyanin on Photosystem I-driven electron transport and phosphorylation was investigated in spinach chloroplasts. Partial inhibition of electron transport and stimulation of plastocyanin-dependent phosphorylation were sometimes observed after adding amounts of antibodies which were in large excess and not related to the plastocyanin content of the chloroplasts. This indicates effects of the antibodies on the membrane. (2) The antibodies against plastocyanin neither directly nor indirectly agglutinated unbroken chloroplast membranes. (3) The plastocyanin content of right-side-out and inside-out thylakoid vesicles isolated by aqueous polymer two-phase partition from chloroplasts disrupted by Yeda press treatment was determined by quantitative rocket electroimmunodiffusion. Right-side-out vesicles retained about 25%, inside-out vesicles none of the original amount of plastocyanin. (4) The effect of externally added plastocyanin on the reduction of P-700 was studied by monitoring the absorbance changes at 703 nm after a long flash. In inside-out vesicles P-700 was reduced by the added plastocyanin but not in right-side-out vesicles and class II chloroplasts. These results provide strong evidence for a function of plastocyanin at the internal side of the thylakoid membrane.     

CNS neurons express two distinct plasma membrane electron transport systems implicated in neuronal viability

Trans-plasma membrane electron transport is critical for maintaining cellular redox balance and viability, yet few, if any, investigations have studied it in intact primary neurons. In this investigation, extracellular reduction of 2,6-dichloroindophenol (DCIP) and ferricyanide (FeCN) were measured as indicators of trans-plasma membrane electron transport by chick forebrain neurons. Neurons readily reduced DCIP, but not FeCN unless CoQ(1), an exogenous ubiquinone analog, was added to the assays. CoQ(1) stimulated FeCN reduction in a dose-dependent manner but had no effect on DCIP reduction. Reduction of both substrates was totally inhibited by epsilon-maleimidocaproic acid (MCA), a membrane-impermeant thiol reagent, and slightly inhibited by superoxide dismutase. Diphenylene iodonium, a flavoenzyme inhibitor, completely inhibited FeCN reduction but had no affect on DCIP reduction, suggesting that these substrates are reduced by distinct redox pathways. The relationship between plasma membrane electron transport and neuronal viability was tested using the inhibitors MCA and capsaicin. MCA caused a dose-dependent decline in neuronal viability that closely paralleled its inhibition of both reductase activities. Similarly capsaicin, a NADH oxidase inhibitor, induced a rapid decline in neuronal viability. These results suggest that trans-plasma membrane electron transport helps maintain a stable redox environment required for neuronal viability.     

Distinct trans-plasma membrane redox pathways reduce cell-impermeable dyes in HeLa cells

Abstract

Trans-plasma membrane electron transport (tPMET) in mammalian cells has been demonstrated using artificial cell-impermeable dyes, but the extent to which reduction of these dyes involves distinct pathways remains unclear. Here we compare the properties of three commonly used dyes, WST-1, FeCN and DCIP. The presence of an intermediate electron carrier (mPMS or CoQ₁) was obligatory for WST-1 reduction, whereas FeCN and DCIP were reduced directly. FeCN reduction was, however, greatly enhanced by CoQ₁, whereas DCIP was unaffected. Superoxide dismutase (SOD) and aminooxyacetate (AOA), a malate/aspartate shuttle inhibitor, strongly inhibited WST-1 reduction and reduced DCIP reduction by 40–60%, but failed to affect FeCN reduction, indicating involvement of mitochondrial TCA cycle-derived NADH and a possible role for superoxide in WST-1 but not FeCN reduction. Reduction of all three substrates was similarly inhibited by dicoumarol, diphenyleneiodonium and capsaicin. These results demonstrate that WST-1 FeCN and DCIP are reduced by distinct tPMET pathways.     

