Electron Transport Kinetics Research Articles

THE CHROMOPHORES AS ENDOGENOUS SENSITIZERS INVOLVED IN THE PHOTOGENERATION OF SINGLET OXYGEN IN SPINACH THYLAKOIDS

Abstract— The photogeneration of singlet oxygen (1O2) from thylakoids and the chromophores involved as endogenous sensitizers were investigated using chloroplasts and thylakoids isolated from spinach. The blue light‐induced inhibition kinetics of photosynthetic electron transport and that of CTvCF, ATPase were also studied. The spectral dependence of the generation of 1O2 from thylakoid membranes, measured by the imidazole plus RNO method, clearly demonstrated that the Fe‐S centers play an important role in 1O2 generation, acting as sensitizers in thylakoids. The photoinhibition of the electron transport in isolated chloroplasts was strikingly depressed by a lipid‐soluble '02 quencher and enhanced by deuterium oxide substitution, indicating that the inhibition processes are mainly mediated by 1O2 which is produced via photodynamic activation. The involvement of chloroplast cytochromes in the production of 1O2 was deduced from the action spectrum for the photodynamic inhibition of the electron carrier chain. The results obtained from the kinetic studies appear consistent with the involvement of some components such as the Fe‐S centers and cytochrome chromophores of the carrier chain in the generation of 1O2.

Simulation of canopy photosynthesis in maize and soybean

In a detailed crop growth model, canopy photosynthetic rates within a changing environment should be accurately estimated. Such estimates require that irradiance distribution through the canopy be calculated so that leaf photosynthetic rates may be estimated and summed for the canopy. Here, a calculation of the distribution and intensity of direct and diffuse irradiance within horizontal layers through corn and soybean canopies is developed from field measurements of irradiance, leaf angle distribution, canopy leaf area and estimated solar angle. The results of these calculations are used as inputs to a leaf photosynthesis model based upon laboratory determinations of carboxylase-oxygenase and electron transport kinetics. The photosynthesis rates estimated in this model are summed over the canopy to estimate canopy gross photosynthetic rates, then adjusted to net photosynthesis by subtracting an estimate of canopy respiration derived from literature coefficients. Comparisons between model estimates and field observations were within 10%, often 5%, when net photosynthesis was between 0 and 75 μmol m −2 s −1 for maize and between −5 and 25 μmol m −2 s −1 for soybean. The model tended to overestimate net photosynthesis when using factors from the literature to calculate diffuse photosynthetically active irradiance from diffuse total irradiance, suggesting that these factors are too high. Also, measured versus modelled canopy net photosynthesis were compared when the irradiance distribution through the canopy was simulated by a simple extinction coefficient while using the same model for leaf photosynthesis. With this substitution, estimated gross photosynthesis was often less than measured net photosynthesis, due possibly to an underestimation of photosynthesis from diffuse irradiance. Accounting for the interception of diffuse irradiance is suggested when irradiance distribution is computed as a simple extinction coefficient.

Reconstitution of energy transfer and electron transfer between solubilised pigment-protein complexes from thylakoid membranes. The role of acyl lipids

Solubilisation of thylakoid membranes from young leaves of Pisum sativum in the presence of Triton X-100 resulted in an almost complete loss of quenching of light-harvesting chlorophyll-protein (LHCP) fluorescence, as measured at 77°K. There were concomitant changes in the kinetics of light-saturation curves of electron transport from 2,6-dichlorophenolindophenol/ascorbate to methyl viologen. These effects were accompenied by a physical dissociation of LHCP polypeptides from photosystem I (PSI) and photosystem II (PSII) polypeptides, as determined by polyacrylamide gel-electrophoresis. Detergent-dialysis in the presence of exogenous purified galactolipids, about 80% of which were linoleoyl molecular species, only partially reversed these effects. However, detergent-dialysis using the phospholipids, phosphatidylglycerol and phosphatidylcholine, resulted in the substantial restoration of 77°K fluorescence quenching and the restoration of both emission spectra and electron transport kinetics of both Photosystems I and II that were typical of native membranes.

