Abbreviations: DCMU, 3-(3′,4′-dichlorophenyl)-1,1-dimethylurea; HEPES, hydroxyethyl-piperazineethanesulfonic acid; MES, (N-morpholino)ethanesulfonic acid; DCIP, 2,4-dichlorophenol-indophenol 相似文献
2. In the presence of methyl viologen, all the blue-green algae except Anabaena flos-aquae show a light-dependent O2 consumption as well as a post-illumination O2 evolution. The rate of O2 consumption is stimulated by 1 mM KCN, an inhibitor of catalase, but the dark O2 evolution becomes suppressed.
3. A. flos-aquae shows a light-dependent methyl viologen-catalyzed O2 uptake which is not affected by 1 mM KCN. Furthermore, there is no release of O2 in the dark following illumination.
4. With C. reinhardi, the cells do not show any net O2 exchange during or after illumination. Addition of 1 mM KCN, however, results in an immediate O2 uptake in the light.
5. Based on the mechanism postulated for the Mehler reaction in isolated chloroplasts, it was deduced that the differences in the kinetics of the O2 exchange catalyzed by methyl viologen reflect differences in the endogenous catalase activity in these algae. Cells of A. flos-aquae are deficient in catalase activity whereas those of the other blue-green algae possess catalase, although at low activity. C. reinhardi, on the other hand, has high catalase activity in vivo.
6. These findings are corroborated by results obtained from O2 electrode measurements of catalase activity in cell-free extracts of these algae.
7. The possible roles of catalase in algae and the implications of these results are also discussed. 相似文献
1. 1. The mechanism of the photooxidation of ascorbate and of Mn2+ by isolated chloroplasts was reinvestigated.
2. 2. Our results suggest that ascorbate or Mn2+ oxidation is the result of the Photosystem I-mediated production of the radical superoxide, and that neither ascorbate nor Mn2+ compete with water as electron donors to Photosystem II nor affect the rate of electron transport through the two photosystems: The radical superoxide is formed as a result of the autooxidation of the reduced forms of low potential electron acceptors, such as methylviologen, diquat, napthaquinone, or ferredoxin.
3. 3. In the absence of ascorbate or Mn2+ the superoxide formed dismutases either spontaneously or enzymatically producing O2 and H2O2. In the presence of ascorbate or Mn2+, however, the superoxide is reduced to H2O2 with no formation of O2. Consequently, in the absence of reducing compounds, in the reaction H2O to low potential acceptor one O2 (net) is taken up per four electrons transported where as in the presence of ascorbate, Mn2+ or other suitable reductants up to three molecules O2 can be taken up per four electrons transported.
4. 4. This interpretation is supported by the following observations: (a) in a chloroplast-free model system containing NADPH and ferredoxin-NADP reductase, methylviologen can be reduced to a free radical which is autooxidizable in the presence of O2; the addition of ascorbate or Mn2+ to this system results in a two fold stimulation of O2 uptake, with no stimulation of NADPH oxidation. The stimulation of O2 uptake is inhibited by the enzyme superoxide dismutase; (b) the stimulation of light-dependent O2 uptake in the system H2O → methylviologen in chloroplasts is likewise inhibited by the enzyme superoxide dismutase.
5. 5. In Class II chloroplasts in the system H2O → NADP upon the addition of ascorbate or Mn2+ an apparent inhibition of O2 evolution is observed. This is explained by the interaction of these reductants with the superoxide formed by the autooxidation of ferredoxin, a reaction which proceeds simultaneously with the photoreduction of NADP. Such an effect usually does not occur in Class I chloroplasts in which the enzyme superoxide dismutase is presumably more active than in Class II chloroplasts.
6. 6. It is proposed that since in the Photosystem I-mediated reaction from reduced 2,4-dichlorophenolindophenol to such low potential electron acceptor as methylviologen, superoxide is formed and results in the oxidation of the ascorbate present in the system, the ratio ATP/2e in this system (when the rate of electron flow is based on the rate of O2 uptake) should be revised in the upward direction.