Superoxide formation in pea chloroplasts by a dioxathiadiaza-2,5-pentalene derivative,a new lipophilic: Photosystem I acceptor

The dioxathiadiaza-2,5-pentalene derivative, HEP II, has herbicidal effects similar to those of methyl viologen. HEP II promotes superoxide formation when added to illuminated pea chloroplasts. Superoxide dismutase, but not catalase, diminished formation of the Superoxide whereas cyanide and azide enhanced its formation, presumably by inhibiting the endogenous superoxide dismutase activity. DCMU, which inhibits photosynthetic electron transport, inhibited Superoxide formation. Rates of superoxide formation and oxygen uptake were very similar when equal concentrations of methyl viologen or HEP II were added. At subsaturating concentrations of electron acceptor, Mg²⁺ decreased the rate of oxygen uptake with methyl viologen but not with HEP II, probably reflecting differences in their interactions with the Photosystem I electron donation site. It is likely that HEP II, by analogy with methyl viologen, is reduced by chloroplast Photosystem I and reoxidised by molecular oxygen, generating superoxide.     

Inhibition of Photosystem II in Isolated Chloroplasts by Lead

Inhibition of photosynthetic electron transport in isolated chloroplasts by lead salts has been demonstrated. Photosystem I activity, as measured by electron transfer from dichlorophenol indophenol to methylviologen, was not reduced by such treatment. However, photosystem II was inhibited by lead salts when electron flow was measured from water to methylviologen and Hill reaction or by chlorophyll fluorescence. Fluorescence induction curves indicated the primary site of inhibition was on the oxidizing side of photosystem II. That this site was between the primary electron donor of photosystem II and the site of water oxidation could be demonstrated by hydroxylamine restoration of normal fluorescence following lead inhibition.     

Effect of surface potential on P-700 reduction in chloroplasts

The effect of salt addition on the rate of reduction of P-700 oxidized by flash illumination was analyzed. In broken chloroplasts, the rate of P-700 reduction was accelerated by salts of mono-, di- and trivalent cations, with the increasing effectiveness in this order, in the presence of various artificial electron donors or acceptors. The rate was not dependent on the concentration and the valence of anions. On the other hand, in Photosystem I-enriched subchloroplast particles, added KCl did not induce the acceleration of direct reduction of P-700 by reduced DCIP.At low KCl concentrations (below 10 mM), the rate of P-700 reduction was also accelerated by added KCl in sonicated chloroplasts to which purified plastocyanin was added. The curves of dependence of the reduction rate on plastocyanin concentration were not of the Michaelis-Menten type, but sigmoidal. The maximal of P-700 reduction was higher at higher salt concentrations and the half-maximal plastocyanin concentration for P-700 reduction became lower with increasing NaCl concentrations.In broken chloroplasts treated with 50 mM glutaraldehyde, the rate of P-700 reduction was not accelerated by added KCl.The Debye-Hückel theory and the Gouy-Chapman theory were applied to our data to analyze the electrostatic interaction between electron tranfer components on thylakoid membranes. It is suggested that the major factor determining the rate of P-700 reduction is the donation of electrons from plastocyanin to P-700. Most of the observed effect is probably due to the increase in the local concentration or accessibility of plastocyanin to the site of P-700 reduction which is expected when the negative surface potential rises when salt is added.     

The mechanism of the oxidation of ascorbate and Mn by chloroplasts: The role of the radical superoxide

1. 1. The mechanism of the photooxidation of ascorbate and of Mn²⁺ by isolated chloroplasts was reinvestigated.

2. 2. Our results suggest that ascorbate or Mn²⁺ oxidation is the result of the Photosystem I-mediated production of the radical superoxide, and that neither ascorbate nor Mn²⁺ compete with water as electron donors to Photosystem II nor affect the rate of electron transport through the two photosystems: The radical superoxide is formed as a result of the autooxidation of the reduced forms of low potential electron acceptors, such as methylviologen, diquat, napthaquinone, or ferredoxin.