Kinetics of the oxygen-evolving complex in salt-washed photosystem II preparations

The kinetics of flash-induced electron transport were investigated in oxygen-evolving Photosystem II preparations, depleted of the 23 and 17 kDa polypeptides by washing with 2 M NaCl. After dark-adaptation and addition of the electron acceptor 2,5-dichloro- p-benzoquinone, in such preparations approx. 75% of the reaction centers still exhibited a period 4 oscillation in the absorbance changes of the oxygen-evolving complex at 350 nm. In comparison to the control preparations, three main effects of NaCl-washing could be observed: the half-time of the oxygen-evolving reaction was slowed down to about 5 ms, the misses and double hits parameters of the period 4 oscillation had changed, and the two-electron gating mechanism of the acceptor side could not be detected anymore. EPR-measurements on the oxidized secondary donor Z + confirmed the slower kinetics of the oxygen-releasing reaction. These phenomena could not be restored by readdition of the released polypeptides nor by the addition of CaCl 2, and are ascribed to deleterious action of the highly concentrated NaCl. Otherwise, the functional coupling of Photosystem II and the oxygen-evolving complex was intact in the majority of the reaction centers. Repetitive flash measurements, however, revealed P +Q − recombination and a slow Z + decay in a considerable fraction of the centers. The flash-number dependency of the recombination indicated that this reaction only appeared after prolonged illumination, and disappeared again after the addition of 20 mM CaCl 2. These results are interpreted as a light-induced release of strongly bound Ca 2+ in the salt-washed preparations, resulting in uncoupling of the oxygen-evolving system and the Photosystem II reaction center, which can be reversed by the addition of a relatively high concentration of Ca 2+.

Comparative study on the kinetics of electron transport and the slow chlorophyll fluorescence changes in bean leaves

Comparative study on the kinetics of electron transport and the slow chlorophyll fluorescence changes in bean leaves

Photosynthetic electron transport in relation to thylakoid membrane composition and organization

Abstract. A review is given of the organization and properties of thylakoid membrane proteins and lipids as a basis for understanding the factors which regulate the light reactions of photosynthesis. Particular emphasis is placed on the lateral organization of the major intrinsic multipeptide complexes and on the importance of diffusional processes in controlling the kinetics of electron transport and the distribution of light energy between photosystems 1 and 2.

Photoinhibition of photosynthesis: effect on chlorophyll fluorescence at 77K in intact leaves and in chloroplast membranes of Nerium oleander

The effect of exposing intact leaves and isolated chloroplast membranes of Nerium oleander L. to excessive light levels under otherwise favorable conditions was followed by measuring photosynthetic CO2 uptake, electron transport and low-temperature (77K=-196°C) fluorescence kinetics. Photoinhibition, as manifested by a reduced rate and photon (quantum) yield of photosynthesis and a reduced electron transport rate, was accompanied by marked changes in fluorescence characteristics of the exposed upper leaf surface while there was little effect on the shaded lower surface. The most prominent effect of photoinhibitory treatment of leaves and chloroplasts was a strong quenching of the variable fluorescence emission at 692 nm (Fv,692) while the instantaneous fluorescence (Fo,692) was slightly increased. The maximum and the variable fluorescence at 734 nm were also reduced but not as much as FM,692 and Fv,692. The results support the view that photoinhibition involves an inactivation of the primary photochemistry of photosystem II by damaging the reaction-center complex. In intact leaves photoinhibition increased with increased light level, increased exposure time, and with decreased temperature. Increased CO2 pressure or decreased O2 pressure provided no protection against photoinhibition. With isolated chloroplasts, inhibition of photosystem II occurred even under essentially anaerobic conditions. Measurements of fluorescence characteristics at 77K provides a simple, rapid, sensitive and reproducible method for assessing photoinhibitory injury to leaves. The method should prove especially useful in studies of the occurrence of photoinhibition in nature and of interactive effects between high light levels and major environmental stress factors.

