Inhibition Of Oxygen Evolution Research Articles

Environmental Modification of Seedling Alfalfa,Medicago sativa, Tolerance to Bromoxynil

Seedling alfalfa exhibited increased phytotoxicity from bromoxynil when applications were made with temperatures greater than 28 C both in controlled environments and in the field. Treatments applied in the morning were more phytotoxic than treatments made in the late afternoon. In laboratory experiments, inhibition of oxygen evolution occurred within 1 h after treatment. The amount of permanent damage sustained was dependent on the rate of bromoxynil and the length of time the alfalfa was exposed to light or darkness immediately after spraying. Bromoxynil treatments in cool weather and/or in the late afternoon should minimize injury to the crop.

Calcium content of oxygen-evolving Photosystem II preparations from higher plants. Effects of NaCl treatment

The abundance of bound Ca 2+ in four Photosystem II oxygen-evolving preparations from spinach and rice were determined after removal of contaminating Ca 2+ with chelex 100 (Kashino, Y., Satoh, K. and Katoh, S. (1986) FEBS Lett. 205, 150–154). (1) Spinach membrane preparations which evolved oxygen at rates of 350–500 μmol per mg chlorophyll (Chl) per h contained, on average, 1.8 Ca 2+ per 200 Chl, whereas a mean value of 2.1 Ca 2+ per 200 Chl was obtained with more active spinach preparations, showing oxygen-evolving rates of 500–800 μmol per mg Chl per h. The most active membrane preparations, which evolved oxygen at rates of 750–1050 μmol per mg Chl per h, were isolated from rice seedlings. The rice preparations also contained 2.1 Ca 2+ per 200 Chl. Thus, different activities of the three preparations cannot be related to difference in their Ca 2+ abundance and the maximum number of Ca 2+ needed to promote high rates of oxygen evolution is 2 Ca 2+ per 200 Chl in higher plants. (2) Highly purified spinach oxygen-evolving complexes (Ikeuchi, M., Yuasa, M. and Inoue, Y. (1985) FEBS Lett. 185, 316–323) contained 1 Ca 2+ per 50 Chl. (3) Treatment of the spinach membrane preparations with high concentrations of NaCl, either in the dark or light, caused no significant loss of the bound Ca 2+, although the treatment strongly inhibited oxygen evolution and the inhibition was considerably reversed by addition of 5 mM CaCl 2. Exposure of the NaCl-washed membranes to EGTA failed to reduce the amount of bound Ca 2+. Thus, inactivation of oxygen evolution by the salt wash is not related to release of Ca 2+. (4) The abundance of Ca 2+ in spinach membrane preparations was not, or only slightly, affected by treatments with 1 mM NH 2OH, 1 M MgCl 2 or 0.8 M Tris, which solubilized either Mn or the three extrinsic proteins associated with the membranes, or both. It is concluded that the three proteins are not involved in the Ca 2+ binding. The number of Ca 2+ functioning in Photosystem II electron transport of higher plants is discussed.

Protection of photosynthetic O2 evolution against heat inactivation: the role of chloride, pH and coupling status

Heat inactivation of photosynthetic O2 evolution was studied in isolated thylakoids from spinach (Spinacia oleracea) and mangrove (Avicennia marina) leaves. Different temperatures, salt, pH and uncoupler effects were investigated. From these results and others in the literature it was concluced that chloride loss from the membrane and, more specifically, the oxygen-evolving complex of photosystem II, may be the cause of inhibition of oxygen evolution during heat inactivation.

Regulation of nitrogenase gene expression in anaerobic cultures of Anabaena variabilis.