3. 3. In the absence of ascorbate or Mn²⁺ the superoxide formed dismutases either spontaneously or enzymatically producing O₂ and H₂O₂. In the presence of ascorbate or Mn²⁺, however, the superoxide is reduced to H₂O₂ with no formation of O₂. Consequently, in the absence of reducing compounds, in the reaction H₂O to low potential acceptor one O₂ (net) is taken up per four electrons transported where as in the presence of ascorbate, Mn²⁺ or other suitable reductants up to three molecules O₂ can be taken up per four electrons transported.

4. 4. This interpretation is supported by the following observations: (a) in a chloroplast-free model system containing NADPH and ferredoxin-NADP reductase, methylviologen can be reduced to a free radical which is autooxidizable in the presence of O₂; the addition of ascorbate or Mn²⁺ to this system results in a two fold stimulation of O₂ uptake, with no stimulation of NADPH oxidation. The stimulation of O₂ uptake is inhibited by the enzyme superoxide dismutase; (b) the stimulation of light-dependent O₂ uptake in the system H₂O → methylviologen in chloroplasts is likewise inhibited by the enzyme superoxide dismutase.

5. 5. In Class II chloroplasts in the system H₂O → NADP upon the addition of ascorbate or Mn²⁺ an apparent inhibition of O₂ evolution is observed. This is explained by the interaction of these reductants with the superoxide formed by the autooxidation of ferredoxin, a reaction which proceeds simultaneously with the photoreduction of NADP. Such an effect usually does not occur in Class I chloroplasts in which the enzyme superoxide dismutase is presumably more active than in Class II chloroplasts.

6. 6. It is proposed that since in the Photosystem I-mediated reaction from reduced 2,4-dichlorophenolindophenol to such low potential electron acceptor as methylviologen, superoxide is formed and results in the oxidation of the ascorbate present in the system, the ratio ATP/2e in this system (when the rate of electron flow is based on the rate of O₂ uptake) should be revised in the upward direction.

Genetic variation in soybean photosynthetic electron transport capacity is related to plastocyanin concentration in the chloroplast

Fifteen ancestral genotypes of United States soybean cultivars were screened for differences in photosynthetic electron transport capacity using isolated thylakoid membranes. Plants were grown in controlled environment chambers under high or low irradiance conditions. Thylakoid membranes were isolated from mature leaves. Photosynthetic electron transport was assayed as uncoupled Hill activity using 2,6-dichlorophenolindophenol (DCIP). Soybean electron transport activity was dependent on genotype and growth irradiance and ranged from 6 to 91 mmol DCIP reduced [mol chlorophyll]^–1 s^–1. Soybean plastocyanin pool size ranged from 0.1 to 1.3 mol plastocyanin [mol Photosystem I]^–1. In contrast, barley and spinach electron transport activities were 140 and 170 mmol DCIP reduced [mol chlorophyll]^–1 s^–1, respectively, with plastocyanin pool sizes of 3 to 4 mol plastocyanin [mol Photosystem I]^–1. No significant differences in the concentrations of Photosystem II, plastoquinone, cytochrome b₆f complexes, or Photosystem I were observed. Thus, genetic differences in electron transport activity were correlated with plastocyanin pool size. The results suggested that plastocyanin pool size can vary significantly and may limit photosynthetic electron transport capacity in certain species such as soybean. Soybean plastocyanin consisted of two isoforms with apparent molecular masses of 14 and 11 kDa, whereas barley and spinach plastocyanins each consisted of single polypeptides of 8 and 12 kDa, respectively.Abbreviations DAP days after planting - DCIP 2,6-dichlorophenolindophenol - LiDS lithium dodecyl sulfate - PPFD photosynthetic photon flux density (mol photons m^–2 s^–1) - PS I Photosystem I - PS II Photosystem II - P700 reaction center of Photosystem I The US Government right to retain a non-exclusive, royalty free licence in and to any copyright is acknowledged.