Transmembrane Electron Transport in Plasma Membrane Vesicles Loaded with an NADH-Generating System or Ascorbate

Sugar beet (Beta vulgaris L.) leaf plasma membrane vesicles were loaded with an NADH-generating system (or with ascorbate) and were tested spectrophotometrically for their ability to reduce external, membrane-impermeable electron acceptors. Either alcohol dehydrogenase plus NAD⁺ or 100 millimolar ascorbate was included in the homogenization medium, and right-side-out (apoplastic side-out) plasma membrane vesicles were subsequently prepared using two-phase partitioning. Addition of ethanol to plasma membrane vesicles loaded with the NADH-generating system led to a production of NADH inside the vesicles which could be recorded at 340 nanometers. This system was able to reduce 2,6-dichlorophenolindophenol-3′-sulfonate (DCIP-sulfonate), a strongly hydrophilic electron acceptor. The reduction of DCIP-sulfonate was stimulated severalfold by the K⁺ ionophore valinomycin, included to abolish membrane potential (outside negative) generated by electrogenic transmembrane electron flow. Fe³⁺-chelates, such as ferricyanide and ferric citrate, as well as cytochrome c, were not reduced by vesicles loaded with the NADH-generating system. In contrast, right-side-out plasma membrane vesicles loaded with ascorbate supported the reduction of both ferric citrate and DCIP-sulfonate, suggesting that ascorbate also may serve as electron donor for transplasma membrane electron transport. Differences in substrate specificity and inhibitor sensitivity indicate that the electrons from ascorbate and NADH were channelled to external acceptors via different electron transport chains. Transplasma membrane electron transport constituted only about 10% of total plasma membrane electron transport activity, but should still be sufficient to be of physiological significance in, e.g. reduction of Fe³⁺ to Fe²⁺ for uptake.     

Electron and proton transport across the plasma membrane

Transplasma membrane electron transport in both plant and animal cells activates proton release. The nature and components of the electron transport system and the mechanism by which proton release is activated remains to be discovered. Reduced pyridine nucleotides are substrates for the plasma membrane dehydrogenases. Both plant and animal membranes have unusual cyanide-insensitive oxidases so oxygen can be the natural electron acceptor. Natural ferric chelates or ferric transferrin can also act as electron acceptors. Artificial, impermeable oxidants such as ferricyanide are used to probe the activity. Since plasma membranes containb cytochromes, flavin, iron, and quinones, components for electron transport are present but their participation, except for quinone, has not been demonstrated. Stimulation of electron transport with impermeable oxidants and hormones activates proton release from cells. In plants the electron transport and proton release is stimulated by red or blue light. Inhibitors of electron transport, such as certain antitumor drugs, inhibit proton release. With animal cells the high ratio of protons released to electrons transferred, stimulation of proton release by sodium ions, and inhibition by amilorides indicates that electron transport activates the Na⁺/H⁺ antiport. In plants part of the proton release can be achieved by activation of the H⁺ ATPase. A contribution to proton transfer by protonated electron carriers in the membrane has not been eliminated. In some cells transmembrane electron transport has been shown to cause cytoplasmic pH changes or to stimulate protein kinases which may be the basis for activation of proton channels in the membrane. The redox-induced proton release causes internal and external pH changes which can be related to stimulation of animal and plant cell growth by external, impermeable oxidants or by oxygen.     

Effects of Helminthosporium maydis Race T Toxin on Electron Transport in Susceptible Corn Mitochondria and Prevention of Toxin Actions by Dicyclohexylcarbodiimide