Derepression of nitrogenase gene expression was studied at the mRNA and enzyme activity levels in anaerobic cultures of Anabaena variabilis 29413. Cells, previously grown with ammonium chloride, were incubated in the absence of fixed nitrogen compounds under an Ar atmosphere with dichlorophenyldimethyl-urea present to inhibit oxygen evolution. The appearance of nitrogenase mRNA (measured by dot blot hybridization analysis) and nitrogenase activity (measured as acetylene-reducing activity) was followed, and the cells were also observed by phase-contrast microscopy. Nitrogenase mRNA could be detected after 1.5 to 2.0 h of nitrogen starvation; enzyme activity appeared about 1 h later. Although enzyme activity increased for many hours, mRNA levels reached a steady state rapidly. Neither heterocysts nor proheterocysts formed under these conditions; however, the cells were observed to shrink and become chlorotic. When anaerobic, derepressed cultures were exposed to oxygen, nitrogenase mRNA levels decreased very rapidly.

Fluoride inhibition of oxygen evolution; new evidence from 35Cl-NMR measurements

35Cl-NMR was used to investigate the nature of F −-induced inhibition of oxygen-evolution in thylakoids from the mangrove, Avicennia marina. These studies showed a correlation between F − inhibition of oxygen evolving activity and increased bulk Cl − relaxation, possibly associated with the formation of a new class of high-affinity Cl −-binding sites, or a change in the nature of the existing binding sites, induced by F −. The presence of added Cl − did not alter the F −-induced inhibition of oxygen evolution. Increased Cl − relaxation and F −-induced inhibition of oxygen evolution occurred at lower concentrations of F − at pH 7.8 than at pH 6.3. In mangrove thylakoids. F −-induced inhibition of oxygen evolution does not appear to be due to competition with Cl − for Cl − binding sites, but instead involves some other interaction close to the oxygen-evolving complex.

Sulfate inhibition of photosystem II oxygen evolving complex

Effect of sulfate on the oxygen evolution in barley Photosystem II membrane fraction was investigated. Purified Photosystem II membrane fraction was exposed to sulfate effect in dark or under dim light illumination. The presence of 10 mM CaCl2 during the sulfate treatments prevented the loss of oxygen evolution. The protection was complete even at the concentration of 50 mM Na2SO4. However, light stimulated sulfate inhibition of oxygen evolution by the Photosystem II. After incubation with sulfate, we found there was depletion of 18 and 23 kDa polypeptides in Photosystem II complex. Here we provide new evidence that sulfate inhibition of oxygen evolution can be caused by depletion of chloride and calcium ions from the water splitting complex rather than by partial depletion of 18 and 23 kDa polypeptides from the complex.

Inhibition of photosynthetic activities under slow water stress measured in vivo by the photoacoustic method

A slow water stress over several days was imposed on tobacco plants (Nicotiana tabacum L. var. Xanthi) by withholding water from the soil. Photosynthesis was measured in leaves from those water‐stressed plants by the photoacoustic method. Slow drought induced marked changes in the photoacoustic signals, which were largely similar to those observed previously in leaves subjected to rapid desiccation in air (over 3–4 h), reflecting two simultaneous changes: 1) Modification of the heat and oxygen diffusion characteristics of the leaves due to changes in their anatomical structure [shown by the change in the slope of the plot of the oxygen (AOX) to photothermal signal (APT) ratio vs the square root of the modulation frequency]; 2) Inhibition of gross photosynthesis measured by the extrapolation of the AOX/APT ratio to zero frequency. However, in contrast to rapid water stress in detached leaves, where it was shown that mainly the oxidizing side of photosystem II (PS II) was damaged, we found a slower and more complex phenomenology having largely biphasic kinetics. During the first 6 days, there was a strong reduction in the photochemical energy storage, but the inhibition of oxygen evolution was relatively mild. The Emerson enhancement in state 1 dropped considerably, indicating lowering of the apparent absorption cross‐section of PS II. Fluorescence measurements suggest that PS II reaction center itseIf may be the primary site of the damage. PS I activity, judged by cytochrome f photooxidation, remained largely intact. The subsequent days were associated with a further spectacular decrease in the oxygen evolution quantum yield with both photosystems damaged. The photochemical energy storage continued to decrease further. The Emerson enhancement ratio of the remaining activities in both State 1 and 2 showed a marked increase, indicating the reestablishment of a strong imbalance in the distribution of excitation energy within the photochemical apparatus in favor of PS II. All the photoacoustic changes observed in response to drought were completely reversible within 2–3 days upon rewatering of the soil.

