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
H2O → IIbhv → C550 → cyt. b559 → PC → IIahv → Fd → NADP+
Photoreaction IIb involves an electron transfer from water to C550 that does not require plastocyanin and is the first known System II photoreaction resistant to inhibition by 3-(3,4-dichlorophenyl)-1,1-dimethyl urea (DCMU) and o-phenanthroline. Cytochrome b559 is reduced by C550 in a reaction that is readily inhibited by DCMU or o-phenanthroline. Thus, the site of DCMU (and o-phenanthroline) inhibition of System II appears to lie between C550 and cytochrome b559. Photoreaction IIa involves an electron transfer from cytochrome b559 and plastocyanin to ferredoxin-NADP+. 相似文献
2. When oxidized plastocyanin is added, a transient is observed when light is first turned on: this is due to a reduction of the plastocyanin before DCIP reduction begins. When reduced plastocyanin is added, a different transient occurs: this is due to a fast photoreduction of DCIP by the plastocyanin and is followed by the slower steady state reduction of DCIP by water. When light is turned off before complete reduction of DCIP, a transient reduction of oxidized plastocyanin by reduced DCIP is seen. Insensitivity of these transients to 3(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) and the greater effectiveness of 710 nm light, along with the known capacity of plastocyanin to mediate electron transfer to System I, prove that an intrinsically fast reduction of DCIP occurs at a site close to the primary photoreduced product of System I.
3. After brief sonication and washing, no residual plastocyanin was detected in chloroplast fragments, and the rate of the slow DCIP reduction (about 50μmoles/mg chl per h) sustained by such fragments was essentially identical to that maintained by fragments of mutants lacking System I activity. Following et al.9, the simplest explanation for this slow DCIP reduction is that is occurs at a site close to System II and the system I is not involved.
4. A very slow transient reduction of DCIP occurs after extinguishing light; this presumably involves another reduction site close to System II, as suggested by 9. 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.
The addition of NaCl to the chloroplast suspension produced a 40–80% increase in fluorescence yield measured at 684 nm at room temperature. The fluorescence increase was completed about 5 min after the addition. The effect saturated at 100 mM NaCl. Low-temperature fluorescence spectra showed that NaCl increased the yields of two fluorescence bands of pigment system II at 684 and 695 nm but decreased that of pigment system I at 735 nm. Similar effects on chlorophyll a fluorescence at room and at low temperatures were obtained with NaBr, NaNO3, Na2SO4, LiCl, KCl, RbCl, CsCl, NH4Cl and CH3NH3Cl.
NaCl suppressed the quantum efficiency of NADP+ reduction supported by the ascorbate-2,6-dichlorophenolindophenol (DCIP) couple as an electron donor system in the presence of 3-(3′,4′-chlorophenyl)-1,1-dimethylurea (DCMU). On the other hand, NaCl only slightly enhanced the quantum yield of photoreaction II measured by the Hill reaction with DCIP.
It is concluded that the monovalent cations tested suppressed the excitation transfer from pigment system II to pigment system I; the effects were the same as those of alkaline earth metals and Mn2+ (refs. 1, 2). 相似文献
Under a stream of nitrogen, whole chloroplasts reduced NADP+ after an osmotic shock, in the absence of added ferredoxin. The resulting ATP/NADPH ratios were high (approx. 2 or 3). They decreased to 1 in the presence of either exogenous ferredoxin, 3-(p-chlorophenyl)-1,1-dimethylurea or limiting light: i.e. high ATP/NADPH ratios were observed only when the terminal step of NADP+ reduction was limiting.
The endogenous anaerobic phosphorylation was inhibited by antimycin A to the same extent as the O2-dependent endogenous non-cyclic phosphorylation.
A direct inhibition of electron transport by antimycin A has never been observed. 相似文献
1. 1. The relationship between temperature and Hill reaction activity has been investigated in chloroplasts isolated from barley (Hordeum vulgare L. cv. Abyssinian).
2. 2. An Arrhenius plot of the photoreduction of 2,6-dichlorophenolindophenol (DCIP) showed no change in slope over the temperature range 2–38 °C. The apparent Arrhenius activation energy (Ea) for the reaction was 48.1 kJ/mol.
3. 3. In the presence of an uncoupler of photophosphorylation, methylamine, the Ea for DCIP photoreduction went through a series of changes as the temperature was increased. Changes were found at 9, 20, 29 and 36 °C. The Ea was highest below 9 °C at 63.7 kJ/mol. Between 9 and 20 °C the Ea decreased to 40.4 kJ/mol and again to 20.2 kJ/mol between 20 and 29 °C. Between 29 and 36 °C there was no further increase in activity with increasing temperature. The temperature-induced changes at 9, 20 and 29 °C were reversible. At temperatures above 36 °C (2 min) a thermal and largely irreversible inactivation of the Hill reaction occurred.
4. 4. Temperature-induced changes in Ea were also found when ferricyanide was substituted for DCIP or gramicidin D for methylamine. The addition of an uncoupler of photophosphorylation was not required to demonstrate temperature-induced changes in DCIP photoreduction following the exposure of the chloroplasts to a low concentration of cations.