The effect of Helminthosporium maydis race T toxin on electron transport in susceptible cytoplasmic male-sterile Texas corn (Zea mays L.) mitochondria was investigated, using dichlorophenol indophenol and ferricyanide as electron acceptors. Succinate-dependent electron transport was stimulated by the toxin, consistent with the well described increase in membrane permeability induced by the toxin. Malate-dependent electron transport was inhibited. This inhibition of electron transport increased as a function of time of exposure to the toxin. Mitochondria from normal-fertile (N) corn were not affected by the toxin. Both the inhibition of electron transport and the increase in ion permeability, such as dissipation of membrane potential and Ca²⁺ gradients, induced by the toxin in T corn was prevented by N,N′-dicyclohexylcarbodiimide, a hydrophobic carbodiimide. A water-soluble carbodiimide, 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide, was ineffective in preventing dissipation of membrane potential by the toxin. These results suggest that the various toxin actions are mediated via interaction of the toxin with one target site, most probably a 13 kilodalton polypeptide unique to T mitochondria. N,N′-dicyclohexylcarbodiimide may confer protection by modifying an amino acid residue in a hydrophobic portion of the target site.     

Electrochromic absorbance changes of photosynthetic pigments in Rhodopseudomonas sphaeroides. I. Stimulation by secondary electron transport at low temperature

Light-induced absorbance changes were measured at temperatures between ?30 and ?55 °C in chromatophores of Rhodopseudomonas sphaeroides. Absorbance changes due to photooxidation of reaction center bacteriochlorophyll (P-870) were accompanied by a red shift of the absorption bands of a carotenoid. The red shift was inhibited by gramicidin D. The kinetics of P-870 indicated electron transport from the “primary” to a secondary electron acceptor. This electron transport was slowed down by lowering the temperature or increasing the pH of the suspension. Electron transport from soluble cytochrome c to P-870⁺ occurred in less purified chromatophore preparations. This electron transport was accompanied by a relatively large increase of the carotenoid absorbance change. This agrees with the hypothesis that P-870 is located inside the membrane, so that an additional membrane potential is generated upon transfer of an electron from cytochrome to P-870⁺.A strong stimulation of the carotenoid changes (more than 10-fold in some experiments) and pronounced band shifts of bacteriochlorophyll B-850 were observed upon illumination in the presence of artificial donor-acceptor systems. Reduced N-methylphenazonium methosulphate (PMS) and N,N,N′,N′-tetramethyl-p-phenylenediamine (TMPD) were fairly efficient donors, whereas endogenous ubiquinone and oxidized PMS acted as secondary acceptor. These results indicate the generation of large membrane potentials at low temperature, caused by sustained electron transport across the chromatophore membrane. The artificial probe, merocyanine MC-V did not show electrochromic band shifts at low temperature.     

Effects of magnesium and chloride ions on light-induced electron transport in membrane fragments from a blue-green alga

The effects of magnesium and chloride ions on photosynthetic electron transport were investigated in membrane fragments of a blue-green alga, Nostoc muscorum (Strain 7119), noted for their stability and high rates of electron transport from water or reduced dichlorophenolindophenol to NADP⁺. Magnesium ions were required not only for light-induced electron transport from water to NADP⁺ but also for protection in the dark of the integrity of the water-photooxidizing system (Photosystem II). Membrane fragments suspended in the dark in a medium lacking Mg²⁺ lost the capacity to photoreduce NADP⁺ with water on subsequent illumination. Chloride ions could substitute, but less effectively, for each of these two effects of magnesium ions. By contrast, the photoreduction of NADP⁺ by DCIPH₂ was independent of Mg²⁺ (or Cl^?) for the protection of the electron transport system in the dark or during the light reaction proper. Furthermore, high concentrations of MgCl₂ produced a strong inhibition of NADP⁺ photoreduction with DCIPH₂ without significantly affecting the rate of NADP⁺ photoreduction with water. The implications of these findings for the differential involvement of Photosystem I and Photosystem II in the photoreduction of NADP⁺ with different electron donors are discussed.     

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.     