Artificial inhibitory effects of sulfite on photosystem II activity measured by oxygen evolution in chloroplasts

In this report we demonstrate sulfite interaction with oxygen and PSII electron acceptors (ferricyanide and para-benzoquinone) during measurement of oxygen evolution in chloroplasts. Redox potentials of oxygen, ferricyanide and para-benzoquinone allow them to compete for sulfite. Without taking this into account, sulfite inhibition of oxygen evolution can be overestimated, since sulfite consumes oxygen and reduces ferricyanide or para-benzoquinone during the measurement. In order to correctly measure the rate of oxygen evolution in chloroplasts, it is necessary to avoid presence of sulfite during the measurement. After overcoming the artifact, mentioned above, we confirm the sulfite inhibition of oxygen evolution in chloroplasts but at a lesser extent than earlier reported. This, however, is a pretreatment effect.

Total recovery of O2 evolution and nanosecond reduction kinetics of chlorophyll-a II + (P-680+) after inhibition of water cleavage with acetate

Oxygen evolution and reduction kinetics of the photooxidized Chl-aII (+) have been measured in oxygen-evolving complexes from the thermophilic cyanobacterium Synechococcus sp. 1. Incubation of PS II particles with acetate resulted in an inhibition of oxygen evolution and a retardation of the Chl-aII (+)=reduction kinetics from the nanosecond range to the microsecond range, indicating a modification of the donor side of photosystem II (PS II). 2. After the first two flashes given to a dark-adapted, acetate treated sample, Chl-aII (+) was re-reduced with a half-life time of 160 μs by a component of the donor side of PS II. Under repetitive excitation Chl-aII (+) was re-reduced in 500 μs by electron back reaction from the primary acceptor QA (-) (X-320(-)). Obviously, in the presence of acetate only two electrons are available from the donor side. 3. Both oxygen evolution and nanosecond reduction kinetics of Chl-aII (+) were restored to the control level when acetate was removed. 4. The results indicate a tight coupling between O2 evolution and nanosecond reduction kinetics of Chl-aII (+). 5. The reversible inhibition is probably due to a replacement of Cl(-) by acetate within the water splitting enzyme. 6. Due to its strongly retarded kinetics, the reversibly modified system may facilitate investigations of the mechanism of the donor side.

Differential Effects of K+ and Na+ on Oxygen Evolution Activity of Photosynthetic Membranes from Two Halophytes and Spinach

KCl reduced oxygen evolution by Sarcocornia quinqueflora thylakoids to well below the level of activity in the presence of NaCI. This inhibition occurred at both high and low pH. On the other hand, both KCl and NaCl induced almost indistinguishable effects on oxygen evolution in spinach thylakoids. Photosystem (PS) Il membranes from Avicennia marina showed a similar inhibition of oxygen evolution by KCl to S. quinqueflora thylakoids, but spinach PS Il particles were not inhibited by KCl. The KCl inhibition of oxygen evolution of halophytic thylakoids was localised to the oxygen-evolving complex and required little KCl, as significant inhibition occurred with only 25 mM. K2SO4 was also inhibitory, but Na2SO4 was not, indicating that K+ was the inhibitory ion. Na2SO4 was partly able to reverse this inhibition caused by KCl, suggesting the inhibition to be a function of the K+ /Na+ ratio. These results have implications concerning the ionic relations of the leaf cells of halophytes.