1. (1) In dark-adapted chloroplasts (i.e. in States S0+S1 according to Kok, B., Forbush, B. and McGloin, M. (1970) Photochem. Photobiol. 11, 457–475), Q, reduced by a flash at low temperature, is reoxidized by a secondary acceptor and the positive charge is stabilized on the Photosystem II donor Z. Although this reaction is strongly temperature dependent, it still occurs very slowly at −60°C.
2. (2) When chloroplasts are placed in the S2+S3 states by a two-flash preillumination at room temperature, the reoxidation of Q− after a flash at low temperature is mainly due to a temperature-independent back reaction which occurs with non-exponential kinetics.
3. (3) Long continuous illumination of a frozen sample at −30°C causes 6–7 reducing equivalents to be transferred to the pool. Thus, a sufficient number of oxidizing equivalents should have been generated to produce at least one O2 molecule.
4. (4) A study of the back reaction in the presence of 3(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) shows the superposition of two distinct non-exponential reactions one temperature dependent, the other temperature independent.
Abbreviations: DCMU; 3(3; 4-dichlorophenyl)-1; 1-dimethylurea 相似文献
2. The initial phase of H+ uptake, analyzed by a flash-yield technique, exhibits linear kinetics (0–3 s) with no sign of transient phenomena such as the very rapid initial uptake (“pH gush”) encountered in the overall Hill reaction with methylviologen. Thus the initial rate of H+ uptake obtained by the flash-yield method is in good agreement with the initial rate estimated from a pH change tracing obtained under continuous illumination.
3. Dibromothymoquinone reduction, observed as O2 evolution by a similar flash-yield technique, is also linear for at least the first 5 s, the rate of O2 evolution agreeing well with the steady-state rate observed under continuous illumination.
4. Such measurements of the initial rates of O2 evolution and H+ uptake yield an H+/e− ratio close to 0.5 for the Photosystem II partial reaction regardless of pH from 6 to 8. (Parallel experiments for the methylviologen Hill reaction yield an H+/e− ratio of 1.7 at pH 7.6.)
5. When dibromothymoquinone is being reduced, concurrent phosphorylation (or arsenylation) markedly lowers the extent of H+ uptake (by 40–60%). These data, unlike earlier data obtained using the overall Hill reaction, lend themselves to an unequivocal interpretation since phosphorylation does not alter the rate of electron transport in the Photosystem II partial reaction. ADP, Pi and hexokinase, when added individually, have no effect on proton uptake in this system.
6. The involvement of a proton uptake reaction with an H+/e− ratio of 0.5 in the Photosystem II partial reaction H2O → Photosystem II → dibromothymoquinone strongly suggests that at least 50% of the protons produced by the oxidation of water are released to the inside of the thylakoid, thereby leading to an internal acidification. It is pointed out that the observed efficiencies for ATP formation (P/e2) and proton uptake (H+/e−) associated with Coupling Site II can be most easily explained by the chemiosmotic hypothesis of energy coupling. 相似文献
1. 1. In fresh chloroplasts ascorbate and other reducing agents, if present in sufficient concentration, fully replace water as the System II oxidant and can sustain maximum rates of 1000–1200 equiv/chlorophyll per h.
2. 2. None of the studied donors proved entirely specific for System II; to a variable extent all could react with the oxidant of System I. We therefore considered only the 3-(3,4-dichlorophenyl)-1,1-dimethylurea-(DCMU)-sensitive fraction of the observed rates as pertinent.
3. 3. Normal fresh chloroplasts contained 3 Mn/200 chlorophyllsII and showed flash yields of approx. 1 O2/1600 chlorophylls. This indicates that each System II trapping and O2-evolving center contains three Mn atoms.
4. 4. O2 evolution capacity is abolished when about 2/3 of the total Mn pool is removed by way of Tris or hydroxylamine extraction, i.e. upon removal of two of the three Mn atoms normally present per reaction center. Between the limits of 1 Mn per trap and 3 Mn per trap O2 evolution capacity is linear with Mn content.
5. 5. Mn removal affects the rates of O2 evolution in strong light and in weak light (quantum yield) in the same fashion. This indicates that complete O2 reaction centers are inactivated.