Photochemical activity and components of membrane preparations from blue-green algae. I. Coexistence of two photosystems in relation to chlorophyll a and removal of phycocyanin

Nostoc muscorum (Strain 7119) cells were disrupted and the accessory pigment phycocyanin was removed from membrane fragments by digitonin treatment. The phycocyanin-depleted membrane fragments retained both Photosystem I and Photosystem II activity, as evidenced by high rates of NADP⁺ photoreduction either by water or by reduced 2,6-dichlorophenolindophenol, indicating that phycocyanin is not an essential component for electron transport activity.No separation of the two photosystems was effected by the digitonin treatment. Even drastic digitonin treatments failed to diminish significantly the remarkably stable electron transport from water to NADP⁺.Action spectra and relative quantum efficiency measurements demonstrated the existence of both Photosystem I and Photosystem II in membrane fragments which contained chlorophyll a as the only significant light-absorbing pigment.     

Electron flow at the polarized mercury-water interface in the presence of membrane fragments rich in Na+−K+-Activated ATPase

Summary Measurements of interfacial electron flow indicate that membrane fragments rich in Na⁺–K⁺-ATPase are capable of absorbing and releasing electrons in the form of random currents at an electrode surface. The electron transporting system, which functions in the presence or absence of substrate and activating ions, may be part of or in contact with the enzyme system, but it is not related to the ATPase activity. The observed electron transport at an electrode surface resembles physiological electron transport processes in being reversible, in extending over the same range of potential, and in being affected by some of the chemicals that interfere with electron transport and oxidative phosphorylation in mitochondria. Our experiments do not provide sufficient evidence to identify the substances that are responsible for the random currents, but the results suggest that the electro-active substances are similar to those which are involved in the reactions at the second phosphorylation site in mitochondria. Experiments with this technique provide a new approach to the study of the mechanism of biological electron transport processes and their possible relation to ATP synthesis and hydrolysis.Supported by U.S. Public Health Service Research Career Development Award K3-GM-8158.     

Effect of NADP+ on light-induced cytochrome changes in membrane fragments from a blue-green alga

The effect of NADP⁺ on light-induced steady-state redox changes of membrane-bound cytochromes was investigated in membrane fragments prepared from the blue-green algae Nostoc muscorum (Strain 7119) that had high rates of electron transport from water to NADP⁺ and from an artificial electron donor, reduced dichlorophenolindophenol (DCIPH₂) to NADP⁺. The membrane fragments contained very little phycocyanin and had excellent optical properties for spectrophotometric assays. With DCIPH₂ as the electron donor, NADP⁺ had no effect on the light-induced redox changes of cytochromes: with or without NADP⁺, 715- or 664-nm illumination resulted mainly in the oxidation of cytochrome f and of other component(s) which may include a c-type cytochrome with an α peak at 549 nm. With 664 nm illumination and water as the electron donor, NADP⁺ had a pronounced effect on the redox state of cytochromes, causing a shift toward oxidation of a component with a peak at 549 nm (possibly a c-type cytochrome), cytochrome f, and particularly cytochrome b₅₅₉. Cytochrome b₅₅₉ appeared to be a component of the main noncyclic electron transport chain and was photooxidized at physiological temperatures by Photosystem II. This photooxidation was apparent only in the presence of a terminal acceptor (NADP⁺) for the electron flow from water.     

Facilitated permeation of methyl viologen into chloroplasts in situ during electric pulse generation in excitable plant cell membranes

Generation of action potential (AP) in plasma membranes of characean algae has a strong impact on photoreactions occurring in chloroplasts. Under physiological conditions, AP suppresses electron transport in alkaline and acidic regions, although to a different extent; these changes are transient and reversible. In the presence of the artificial electron acceptor, methyl viologen (MV²⁺), AP-induced changes in electron transport in photosystem II become irreversible. Incubation of Chara corallina internodal cells with MV²⁺ has no effect on the chlorophyll P700 photooxidation kinetics in photosystem I reaction centers, suggesting that MV²⁺ is inaccessible for interactions with photosystem I, because its permeation into chloroplasts of a resting cell is hindered by membrane barriers. At the same time, AP generation in the presence of MV²⁺ is accompanied by irreversible modification of P700 photooxidation kinetics, as can be evidenced from differences in absorption changes at 810 and 870 nm (ΔA ₈₁₀ signals). These findings suggest that MV²⁺ permeation into chloroplasts in situ is facilitated during or after the AP generation. Similar to the ΔA ₈₁₀ signals, light-induced changes in membrane potential do not depend on the presence of MV²⁺ in the external medium until the first excitatory stimulus is applied. Electric photoresponses of the cell are irreversibly modified by AP generated in the presence of MV²⁺ at the expense of non-cyclic photosynthetic electron transport redirected to the MV²⁺ reduction. It is concluded that AP effects on chloroplast photosynthesis in situ are complex and involve permeability changes for MV²⁺ in membrane barriers of the “plasmalemma-chloroplast envelope” system.     