Characteristics of the Photosystem II reaction centre. II. Electron donors

The properties of Photosystem II electron donation were investigated by EPR spectrometry at cryogenic temperatures. Using preparations from mutants which lacked Photosystem I, the main electron donor through the Photosystem II reaction centre to the quinone-iron acceptor was shown to be the component termed Signal II. A radical of 10 G line width observed as an electron donor at cryogenic temperatures under some conditions probably arises through modification of the normal pathway of electron donation. High-potential cytochrome b-559 was not observed on the main pathway of electron donation. Two types of PS II centres with identical EPR components but different electron-transport kinetics were identified, together with anomalies between preparations in the amount of Signal II compared to the quinone-iron acceptor. Results of experiments using cells from mutants of Scenedesmus obliquus confirm the involvement of the Signal II component, manganese and high-potential cytochrome b-559 in the physiological process leading to oxygen evolution.

Biophotolytic membranes: Simplified kinetic model of photosynthetic electron transport

The ability to isolate and stabilize thylakoid membranes from photosynthetic organisms (cyanobacteria, green algae, or higher plants) facilitates their study as photocatalysts in processes for the production of hydrogen from water using solar energy (biophotolysis). The feasibility of immobilizing photosynthetic membranes with retention of their electron transport capability was explored in this study. The method of immobilizing chloroplast thylakoid membranes which was observed to best preserve their photochemical activity involved the following steps: 1. (1) mixing an aqueous suspension of thylakoid membranes (isolated from Spinacia oleracea) with an aqueous suspension of 1.5% (w/v) collagen; 2. (2) adding glutaraldehyde to a concentration of 0.01% (w/v) and cross-linking for 20 min at 0 °C; 3. (3) casting the collagen-thylakoid mixture on a flat surface; and 4. (4) freeze-drying the cast mixture to form a macroporous collagen-thylakoid film composite. Films prepared by this immobilization technique had a very open, sponge-like fibrous structure with a bulk density of 0.049 g/cm 3 of film. Photosynthethic electron transport activities of free and immobilized thylakoid membranes were determined by measuring the photoproduction of oxygen with a Clark-type polarographic probe using potassium ferricyanide as the electron acceptor (Hill reaction). A simplified kinetic model of the photosynthetic electron transport system (PETS) was formulated based on a reaction scheme which is consistent with current concepts of photosynthesis. The kinetic model relating electron transport rate (O 2 evolution) to incident light intensity is a rectangular hyperbolic function, in agreement with the observed behavior of both immobilized The photocatalytic properties of the collagen-thylakoid film were characterized with respect to electron transport kinetics. A simplified kinetic model of the photosynthetic electron transport system was formulated based on a reaction scheme which is consistent with the current concept of photosynthesis. The model accounts for the significant features of photosynthetic electron transport and yet has sufficient simplicity to be useful in the eprocess engineering of photosynbetic reactor systems. The dependence of initial reaction rates on incident light intensity predicted by the kinetic model was experimentally verified for both free and immobilized thylakoid membranes. Initial-rate data were used to evaluate the parameters of the kinetic model. Future kinetic studies might fruitfully focus on dissolved O 2 catalyst-poisoning effects, as well as photoinactivation and/or photoinhibition effects.

On the mechanism of photosynthetic electron transfer in Rhodopseudomonas capsulata and Rhodopseudomonas sphaeroides

