Artificial Electron Donors Research Articles

Effects of redox agents on the Ca 2+-activated K + channel

The sensitivity to Ca 2+ of the Ca 2+-dependent K + channel can be increased by the artificial electron donor system ascorbate + phenazine-methosulphate in a variety of animal cells. In the human erythrocyte the shift from the ‘low’ to the ‘high-affinity’ state seems to depend on the reduction of a membrane component accepting 2 electrons and with an standard redox potential (pH 7.5) of about 47 mV. The relevance of this redox modulation under physiological circumstances is unknown at the moment.

Isolation and characterization of an Escherichia coli mutant lacking cytochrome d terminal oxidase.

A screening procedure was devised which permitted the isolation of a cytochrome d-deficient mutant by its failure to oxidize the artificial electron donor N,N,N',N'-tetramethyl-p-phenylenediamine. Cytochrome a1 and probably cytochrome b558 were also missing in the mutant. Growth and oxygen uptake rates were similar for both parent and mutant strains. However, the strain lacking cytochrome d had an increased sensitivity to cyanide, indicating that cytochrome d confers some resistance to this respiratory inhibitor. The gene responsible for these phenotypes has been named cyd and maps between tolA and sucB.

The distribution of redox equivalents in the anaerobic respiratory chain of Paracoccus denitrificans.

The branching of the electron flow to individual teminal acceptors NO3−, NO2− and N2O was investigated in denitrifying cells of Paracoccus denitrificans. It was found that the extent of electron flow to nitrate depends on the activity of electron flow through the terminal part of the respiratory chain comprising cytochrome c.NO2− and N2O either endogenously formed or exogenously added decreased the rate of nitrate reduction by oxidizing the respiratory chain via nitrite reductase and nitrous oxide reductase. The inhibitory effect of both intermediates of denitrification was released by antimycin. The rate of the first reaction of the sequence NO3−→ NO2−→ N2O → N2 is in the type of cells used under feedback redox control exerted by concentration of intermediates. This regulation can be designated as ‘inhibition by product via respiratory chain’.The reaction rate of nitrate denitrification could also be influenced by changing the portion of electron flow from substrate to cytochrome c by artificial electron acceptors and donors. By varying the concentration of N,N,N′,N′‐tetramethyl‐p‐phenylenediamine in the presence of ascorbate, the desired redox state of cytochrome c and consequently various rates of nitrate reductase activity could be established. The basis of the enhancing effect of dithionite on the rate of nitrate reduction could also be explained by its functioning as an electron donor for the terminal part of respiratory chain.The quantitative evalution of the extent of electron flow revealed that both branches of the respiratory chain (to nitrate reductase and to cytochrome c) compete for a constant limited flow of redox equivalents supplied from dehydrogenases. A strong evidence exists that the same mechanism is implied in the inhibition of nitrate reduction brought about by oxygen.

Elaboration of mitochondrial function during Trypanosoma brucei differentiation

The development of various mitochondrial functions has been studied using an in vitro system in which Trypanosoma brucei LUMP 1026 bloodstream trypomastigotes differentiate to procyclic trypomastigotes. During differentiation, antimycin A sensitive respiration begins to develop prior to cyanide sensitive respiration, with sensitivity levels equivalent to established procyclic trypomastigotes attained by 20–24 days in culture. In the early stage of differentiation, inhibitor sensitivity is dependent upon the order of inhibitor addition to the cells. Stimulation of oxygen uptake by the artificial electron donor ascorbate (plus tetramethyl- p-phenylenediamine as carrier) closely parallels the development of cyanide sensitivity. Cytochrome aa 3 is not observed spectroscopically until nine days in culture. Specific activities of two mitochondrial enzymes, succinate dehydrogenase and succinate cytochrome c reductase, increase from nil in bloodstream trypomastigotes to significant levels in established procyclic trypomastigotes by 24–30 days in culture.