Sugar transport and potassium permeability in yeast plasma membrane vesicles

Plasma membrane vesicles were isolated from homogenised yeast cells by filtration, differential centrifugation and aggregation of the mitochondrial vesicles at pH 4. As judged by biochemical, cell electrophoretic and electron microscopic criteria a pure plasma membrane vesicle preparation was obtained.The surface charge density of the plasma membrane vesicles is similar to that of intact yeast cells with an isoelectric point below pH 3. The mitochondrial vesicles have a higher negative surface charge density in the alkaline pH range. Their isoelectric point is near pH 4.5, where aggregation is maximal.The yield of vesicles sealed to K⁺ was maximal at pH 4 and accounted for about one third of the total vesicle volume.The plasma membrane vesicles demonstrate osmotic behaviour, they shrink in NaCl solutions when loosing K⁺.As in intact yeast cells the entry and exit of sugars like glucose or galactose in plasma membrane vesicles is inhibited by UO₂²⁺.Counter transport in plasma membrane vesicles with glucose and mannose and iso-counter transport with glucose suggests that a mobile carrier for sugar transport exists in the plasma membrane.After galactose pathway induction in the yeast cells and subsequent preparation of plasma membrane vesicles the uptake of galactose into the vesicles increased by almost 100% over the control value without galactose induction. This increase is explained by the formation of a specific galactose carrier in the plasma membrane.     

New approach of monitoring changes in chlorophyll a fluorescence of single guard cells and protoplasts in response to physiological stimuli

A new type of microfluorometer was applied to assess photosynthesis at the single-cell level by chlorophyll fluorescence using the saturation pulse method. A microscopy–pulse amplitude modulation (PAM) chlorophyll fluorometer was combined with a Zeiss Axiovert 25 inverted epifluorescence microscope for high-resolution measurements on single mesophyll and guard cells and the respective protoplasts. Available information includes effective quantum yield of photosystem II, relative electron transport rate and energization of the thylakoid membrane due to the transthylakoidal proton gradient. Dark–light induction curves of guard cell (GCPs) and mesophyll cell protoplasts (MCPs) displayed very similar characteristics, indicating similar functional organization of thylakoid membranes in both types of chloroplasts. Light response curves, however, revealed much earlier saturation of photosynthetic electron flow in GCPs than in MCPs. Under anaerobiosis, photosynthetic electron flow and membrane energization were severely suppressed. A similar effect was observed in guard cells when epidermal peels were incubated with the fungal toxin fusicoccin which activates the plasma membrane H⁺-ATPase and causes irreversible opening of stomata. The drop in electron transport rate was prevented by blocking ATP consumption of the H⁺ pump or by glucose addition. These results show that chlorophyll fluorescence quenching analysis allows profound insights into stomatal physiology.     

Membrane mutation affecting energy-linked functions inEscherichia coli K 12

A small-colony forming variant ofEscherichia coli with a mutation in thenof gene was analysed. The alternation of the protein composition in the cytoplasmic membrane and the interaction with K and E group colicina indicated a membrane mutation. The effect of this mutation on some membrane-bound processes, the activity of Mg²⁺-activated ATPase, the growth on different carbon sources and the active transport of amino acids, is described. This mutation does not exert any effect on the electron transport system.     