(1) Current models for the mechanism of cyclic electron transport in Rhodopseudomonas sphaeroides and Rhodopseudomonas capsulata have been investigated by observing the kinetics of electron transport in the presence of inhibitors, or in photosynthetically incompetent mutant strains. (2) In addition to its well-characterized effect on the Rieske-type iron sulfur center, 5-( n-undecyl)-6-hydroxy-4,7-dioxobenzothiazole (UHDBT) inhibits both cytochrome b 50 and cytochrome b −90 reduction induced by flash excitation in Rps. sphaeroides and Rps. capsulata. The concentration dependency of the inhibition in the presence of antimycin (approx. 2.7 mol UHDBT/mol reaction center for 50% inhibition of extent) is very similar to that of its inhibition of the antimycin-insensitive phase of ferricytochrome c re-reduction. UHDBT did not inhibit electron transfer between the reduced primary acceptor ubiquinone (Q ⨪ I) and the secondary acceptor ubiquinone (Q II) of the reaction center acceptor complex. A mutant of Rps. capsulata, strain R126, lacked both the UHDBT and antimycin-sensitive phases of cytochrome c re-reduction, and ferricytochrome b 50 reduction on flash excitation. (3) In the presence of antimycin, the initial rate of cytochrome b 50 reduction increased about 10-fold as the E h(7.0) was lowered below 180 mV. A plot of the rate at the fastest point in each trace against redox potential resembles the Nernst plot for a two-electron carrier with E m(7.0) ≈ 125 ± 15 mV. Following flash excitation there was a lag of 100–500 μs before cytochrome b 50 reduction began. However, there was a considerably longer lag before significant reduction of cytochrome c by the antimycin-sensitive pathway occurred. (4) The herbicide ametryne inhibited electron transfer between Q ⨪ I and Q II. It was an effective inhibitor of cytochrome b 50 photoreduction at E h(7.0) 390 mV, but not at E h(7.0) 100 mV. At the latter E h, low concentrations of ametryne inhibited turnover after one flash in only half of the photochemical reaction centers. By analogy with the response to o-phenanthroline, it is suggested that ametryne is ineffective at inhibiting electron transfer from Q ⨪ I to the secondary acceptor ubiquinone when the latter is reduced to the semiquinone form before excitation. (5) At E h(7.0) > 200 mV, antimycin had a marked effect on the cytochrome b 50 reduction-oxidation kinetics but not on the cytochrome c and reaction center changes or the slow phase III of the electrochromic carotenoid change on a 10-ms time scale. This observation appears to rule out a mechanism in which cytochrome b 50 oxidation is obligatorily and kinetically linked to the antimycin-sensitive phase of cytochrome c reduction in a reaction involving transmembrane charge transfer at high E h values. However, at lower redox potentials, cytochrome b 50 oxidation is more rapid, and may be linked to the antimycin-sensitive reduction of cytochrome c. (6) It is concluded that neither a simple linear scheme nor a simple Q-cycle model can account adequately for all the observations. Future models will have to take account of a possible heterogeneity of redox chains resulting from the two-electron gate at the level of the secondary quinone, and of the involvement of cytochrome b −90 in the rapid reactions of the cyclic electron transfer chain

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.

Mitrochondrial NADH dehydrogenase in cystic fibrosis.

We have shown that skin fibroblast from patients with cystic fibrosis (CF) and from carriers for CF [heterozygotes (HZ)] consume more O2 than do their controls. When the mitochondrial electron transport inhibitor rotenone was added to the cells, the relative inhibition of O2 consumption was CF greater than HZ greater than controls (P less than 0.005 in both comparisons). Because rotenone specifically inhibits NADH dehydrogenase, [NADH: (acceptor) oxidoreductase, EC 1.6.99.3], which is the enzyme of energy-conserving site 1 of the mitochondrial electron transport system, activity and kinetics of this enzyme system were studied in fibroblast homogenates. NADH dehydrogenase activity was equal in cells from the three genotypes. At pH 8.0, affinity of the enzyme for its substrate was CF greater than HZ = controls; at pH 8.6, affinity was CF greater than HZ = controls (P less than 0.005 for the differences). pH optima for the genotypes were without exception 8.6 (CF), 8.3 (HZ), and 8.0 (control). HZ and control lines were distinguished unequivocally in a blind test on the basis of differences in pH optima. Purified mitochondrial preparations revealed pH optima identical to those found in whole cell homogenates. These data suggest that the mutant gene responsible for CF is expressed in the complex mitochondrial NADH dehydrogenase system.