Isolation and characterisation of metribuzin-resistant Chlamydomonas reinhardii cells

Chlamydomonas reinhardii cells were treated with 5-fluorodeoxyuridine and ethylmethanesulfonate to induce mutagenesis. The mutant cells were analyzed for resistance against metribuzin (4-amino-6-( t-butyl)-3-methylthio-1,2,4-triazine-5-one). Clones with normal growth were isolated and the mutant cells further characterized. The photosynthetic rates of the mutant cells were about 20% lower than those of wild-type cells. The mutant cells were not only resistant against metribuzin (p I 50 lowered from 6.65 to 3.41) but also against bromacil, atrazine, phenisopham and tolerant against 3-(3,4-dichlorophenyl)-1,1-dimethylurea. However, the mutant was more susceptible to phenolic electron-transport inhibitors like bromonitrothymol, ioxynil and i-dinoseb. 2,4-Dinitrophenyl-2′-iodo-3′-methyl-4′-nitro-6′-isopropyl phenyl ether inhibited the wild-type thylakoids more than the mutant. The analysis of the electron transport with artificial electron donors and acceptors showed that only Photosystem II was affected by the mutation and not Photosystem I. Binding experiments with isolated thylakoids of resistant and susceptible cells using [ 14C]metribuzin and [ 3H]-i-dinoseb revealed that metribuzin did not bind specifically to the thylakoids of the mutant cells, but that i-dinoseb did bind to the thylakoids of the mutant, and even better than to the thylakoids of the wild-type cells. Fluorescence studies confirmed these results.

Formation of a Membrane Potential by Reconstituted Liposomes Made with Cytochrome b562-o Complex, a Terminal Oxidase of Escherichia coli*

A terminal oxidase of the Escherichia coli K12 respiratory chain (cytochrome b562-o complex) was reconstituted into liposomes by freeze-thaw/sonication method. Formation of a membrane potential (-145 mV) by the reconstituted cytochrome b562-o complex was observed with the fluorescent dye 3,3'-dipropylthiodicarbocyanine iodide on addition of an artificial electron donor ubiquinol-1 or ascorbate-phenazine methosulfate. The membrane potential formed was inhibited by the protonophore uncouplers 3,5-di-tert-butyl-4-hydroxybenzylidenemalononitrile, carbonyl cyanide p-trifluoromethoxyphenylhydrazone, and carbonylcyanide m-chlorophenylhydrazone, and the inhibitors of the oxidase system zinc sulfate, potassium cyanide, and 2-n-heptyl-4-hydroxyquinoline-N-oxide. This is the first indication that there is a coupling site in an E. coli terminal oxidase, which consists of b-type cytochromes.

EPR-detectable magnetically interacting manganese ions in the photosynthetic oxygen-evolving system after continuous illumination

Freezing of spinach or barley chloroplasts during continuous illumination results in the trapping of a paramagnetic state or a mixture of such states characterized by a multiline EPR spectrum. Added Photosystem II electron acceptor enhances the signal intensity considerably. Treatments which abolish the ability of the chloroplasts to evolve oxygen, by extraction of the bound manganese, prevent the formation of the paramagnetic species. Restoration of Photosystem II electron transport in inhibited chloroplasts with an artificial electron donor (1,5-diphenylcarbazide) does not restore the multiline EPR spectrum. The presence of 3-(3,4-dichlorophenyl)-1, 1-dimethylurea (DCMU) results in a modified signal which may represent a second paramagnetic state. The paramagnetic forms appear to originate on the donor side in Photosystem II and are dependent on a functional oxygenevolving site and bound, intact manganese. It is suggested that magnetically interacting manganese ions in the oxygen-evolving site may be responsible for the EPR signals. This suggestion is supported by calculations.

Effect of 6-Benzyl Aminopurine on the Stabilization of Absorption Spectrum and Hill Activity of Isolated Chloroplasts

Summary Beet spinach chloroplasts isolated in the presence or absence of BAP (6-benzyl aminopurine) were suspended in sugar or salt medium and were allowed to age in light (1,000 JM -2 s -1 ). Loss in the Hill activity (H 2 0 to ferricyanide (Fe (CN 6 ) 3- ) due to light-induced ageing could be partly protected by the inclusion of the hormone (BAP). The extent and pattern of protection of chloroplast function by the hormone is similar for both salt and sugar media. During light-induced ageing, chloroplasts also lose the capacity to use the artificial electron donor DPC (diphenyl carbazide) for photoreduction of DCPIP. This loss is fast er for chloroplasts suspended in salt medium than for chloroplasts incubated in sugar medium. BAP could not provide any protection for this loss in DPC supported DCPIP photoreduction ability of chloroplasts. Loss in chlorophyll contents during light induced ageing was found to be negligible. However, absorption spectrum for chloroplasts showed changes in t he blue to red peak ratio upon ageing in light. This was found to be more pronounced in chloroplasts incubated in salt medium wih or without BAP treatment than in chloroplasts incubated in sugar medium. Accompanied this change in peak ratio, there is a small blue shift (1-2 nm) in the position of red absorption peak. BAP seems to arrest this shift.