The release of polypeptides and manganese from oxygen-evolving photosystem II preparations following zinc-treatment

Inhibition of oxygen evolution in photosystem II membrane fragments from pea chloroplasts by washing with Zn 2+ causes appearance of the EPR signal of Mn(H 2O) 6 2+. This Mn 2+ remains associated with the membrane fraction. Release of Mn 2+ into the medium was correlated with the amount of the 23 kDa protein removed from the membrane. This suggests that this protein may function as a ‘gate’ to an aqueous compartment into which Mn 2+ is released. Inhibition by Zn 2+ correlated with the release of 1 Mn 2+ per reaction centre, out of a total stoichiometry of 4 Mn atoms per reaction centre. By comparing the release of Mn following Zn-treatment of NaCI or CaC1 2 washed membranes, it is concluded that the 33 kDa protein is involved in binding of 2 Mn.

Inhibition of Photosynthetic Electron Transport by Halogenated 4-Hydroxy-pyridines

Abstract Herbicidal halogen substituted 4-hydroxypyridines are inhibitors of photosynthetic electron flow in isolated thylakoid membranes by interfering with the acceptor side of photosystem II. Tetrabromo-4-hydroxypyridine, the most active compound found, has a pI50-value of 7.6 in the inhibition of oxygen evolution in both the reduction of an acceptor of photosystem I and an acceptor of photosystem II. The new inhibitors displace both metribuzin and ioxynil from the membrane. The 4-hydroxypyridines, like ioxynil, have unimpaired inhibitor potency in Tristreated chloroplasts, whereas the DCMU-type family of herbicides does not. It is suggested that 4-hydroxypyridines are complementary to phenol-type inhibitors, and a common essential element is proposed. The 4-hydroxypyridines do not inhibit photosystem I or non-cyclic electron flow through the cytochrome b/f complex. But they do have a second inhibition site in photosynthetic electron transport since they inhibit ferredoxin-catalyzed cyclic electron flow, indicating an antimycin-like property. A comparison of the in vitro potency of the compounds with the in vivo potency shows no correlation. A major herbicidal mode of action of the group is related to the inhibition of carotenoid synthesis, and access to the chloroplast lamellae in vivo for inhibition of electron transport may be restricted.

The roles of the extrinsic subunits in Photosystem II as revealed by EPR

The effects of selective removal of extrinsic proteins on donor side electron transport in oxygen-evolving PS II particles were examined by monitoring the decay time of the EPR signal from the oxidized secondary donor, Z +, and the amplitude of the multiline manganese EPR signal. Removal of the 16 and 24 kDa proteins by washing with 1 M NaCl inhibits oxygen evolution, but rapid electron transfer to Z + still occurs as evidenced by the near absence of Signal II f. The absence of a multiline EPR signal shows that NaCl washing induces a modification of the oxygen-evolving complex which prevents the formation of the S 2 state. This modification is different from the one induced by chloride depletion of PS II particles, since in these a large multiline EPR signal is found. After removal of the 33 kDa protein with 1 M MgCl 2, Signal II f is generated after a light flash. Readdition of the 33 kDa component to the depleted membranes accelerates the reduction of Z +. Added calcium ions show a similar effect. These findings suggest that partial advancement through the oxygen-evolving cycle can occur in the absence of the 16 and 24 kDa proteins. The 33 kDa protein, on the other hand, may be necessary for such reactions to take place.

The high affinity binding site for calcium on the oxidizing side of photosystem II

Preparations of photosystem II complex from spinach chloroplasts with Triton X-100 were treated with 1 M KCl to release 17 KDa and 23 KDa polypeptides. The inhibited oxygen evolution activity could be reactivated by adding high concentration (mM) of Ca ++ or by reconstituting 17 KDa and 23 KDa polypeptides which were found to promote high affinity binding of Ca ++ to the reconstituted membranes (Ghanotakis et al. FEBS (1984) 170, 169–173). Inclusion of 50 mM Ca ++ during KCl treatment did not prevent the release of 17 KDa and 23 KDa polypeptides but protected oxygen evolution from being inactivated. It is explained by preservation of the high affinity binding site for Ca ++ if. Ca ++ is present during the salt treatment even though depletion of 17 KDa and 23 KDa polypeptides usually results in replacement by a low affinity (mM) binding site for Ca ++. It also implies that the high affinity binding site is not located on 17 KDa and 23 KDa polypeptides.