6. 6. With Mn removal the capacity for donor (ascorbate or p-phenylenediamine) photooxidation in strong light declines in a very similar fashion as the O2 evolving capacity. However, after removal of 2/3 of the Mn pool (by Tris or hydroxylamine extraction) 15–20% of the maximum rate remains (100–250 equiv/chlorophyll per h) as previously noticed by other workers. Secondly, the rate in weak light (quantum yield) of these photooxidations remains unaffected by Mn removal. This shows that for donor photooxidation the larger of the two Mn pools is not essential.
7. 7. Complete removal of Mn (< 1 Mn/4000 chlorophylls) led to 90–95% loss of donor photooxidation in strong light.
8. 8. Removal of 2/3 of the Mn left a low fluorescence yield (variable fraction = 0) which could be fully restored by adding DCMU. After complete removal of Mn (< 1 Mn/4000 chlorophylls) DCMU enhanced the yield of the variable fluorescence to only 1/2 the maximum level but the full maximum could be restored by chemical reduction. This indicates that fluorescence quencher of System II, Q, is not affected by Mn removal.
9. 9. Of the three Mn associated with each trapping center, one is linked more closely to the center than the other two. While all three are essential for O2 evolution, artificial donors can enter with various rate constants at several loci on the oxidant side of System II.
Abbreviations: DCMU, 3-(3,4-dichlorophenyl)-1,1-dimethylurea; Q, the quencher of System II fluorescence; F0, the invariant low level of fluorescence observed at onset of illumination; Fmax, maximum level of fluorescence; DCIPH2, 2,6-dichlorophenolindophenol, reduced form; DH2, a reductant capable of donating electrons to light-induced oxidants; A pool, the large electron acceptor pool in association with Q of System II; PMS, N-methylphenazonium ion (phenazine methosulfate) 相似文献
2. According to Den Haan et al. ((1974) Biochim. Biophys. Acta 368, 409–421), hydroxylamine destroys the secondary donor responsible for the fast reduction of Chl+. In the presence of 3 mM hydroxylamine, only the secondary donor D is functional and a flash induces mainly a single hit process.
3. The saturation curves for the fast and the slow rises have been studied in the presence of 3(3,4-dichlorophenyl)-1,1-dimethylurea for a second actinic flash given 2.5 s after a first saturating one. The large decrease in the half-saturating energy indicates the existence of efficient energy transfer occuring between photosynthetic units.
4. Two alternate hypotheses are discussed (a) in which D is an auxiliary donor and (b) in which D is included in the main electron transfer chain. 相似文献
The influence of O2 concentrations on the Hill reactions in the presence of p-benzoquinone, ferricyanide, NADP+, NADP+ plus ferredoxin has been studied with isolated spinach chloroplasts.
Because of the partial reoxidation of the hydroquinone, which is depending upon the O2 concentration, it does not seem possible to localize a site of action for O2.
With ferricyanide the influence of O2 is weak. However, the rate of ferricyanide reduction is increased in the presence of O2. The observed stimulation is greater for 21% O2 than for 70% O2. Bicarbonate stimulates the ferricyanide reduction and decreases the stimulating effect of 21% O2.
O2 decreases the rate of NADP+ reduction. Ferredoxin as well as bicarbonate stimulate the NADP+ reduction and reduce the O2 inhibition.
These results seem to indicate that O2 may enter the electron transport chain at a site situated near Photosystem I and before the ferredoxin's site.
The inhibitory effect of O2 on the Hill reactions with p-benzoquinone and NADP+ is depending upon the plants' growth conditions. It is greater with plants grown under weak light. 相似文献
1. 1. Small particles prepared from spinach chloroplasts after treatment with digitonin, exhibited Photosystem I reactions, including phosphorylation, at rates as high as those in chloroplasts, whereas electron flow from water to NADP+ or ferricyanide through Photosystem II was completely lost. Mediators of cyclic electron flow, such as pyocyanine, or N-methylphenazonium methosulfate in red light, had to be reduced to support photophosphorylation.Diaminodurene at high concentrations catalyzed cyclic phosphorylation under anaerobic conditions without addition of a reductant. In fact, addition of ascorbate gave rise to a marked inhibition which was released by addition of a suitable electron acceptor such as methylviologen.