Photosynthetic electron transport by Zwittergent-extracted chloroplasts requires solubilized plastocyanin

Evidence from numerous laboratories had demonstrated that light plays a regulatory role in photosynthesis through the modulation of key enzymes [1-3]. One system that has been proposed to account for light-dependent enzyme modulation involves membrane-bound cyst(e)ine-containing proteins, designated 'light effect mediators' or 'LEMs'-components proposed to occur in the oxidized (disulfide) state in the dark and in the reduced (sulfhydryl) state in the light [2,4]. It is visualized that chloroplasts contain two LEMs (both on the acceptor side of photosystem I) that, following reduction by non-cyclic electron transport, modulate enzymes of the stroma. One of the proposed LEM components can be solubilized from peas by extracting chloroplast membranes with a Zwittergent detergent [5,6]. In the presence of an artificial electron donor, ascorbate/ 2,6-dichlorophenolindophenol (DCPIP), the Zwittergent-treated chloroplast membranes promoted the photoregulation of NADP-malate dehydrogenase of the stroma. Photoregulation in this system was dependent on the solubilized protein fraction that, based on SDS gel electrophoresis, contained several protein components. In experiments designed to determine the effect of Zwittergent extraction on enzyme photoregulation by systems under investigation in our laboratory (i.e., the ferredoxin/thioredoxin and ferralterin systems

Bacterial cell envelopes with functional flagella.

Our aim was to isolate from bacteria a flagellated, subcellular system whose content could be changed at will. Because the control of bacterial chemotaxis resides in the direction of rotation of the flagella, such a system would be ideal for the study of this control mechanism. By incubating bacteria with penicillin and then lysing them osmotically, we were able to isolate cell envelopes from Escherichia coli and Salmonella typhimurium. These envelopes have the same sidedness and similar shape and dimensions as the original bacteria; they are practically free of cytoplasm; they are osmotically sensitive, having intact the cytoplasmic membrane and at least part of the cell wall; and they have flagella. This preparation was used to find out what is required to restore flagellar rotation, which had been lost during osmotic lysis. By visualizing the image of individual flagella with high intensity light microscopy or by tethering the cell envelopes, we found that adding artificial electron donors as an energy source is enough to restore rotation. This seems to indicate that no cytoplasmic components are required and that the proton electrochemical potential is indeed the driving force for flagellar rotation. However, the rotation was almost entirely counterclockwise, while in intact bacteria the flagella rotate in both directions. This may indicate that a cytoplasmic component is required to allow clockwise rotation. The significance of these results for the study of chemotaxis is discussed.

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.

Inhibition of electron flow and energy transduction in isolated spinach chloroplasts by the herbicide dinoseb

The mode of action of dinoseb (2-sec-butyl-4-6-dinitrophenol) on chloroplast reactions was studied. Electron flow from water or from an artificial electron donor, diphenylcarbazide (DPC), to dichlorophenol indophenol (DCPIP) was inhibited at low concentrations of the herbicide (5–10 μM) suggesting a site for dinoseb inhibition at the oxidizing side of photosystem II (PS II). Ferricyanide photoreduction was also inhibited by dinoseb. Cyclic and non-cyclic photophosphorylation and Mg 2+-ATPase activity were inhibited by dinoseb, which indicates that this herbicide also acts as an energy transfer inhibitor. Among the above mentioned activities, non-cyclic photophosphorylation was the most sensitive to the inhibition by dinoseb. Ca 2+-ATPase activity of solubilized heat activated chloroplast coupling factor 1 (CF 1) was stimulated by dinoseb. However, the same activity was inhibited in chloroplasts, which perhaps reflect a difference in the mode of interaction of dinoseb with solubilized and membrane bound coupling factor.

Sodium-ion stimulated amino acid uptake in membrane vesicles of alkalophilic Bacillus no. 8-1.

Membrane vesicles were isolated from alkalophilic Bacillus No. 8-1, and the active transport of amino acids was studied. The transport of amino acids was dependent upon substrate oxidation and the presence of Na+. Concentrative uptake of amino acids was stimulated by the addition of an artificial electron donor system, ascorbate-phenazine methosulfate (PMS), and to a lesser extent by NADH, while succinate, L-lactate, and alpha-glycerol-phosphate did not stimulate the uptake. N,N,N',N'-Tetramethyl-p-phenylenediamine (TMPD) and cytochrome c were able to replace PMS, and reduced forms of these compounds were also very efficient electron donors. Amino acid transport was dependent on electron transfer, and inhibition of NADH oxidation by cyanide, 2-heptyl-4-hydroxyquinoline-N-oxide (HOQNO), and sodium azide directly prohibited serine transport. The pH optima for serine transport lay between pH 8 and 9 for all energy sources. Sodium ion stimulated serine transport in the presence of NADH, NADH plus cytochrome c or succinate plus PMS, but had no stimulatory effect on the corresponding dehydrogenase activities. Sodium ion was also required for accumulation of serine in response to an artificial membrane potential where the respiratory chain was not operative. These results indicated that the stimulatory effect of Na+ on amino acid uptake was on the transport process itself.