EPR detection of a cryogenically photogenerated intermediate in photosynthetic oxygen evolution

In Photosystem II preparations at low temperature we were able to generate and trap an intermediate state between the S 1 and S 2 states of the Kok scheme for photosynthetic oxygen evolution. Illumination of dark-adapted, oxygen-evolving Photosystem II preparations at 140 K produces a 320-G-wide EPR signal centered near g = 4.1 when observed at 10 K. This signal is superimposed on a 5-fold larger and somewhat narrower background signal; hence, it is best observed in difference spectra. Warming of illuminated samples to 190 K in the dark results in the disappearance of the light-induced g = 4.1 feature and the appearance of the multiline EPR signal associated with the S 2 state. Low-temperature illumination of samples prepared in the S 2 state does not produce the g = 4.1 signal. Inhibition of oxygen evolution by incubation of PS II preparations in 0.8 M NaCl buffer or by the addition of 400 μM NH 2OH prevents the formation of the g = 4.1 signal. Samples in which oxygen evolution is inhibited by replacement of Cl − with F − exhibit the g = 4.1 signal when illuminated at 140 K, but subsequent warming to 190 K neither depletes the amplitude of this signal nor produces the multiline signal. The broad signal at g = 4.1 is typical for a S = 5 2 spin system in a rhombic environment, suggesting the involvement of non-heme Fe in photosynthetic oxygen evolution.

EPR studies on the photosystem II donor side in salt-washed and reconstituted inside-out thylakoids

EPR measurements on inside-out thylakoids revealed that salt-washing, known to inhibit oxygen evolution and release a 23 and a 16 kDa protein, induced a Signal II f and decreased the EPR signal from state S 2. Readdition of the released 23 kDa protein restored the oxygen evolution and decreased the Signal II f, but did not relieve the decrease in the state S 2 signal. It is suggested that salt-washing inhibits the electron transfer from the oxygen-evolving site to Z, the physiological donor to P680. In inhibited photosystem II units lacking Signal II f, Z + is rapidly reduced, possibly by a modified S-cycle unable to evolve oxygen.

The chloride requirement for photosynthetic oxygen evolution. Analysis of the effects of chloride and other anions on amine inhibition of the oxygen-evolving complex

In the presence of Cl −, the severity of ammonia-induced inhibition of photosynthetic oxygen evolution is attenuated in spinach thylakoid membranes (Sandusky, P.O. and Yocum, C.F. (1983) FEBS Lett. 162, 339–343). A further examination of this phenomenon using steady-state kinetic analysis suggests that there are two sites of ammonia attack, only one of which is protected by the presence of Cl −. In the case of Tris-induced inhibition of oxygen evolution only the Cl − protected site is evident. In both cases the mechanism of Cl − protection involves the binding of Cl − in competition with the inhibitory amine. Anions (Br − and NO − 3) known to reactive oxygen evolution in Cl −-depleted membranes also protect against Tris-induced inhibition, and reactivation of Cl −-depleted membranes by Cl − is competitively inhibited by ammonia. Inactivation of the oxygen-evolving complex by NH 2OH is impeded by Cl −, whereas Cl − does not affect the inhibition induced by so-called ADRY reagents. We propose that Cl − functions in the oxygen-evolving complex as a ligand bridging manganese atoms to mediate electron transfer. This model accounts both for the well known Cl − requirement of oxygen evolution, and for the inhibitory effects of amines on this reaction.