Bioelectrical response of the Nitella flexilis cell to illumination: A new possible state of plasmalemma in a plant cell

Transient hyperpolarization of the external cytoplasmatic membrane may be observed on rapid illumination of the Nitella flexilis cell. Several important properties of that response make the latter similar to a considerable degree to the excitation response.The condition for transient hyperpolarization is the normal functioning of the electron transport chain conjugated with non-cyclic photophosphorylation.The value of the membrane potential at the moment of hyperpolarization of the external cytoplasmic membrane, is determined by the difference in the electrochemical potential of HCO₃^? or H⁺. This state of the plasmalemma supplements the two other known states: normal and depolarized (excited), when the main ions determining membrane potential are K⁺ and Cl^?.     

Effect of N,N'-dicyclohexylcarbodiimide (DCCD) on electron transport particles of Mycobacterium phlei

N,N′-dicyclohexylcarbodiimide (DCCD) was found to uncouple phosphorylation from oxidation with succinate and NAD⁺-linked substrates in the system from Mycobacterium phlei. However, in contrast to the effect of this agent in mammalian mitochondria, DCCD was found to stimulate oxidation with succinate as an electron donor and to inhibit the oxidation of NAD⁺-linked substrates. Furthermore, in the M. phlei system DCCD was found to inhibit the membrane bound latent ATP-ase but had no effect on this activity when the latent ATPase was removed from the membrane vesicles. Reconstitution with the fraction containing latent ATPase activity and the membrane vesicles resulted in inhibition of latent ATPase by DCCD. Studies of the effect of DCCD on the resolved system indicated that DCCD may be associated with membrane vesicles or causes secondary changes in conformation of membrane vesicles. Although DCCD inhibited membrane bound ATPase it did not prevent the addition of the solubilized ATPase to the membrane vesicles. DCCD was found to have no effect on purified succinic dehydrogenase activity but stimulated this activity in the electron transport particles.     

Generation of membrane potential during photosynthetic electron flow in chromatophores from Rhodopseudomonas capsulata

1. When cytochrome c₂ is available for oxidation by the photosynthetic reaction centre, the decay of the carotenoid absorption band shift generated by a short flash excitation of Rhodopseudomonas capsulata chromatophores is very slow (half-time approximately 10 s). Otherwise the decay is fast (half-time approximately 1 s in the absence and 0.05 s in the presence of 1,10-ortho-phenanthroline) and coincides with the photosynthetic back reaction.2. In each of these situations the carotenoid shift decay, but not electron transport, may be accelerated by ioniophores. The ionophore concentration dependence suggests that in each case the carotenoid response is due to a delocalised membrane potential which may be dissipated either by the electronic back reaction or by electrophoretic ion flux.3. At high redox potentials, where cytochrome c₂ is unavailable for photo-oxidation, electron transport is believed to proceed only across part of the membrane dielectric. Under such conditions it is shown that the driving force for carbonyl cyanide trifluoromethoxyphenyl hydrazone-mediated H⁺ efflux is nevertheless decreased by valinomycin/K⁺; demonstrating that the [BChl]₂ → Q electron transfer generates a delocalised membrane potential.     

Genomic,Proteomic, and Biochemical Analysis of the Organohalide Respiratory Pathway in Desulfitobacterium dehalogenans