Respiratory properties of cytochrome-c-deficient mutants of Azotobacter vinelandii.

Cytochrome-c-deficient mutants of Azotobacter vinelandii have been isolated following mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine. These mutants grow well under nitrogen-fixing conditions and studies of the physiology and energy conservation efficiency show no apparent differences from those of the parent strain. Under oxygen-limited growth conditions, the growth rate of the cytochrome-c-deficient mutant was slightly slower (approx. 15%) than that of the parent strain. Cytochromes of the c-type are required for the oxidation of artificial electron donors such as reduced N,N,N',N'-tetramethyl-p-phenylenediamine [Ph(NMe2)2]. This study could not demonstrate a physiological role for the c-type cytochromes which supports the idea that the minor Ph(NMe2)2-oxidizing pathway of the electron transport chain may be independent of the major pathway terminated by cytochrome d.

Purification and some properties of l-ascorbic acid-specific peroxidase in Euglena gracilis z

l-Ascorbic acid-specific peroxidase was purified approximately 280-fold from a cell extract of Euglena gracilis. l-Ascorbic acid was virtually the only natural electron donor; the activity with glutathione was less than 1.1% of that with l-ascorbic acid. Cytochrome c, NADH, NADPH, palmitic acid, and triose reductone did not donate electrons to this enzyme. Among artificial electron donors d-araboascorbic acid and pyrogallol had considerable activities but guaiacol, iodide, and reductic acid showed only weak activities. The Euglena peroxidase showed an oxidase activity only with triose reductone. The enzyme had a molecular weight of about 76,000 as estimated from gel filtration data. The K m values of the enzyme for l-ascorbic acid and hydrogen peroxide were 0.41 and 0.056 m m, respectively, while those for pyrogallol and hydrogen peroxide were 8.6 and 0.22 m m, respectively. These results indicate that the Euglena peroxidase belongs to a new type of peroxidase to be designated as l-ascorbic acid peroxidase. The inhibition of the enzyme by cyanide and azide as well as absorption spectra of the enzyme alone and in complex with hydrogen peroxide showed that it is a hemoprotein. Organic hydroperoxides were also reduced by the Euglena peroxidase. Its physiological function to decompose lipid peroxides as well as hydrogen peroxide generated in cells for the protection of cellular integrity was discussed.

Photosynthetic activities of cadmium‐treated tomato plants

AbstractTomato plants (Lycopersicum esculentum Mill. cv. Moneymaker) grown on nutrient medium containing cadmium exhibit reduced net photosynthesis and reduced contents of chlorophyll and accessory pigments. In chloroplasts isolated from cadmiumtreated plants photosystem II activity, as measured by 2,6‐dichlorophenolindophenol photoreduction, and photosystem II + I activity (H2O → methyl viologen) were both inhibited to about 60%. When 1,5‐diphenylcarbazide was used as artificial electron donor, no significant cadmium effect was observed. Photosystem I activity was not affected by cadmium. The fine structure of chloroplasts in cadmium‐treated plants was degenerated, similarly to senescence response. The principal symptom of cadmium action was the occurrence of large plastoglobules and a disorganization of the lamellar structure, mainly grana stacks. Transfer of cadmium‐treated plants into a medium with increased manganese level caused grana stacking and restoration of photosystem II activity.

A mutant defective in electron transfer to nitrate in Escherichia coli K12.

A mutant of Escherichia coli K12 is described which is unable to reduce nitrate with a variety of physiological electron donors but which retains nitrate reductase activity with the artificial electron donor benzyl viologen. It is suggested that the affected gene, chlI, located close to chlC, encodes the cytochrome bNR apoprotein.