Immunological studies on the organization of proteins in photosynthetic oxygen evolution

Studies on inside-out thylakoid vesicles and several Photosystem-II particles have suggested the involvement of three proteins of 33, 23 and 16 kDa in photosynthetic oxygen evolution. In this study, monospecific antibodies were raised against the purified 33, 23 and 16 kDa proteins. The antibodies were used to investigate the organization and function of these proteins in the oxygen-evolving complex. Quantification of the 33, 23 and 16 kDa proteins by rocket immunoelectrophoresis revealed one copy of each polypeptide per some 200 chlorophylls in unfractionated thylakoids. Isolated inside-out thylakoids, derived from the grana partitions, showed 6–8 times more of the 33, 23 and 16 kDa proteins, on a chlorophyll basis, compared to the stroma lamellae vesicles. Agglutination studies revealed that the proteins are exposed on the lumenal side of the thylakoid membrane. An obligatory role for the 23 kDa protein in the photosynthetic water oxidation is suggested from the close correlation obtained between the inhibition of oxygen evolution and the release of this protein as caused by washing the inside-out thylakoids with increasing concentrations of NaCl. For the 16 kDa protein no such correlation was obtained. Rebinding experiments, using both salt-washed and alkaline Tris-washed inside-out thylakoids revealed that the 33 kDa protein was required for the binding of the 23 kDa protein, which in turn enhanced the binding of the 16 kDa protein. It is concluded that the three proteins are closely organized as a complex at the inner thylakoid surface in association with membrane spanning proteins of Photosystem II.

Effect of partial removal and readdition of a 23 kilodalton protein on oxygen yield and flash-induced absorbance changes at 320 nm of inside-out thylakoids

Washing inside-out spinach thylakoids with 250 mM NaCl at pH 7.4 causes up to 75% inhibition of oxygen evolution which has been shown by reconstitution experiments to be due mainly to the removal of a 23 kDa protein (Åkerlund, H.E., Jansson, C. and Andersson, B. (1982) Biochim. Biophys. Acta 681, 1–10). Here we have found the same degree of inhibition and reconstitution of oxygen evolution measured either in flash light or in continuous light at different light intensities, which suggests that the salt-washing causes a complete block of electron transport in certain chains rather than a decreased rate in all chains. Flash-induced absorbance changes at 320 nm, reflecting mainly the electron transfer at the Photosystem-II acceptor side, from the primary to the secondary plastoquinone in their semiquinone form (PQ − APQ − B → PQ APQ 2− B) and to minor extent the donor side of Photosystem II (Renger, G. and Weiss, W. (1983) Biochim. Biophys. Acta 722, 1–11), were measured under repetitive excitation conditions. Salt-washing the inside-out thylakoids caused a change in decay rate from 500–600 μs down to 200 μs, interpreted as an increased proportion of recombination between PQ − A and chlorophyll- a + II. Addition of the 23 kDa protein restored the kinetics up to some 400 μs. Measurements made on dark-adapted material gave evidence that salt-washing also affected S-state turnover. We conclude that the 23 kDa protein functions on the water-splitting side of Photosystem II and is indispensible for the photosynthetic water oxidation.

The effect of thylakoid lipids on an oxygen-evolving Photosystem II preparation

The lipid content of a Photosystem II preparation, derived by Triton X-100 fractionation of spinach thylakoid membranes, which retained high rates of oxygen evolution was examined. It was found that the detergent treatment resulted in a preferential loss of digalactosyldiacylglycerol. The effect on this preparation of exogenously added lipids, or lipid mixtures, purified from thylakoids was investigated. Addition of total polar lipid extract stimulated the rates of oxygen evolution, the same effect being achieved with isolated digalactosyldiacylglycerol or phosphatidylcholine alone. The stimulation of oxygen evolution was dependent on the degree of unsaturation of the lipids used and on the relative amounts of acidic lipids present in the mixtures. Addition of isolated acidic lipids alone resulted in a complete inhibition of oxygen evolution. The results are discussed in terms of the possible involvement of thylakoid lipids in the molecular organisation of the Photosystem II oxygen-evolving system.