Desulfitobacterium dehalogenans is able to grow by organohalide respiration using 3-chloro-4-hydroxyphenyl acetate (Cl-OHPA) as an electron acceptor. We used a combination of genome sequencing, biochemical analysis of redox active components, and shotgun proteomics to study elements of the organohalide respiratory electron transport chain. The genome of Desulfitobacterium dehalogenans JW/IU-DC1^T consists of a single circular chromosome of 4,321,753 bp with a GC content of 44.97%. The genome contains 4,252 genes, including six rRNA operons and six predicted reductive dehalogenases. One of the reductive dehalogenases, CprA, is encoded by a well-characterized cprTKZEBACD gene cluster. Redox active components were identified in concentrated suspensions of cells grown on formate and Cl-OHPA or formate and fumarate, using electron paramagnetic resonance (EPR), visible spectroscopy, and high-performance liquid chromatography (HPLC) analysis of membrane extracts. In cell suspensions, these components were reduced upon addition of formate and oxidized after addition of Cl-OHPA, indicating involvement in organohalide respiration. Genome analysis revealed genes that likely encode the identified components of the electron transport chain from formate to fumarate or Cl-OHPA. Data presented here suggest that the first part of the electron transport chain from formate to fumarate or Cl-OHPA is shared. Electrons are channeled from an outward-facing formate dehydrogenase via menaquinones to a fumarate reductase located at the cytoplasmic face of the membrane. When Cl-OHPA is the terminal electron acceptor, electrons are transferred from menaquinones to outward-facing CprA, via an as-yet-unidentified membrane complex, and potentially an extracellular flavoprotein acting as an electron shuttle between the quinol dehydrogenase membrane complex and CprA.     

Correlation between thylakoid stacking and cation-induced decrease in photosystem I electron transport at saturating light intensity

A Mg²⁺-induced decrease of the rate of photosystem I (PS I) electron transport (DCIPH₂ → methyl viologen) in thylakoids under saturated light intensities has been reported earlier (S. Bose, J. E. Mullet, G. E. Hoch, and C. J. Arntzen, 1981, Photobiochem. Photobiophys.2, 45–52). A similar effect is observed with Na⁺, although the concentration required for half-maximal inhibition was higher by about two orders of magnitude. The cation effect was gradually abolished as the thylakoids were aged by incubation at 30 °C for 6 h. The loss of cation effect on PS I electron transport rate during aging was parallel to the corresponding loss of cation effect on thylakoid stacking. The cation concentration required for thylakoid stacking and the degree of inhibition as a function of cation concentration correlated strongly with the degree of thylakoid stacking. These observations indicated that the inhibition of the rate of PS I electron transport by cations is a consequence of cation-induced stacking of thylakoid membranes. The observed inhibition of the rate of PS I electron transport is discussed in terms of two hypotheses: (i) a fraction (20–30%) of the PS I complexes is trapped in the appressed region of grana and becomes unavailable to the electron donor (DCIPH₂) and (ii) the membrane structure is altered by the cations in such a manner that the rate constant of electron donation by the donor to the electron transport chain in the thylakoid is decreased.     

Degradation of proteins from thylakoid membranes in senescing wheat leaves at high temperature

This investigation determined whether thylakoid proteins would be degraded more rapidly or not in senescing wheat (Triticum aestivum L. em. Thell.) leaves concurrently exposed to high temperatures. Excised leaves were pulse-labelled with [³⁵S]-methionine for a 12 h period, and then incubated at 22,32 or 42°C for 0, 1, 2, or 3 d, before extracting a thylakoid enriched membrane sample. After electrophoretic separation, two prominent [³⁵S]-labelled protein bands were chosen for further analyses. Band A contained the D-1 thylakoid protein and band B contained thylakoid proteins of the light harvesting complex (LHCII) associated with photosystem II (PSII). Total protein, [³⁵S]-labelled protein, band A protein, and band B protein within the thylakoid enriched membrane samples were measured. Unlabelled thylakoid enriched membrane samples, extracted from leaves given similar treatments, were used to measure uncoupled whole-chain and photosystem II (PSII) electron transport and chlorophyll fluorescence. Accentuated decline in whole-chain and PSII electron transport, increasing F_o values, and decreasing F_max values were a result of high temperature injury in leaves treated at 42°C. None of the thylakoid enriched membrane protein fractions were degraded more rapidly in high-temperature treated leaves. Degradation of the total [³⁵S]-labelled membrane proteins and band B was inhibited by the 42°C treatment. The results indicate that high temperature stress may disrupt some aspects of normal senescence.