2. 2. Under aerobic conditions a low O2 uptake, observed in the presence of diaminodurene, was stimulated several-fold upon addition of methylviologen and was stimulated again several-fold on further addition of ascorbate. The rate of phosphorylation, however, remained the same. The low P/2e ratio obtained under these conditions was not decreased at lower light intensities.
3. 3. These findings suggest a phosphorylation site associated with cyclic electron flow through Photosystem I without participation of the electron carriers of Photosystem II. A non-cyclic electron flow to O2 can be induced in this system by addition of methylviologen which effectively competes with the electron acceptors of cyclic flow. This non-cyclic electron flow still involves the same phosphorylation site. A scheme for electron transport and for the location of phosphorylation sites in chloroplasts is proposed.
Abbreviations: PMS, N-methylphenazonium methosulfate; DCIP, dichlorophenolindophenol; DCMU, dichlorophenyl-1,1-dimethylurea; Tricine, tris(hydroxymethyl)methylglycine 相似文献
The results can be explained by the following hypotheses: (a) The primary donor of Photosystem II in its oxidized state, P+, is a fluorescence quencher. (b) Hydroxylamine prevents the secondary electron donor Z from reducing the oxidized reaction center pigment P+ rapidly. This inhibition is dependent on hydroxylamine concentration and is complete at a concentration of 10−2 M. (c) A second donor (not transporting electrons from water) transfers electrons to P+ with a half time of roughly 25 μs. 相似文献
1. (1) MgCl2 (1–10 mM range) decreases the intersystem transfer but does not modify the partition of absorbed photons between the photosystems. MgCl2 addition causes a simultaneous increase of excitation life time (τ) and of fluorescence intensity (F). The same linear relationship is obtained with or without added Mg2+.
2. (2) The deactivation of Photosystem II by the Photosystem II to Photosystem I transfer increases with the level of reduced Photosystem II traps. When all Photosystem II traps are closed, half of Photosystem II excitons are deactivated by transfer to Photosystem I.
3. (3) From the relative values of the 685-nm fluorescence yield and System II electron transport rate in limiting light, measured with and without MgCl2, the values of rate constants of Photosystem II deactivation were calculated.
4. (4) The intersystem transfer determines a 715-nm variable fluorescence, which is lowered by MgCl2 addition. When this transfer is decreased by MgCl2 the efficiency of the transfer between Photosystem II-connected units is enhanced, and a more sigmoidal fluorescence rise is obtained.
A double-layer model of the thylakoid membrane where each photosystem is restricted to one leaflet is proposed to explain the decrease of the intersystem transfer after adding cations. It is suggested that MgCl2 decreases the thickness of the Photosystem I polar region, increasing the distance between the pigments of the two photosystems. 相似文献
1. 1. Alternation or superimposition of light 1 and light 2 illumination induces both fast and slow changes in fluorescence and rate of O2 evolution. The fast changes are ascribed to changes in conditions of the reaction centers in the context of the 1 model and the kinetic analysis of 2. The slow changes are interpreted as adaptations to the intensity and wavelength of illumination. The adaptive mechanism is described in terms of slow variation in fraction () of total absorbed quanta delivered to System 2. At low intensities, the calculated value of for cells adapted to light 2 illumination (light 2 state) is approx. 0.9 of for cells adapted to light 1 illumination (light 1 state).
2. 2. An increase in fluorescence yield was found to accompany the decrease in O2 yield at the onset of light saturation with either light 1 or light 2 excitation. Variation in is proposed to account for the differences between the maximum fluorescence yield observed in steady-state conditions and the 1.5 times higher maximum yield observed in transient conditions or in cells inhibited by 3(3,4-dichlorophenyl)-1,1-dimethylurea. Variation in can also explain the observation of a higher rate of fluorescence emission with light 1 excitation than with light 2 excitation for a given steady-state rate of O2 evolution.
3. 3. A model for energy conversion by System 2 is proposed to account for our observations. The model proposes competitive dissipation of absorbed energy by photochemical trapping at reaction centers and by fluorescence and radiationless de-excitation from both the pigment bed and reaction centers of System 2.
Abbreviations: DCMU, 3(3,4-dichlorophenyl)-1,1-dimethylurea; Fluorescence, denotes total emission and does not imply knowledge of an exponential decay 相似文献