The Role of Ubiquinone-10 in Cyclic Electron Transport in Rhodopsendomonas capsulata Ala pho+: Effects of Lyophilization and Extraction

Abstract 1. The effects of lyophilization and the extraction of ubiquinone-10 on the kinetics of electron transport in Rhodopseudomonas capsulata Ala pho+ have been investigated. 2. Lyophilization reduced the amount of ferrocytochrome c2 photo-oxidized on a microsecond time scale following a single excitation. 3. Lyophilization increased the reactivity of the electron transfer components with redox mediators, particularly N-methyl phenazonium methosulphate (PMS). At a concentration of 1 µᴍ , PMS accelerated reaction center re-reduction, ferricytochrome c2 re-reduction and ferrocytochrome b50 oxidation. The cytochrome c2 re-reduction stimulated by PMS was antimycin A insensitive but the cytochrome b50 oxidation was partially antim ycin sensitive. 4. Removal of 25- 30 molecules of ubiquinone 10 per reaction center removed a secondary acceptor pool, had very little effect on the kinetics of ferricytochrome b50 reduction and ferricyto­ chrome c2 re-reduction, but markedly inhibited ferrocytochrome b50 oxidation. Ubiquinone extraction also caused an increased stimulation of ferrocytochrome b50 oxidation by PMS. 5. The involvement of tightly bound ubiquinone in cytochrome b reduction and in the cytochrome b-c2 oxido-reductase, and the role of semiquinone species is discussed.

Internal pH, pH, and the kinetics of electron transport in chloroplasts.

The relation between the rate of electron transport, ΔpH, and internal pH was studied in lettuce chloroplasts, by following, in parallel, the fluorescence quenching of 9‐aminoacridine and the rate of electron transport. At low external pH (< 8.0) there was an initial burst in the rate of electron transport (R1) followed by a slower steady‐state rate (R2). However, at high external pH (> 8.5) R1 was slower than R2. Nevertheless, when analyzed as a function of internal pH, both R1 and R2 showed the same dependence with a maximal rate observed around internal pH 5.0. Thus, at low external pH, the initial pH is initially around 5.0 and, therefore, R1 is high, but as ΔpH is increased, the internal pH falls below 5.0 and R2 is lower. At high external pH, the internal pH is initially above 5.0 and, therefore, R1 is relatively low. As ΔpH increases the internal pH is lowered toward 5.0 and R2 becomes faster. A similar conversion of the ratio R1/R2 from R1/R2 > 1 to R1/R2 < 1 was also observed at constant external pH by varying the light intensity, or concentration of the uncoupler nigericin.In the presence of nigericin or at low light intensities, the maximal rate was shifted to higher internal pH values. At internal pH values above 5.0 in the presence of nigericin, the rate of electron transport was inversely proportional to ΔpH. These results indicate that the internal pH is not the only parameter that controls the rate of electron transport. It is shown that when all these diverse results are plotted as a function of an average pH (between the internal and external pH) they show a single optimum. Thus, it is suggested that the rate controlling site is embedded in the thylakoid membrane, and is a function of both the internal and external pH. This membrane pH and the size of ΔpH seem to be the major factors controlling the rate of electron transport in chloroplasts.

Tropical pulmonary eosinophilia.

<h3>Abstract</h3> Carbon nanotubes (CNTs) are suited for neurochemistry because of their biological inertness, ability to withstand biofouling, and superior electron transport kinetics. Dopamine, the canonical monoaminergic neuromodulator, contributes to reward, cognition and attention, however, its detection in real-time is challenging due to its low basal concentration in the brain (100nM L^-1). In our present work, we fabricate pyrolytic carbon electrodes and perform a CNT coating to improve the electrochemical kinetics of dopamine. Upon CNTs coating, dopamine shows a sensitivity of 9±18nA/<i>μ</i>M for a cylindrical electrode having a mean surface diameter of 8±4μm. Increasing the scan frequency from 10-100 Hz shows that dopamine electron transfer kinetics improves; wherein dopamine is oxidized at 0.35±0.09V and reduced to -0.10±0.05V for 10 Hz. Increasing the frequency results in a shift of oxidation peak towards the anodic region, wherein dopamine oxidizes at 0.08±3V and reduces at -0.1±0.05V for 100 Hz, thus showing that dopamine redox is reversible which can be attributed to the superior electron transport kinetics of CNTs. The sensor was able to distinguish dopamine signals against other neurochemicals like serotonin and foulant 3,4-Dihydroxyphenylacetic acid (DOPAC). The minimum chemical detection that can be performed using these nanopipettes is 50±18nM L^-1, which is well below the physiological concentrations of dopamine in the brain. <h3>Graphical Abstract</h3>