Trichloroethylene: Its interaction with hepatic microsomal cytochrome P-450 in vitro

The effects of inducing agents on the binding and metabolism of trichloroethylene by hepatic microsomal cytochrome P-450 are reported. The binding constant ( K s) for the interaction of trichloroethylene with hepatic microsomal cytochrome P-450 was not altered by induction with phenobarbital, 3-methylcholanthrene or spironolactone, while the maximum extent of binding ( ΔA max) was increased only following phenobarbital induction. The K s values ( ca. 1 mM) obtained for the binding of trichloroethylene to cytochrome P-450 were similar whether the enzyme was partially purified or an integral part of hepatic microsomes. The Michaelis constant ( K m) for the production of chloral hydrate from trichloroethylene by hepatic microsomal cytochrome P-450 was not altered by induction of different forms of cyfochrome P-450. V max for the production of chloral hydrate and the rate of hepatic microsomal NADPH oxidation in the presence of excess trichloroethylene were increased by phenobarbital induction, but not by spironolactone or 3-methylcholanthrene induction. The artificial electron donors NaClO 2 and H 2O 2, but not NaIO 4, supported the metabolism of trichloroethylene by partially purified cytochrome P-450 from phenobarbital-induced rat liver microsomes. Incubation of hepatic microsomes with NADPH and trichloroethylene resulted in decreased levels of cytochrome P-450 and heme, but did not alter the levels of NADPH-cytochrome c reductase, cytochrome b 5 or glucose-6-phosphatase. The degradation of the heme moiety of cytochrome P-450 by trichloroethylene was not supported by NADH and was not inhibited by reduced glutathione (GSH). The inhibitors of cytochrome P-450—SKF 525-A, metyrapone and CO—inhibited the binding and metabolism of trichloroethylene and the trichloroethylenemediated degradation of cytochrome P-450. It is concluded that the form of cytochrome P-450 which is induced by phenobarbital, binds and metabolizes trichloroethylene, whereas other forms of the enzyme, such as cytochrome P-448, do not. Trichloroethylene appears to be activated by the phenobarbital-induced form of cytochrome P-450 to a reactive species which can then chemically alter the heme moiety of cytochrome P-450.

Development of photosynthetic electron transport in Pinus silvestris

AbstractPhotosynthetic electron transport activity has been measured in chloroplasts isolated from dark‐grown seedlings of Pinus silvestris L. and in chloroplasts isolated from seedlings subjected to illumination for periods of up to 48 h. Activities of photosystem 2, photosystem 1 and photosystem 2 plus 1 have been measured. Chloroplasts isolated from dark‐grown seedlings showed significant electron transport activity through both photosystems and through the entire electron transport chain from water to NADP. Illumination of the seedlings for only 5 min markedly promoted photosystem 2 activity. The artificial electron donor, diphenylcarbazide. promoted activity in chloroplasts from dark‐grown seedlings and in chloroplasts from seedlings illuminated for up to 30 min. In comparison to photosystem 2 and overall electron transport from water to NADP, photosystem 1 activity increased only slightly during illumination. Measurements of electron transport and fluorescence kinetics have confirmed that photosynthetic electron transport capacity is limited on the water splitting side of photosystem 2 in dark‐grown seedlings, whereas the primary and secondary electron acceptors of photosystem 2 are fully synthesized and functioning in darkness.Polyethylene glycol must be used as a protective agent when isolating photoactive chloroplasts from secondary needles of conifers. However, the presence of polyethylene glycol, when isolating chloroplasts from dark‐grown pine cotyledons, caused a total inhibition of the activity of photosystem 2. The failure of others to show a substantial electron transport activity in chloroplasts from dark‐grown Pinus silvestris might depend on their use of polyethylene glycol in the preparation medium and/or on their use of suboptimal reaction conditions for the electron transport measurements.

Rate-limiting step in oxidation of physiological and artificial reductants by Azotobacter vinelandii membrane vesicles

The respiration of Azotobacter vinelandii membrane vesicles was investigated in order to determine the partial rates of electron fluxes at each segment of its branched respiratory chain. It is concluded that under physiological conditions only 20 to 30% of the total flux is carried through the c 4, c 5 → a 1, o chain. Steady state analysis indicates that the limited capacity of the chain is due to the slow rate of oxidation of the cytochromes c by the a 1, o oxidases. This rate-limiting step is bypassed by the artificial electron donors, ascorbate-2,6-dichlorophenol indophenol and ascorbate- N,N,N′,N′-tetramethyl- p-phenylenediamine, which directly reduce the highly active a 1, o oxidases. During the oxidation of ascorbate- N,N,N′,N′-tetramethyl- p-phenylenediamine by the membrane vesicles, an accumulation of oxidized N,N,N′,N′-tetramethyl- p-phenylenediamine occurs. Such accumulation of positively charged molecules should lead to a generation of a membrane potential. This fact and previous data concerning coupling site III of A. vinelandii are discussed.