5. 5. The photoreduction of the lipophilic acceptor, oxidized 2, 3, 5, 6-tetramethyl-p-phenylenediamine, also showed changes in Ea in the absence of an uncoupler.
6. 6. The temperature-induced changes in Hill activity at 9 and 29 °C coincided with temperature-induced changes in the fluidity of chloroplast thylakoid membranes as detected by measurements of electron spin resonance spectra. It is suggested that the temperature-induced changes in the properties and activity of chloroplast membranes are part of a control mechanism for regulation of chloroplast development and photosynthesis by temperature.
Abbreviations: DADox, oxidized 2,3,5,6-tetramethyl-p-phenylenediamine; DCIP, 2,6-dichlorophenolindophenol; 16NS, 3-oxazolidenyloxy-2-(14-carbmethoxytetradecyl)-2-ethyl-4,4-dimethyl; Ea, Arrhenius activation energy 相似文献
At temperatures below −100 °C, the primary reaction of Photosystem II is irreversible. However, at temperatures between −100 and −20 °C a back reaction that is insensitive to 3-(3′,4′-dichlorophenyl)-1,1′-dimethylurea (DCMU) occurs between P680+ and the reduced acceptor.
The amount of reduced acceptor and P680+ present under steady-state illumination at temperatures between −100 and −20 °C is small unless high light intensity is used to overcome the competing back reaction. The amount of reduced acceptor present at low light intensity can be increased by adjusting the oxidation-reduction potential so that P680+ is reduced by a secondary electron donor (cytochrome b559) before P680+ can reoxidize the reduced primary acceptor. The photooxidation of cytochrome b559 and the accompanying photoreduction of C-550 are inhibited by DCMU. The inhibition of C-550 photoreduction by DCMU, the dependence of P680 photooxidation and C-550 photoreduction on light intensity, and the effect of the availability of reduced cytochrome b559 on C-550 photoreduction are unique to the temperature range where the Photosystem II primary reaction is reversible and are not observed at lower temperatures. 相似文献
Between 525 and 690 mμ, the phosphorylation yield for the normal system is constant ( = 0.15 ATP/hv), maintaining a constant P/2e ratio of unity. The P/2e ratios indicate a tight coupling between phosphorylation and electron transport encompassing a single phosphorylation site for the transfer of two electrons.
Between 525 and 680 mμ, the phosphorylation yield for the donor system is constant ( approx. 0.04 ATP/hv), maintaining a P/2e ratio of approx. 0.5. At longer wavelengths (>690 mμ) the phosphorylation yield of the donor system rises ( approx. 0.07–0.08 ATP/hv) concomitant with the rise in the yield of electron flow.
These experiments suggest the possibility that two types of phosphorylation processes operate in chloroplasts, (1) a short-wavelength process coupled to the normal O2-evolving activity, and (2) a long-wavelength process coupled to the electron-donor activity of reagents such as DCIP. 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
The membrane fragments prepared from Anabaena cells grown in the presence of diphenylamine have the activities of both Photosystem 1 (NADP+ reduction with DCIP-ascorbate as electron donor) and Photosystem 2 (DCIP reduction with 1,5-diphenylcarbazide as electron donor).
The fluroescence spectra of these cells at 77°K show peaks at 696 and 731 nm and a shoulder around 687 nm. The fluorescence intensity at 687 and 696 nm is higher in these cells than in normal-Anabaena cells. 相似文献
2. Differences in the flurescence yield of chlorophyll a in flowing and stationary suspensions of untreated chloroplasts and of the large fragments are indicative of light-induced photoreduction of the quencher Q of chlorophyll a, associated with pigment System 2 (chlorophyll a2). The relatively low constant fluorescence yield of chlorophyll a in the small fragments indicates the absence of fluorescent chlorophyll a2 from these fragments and suggests that the low fluorescence is due to chlorophyll a, associated with pigmen System 1 (chlorophyll a1). The ratio of the fluorescence yields of chlorophyll a1 and chlorophyll a2 is 0.45:1. In the large particles the concentration ratio of pigment System 1 and System 2 is 1:3.
3. The efficiencies of quanta absorbed at 673, 683 and 705 nm for NADP+ reduction and P 700 oxidation in untreated chloroplasts and chloroplast fragments indicate that digitonin treatment results in a separation of System 2 from System 1 in the small fragments. Sonication does not cause such a separation. Under the conditions used P 700 oxidation and NADP+ reduction in the small fragments separated after digitonin treatment, occurred with maximal efficiency of 0.7 to 1.0 and 0.7, respectively.
4. The constancy of the fluorescence yield of chlorophyll a1 in the small fragments, under conditions at which P 700 is oxidized and NADP+ is reduced, is interpreted as evidence either for the hypothesis that the fluorescence of chlorophyll a1 is controlled by the redox state of the primary photoreductant XH, or alternatively for the hypothesis that energy transfer from fluorescent chlorophyll a1 to P 700 goes via an intrinsically weak fluorescent, still unknown, chlorophyll-like pigment.
5. The low-temperature emission band around 730 nm is argued not to be due to excitation by System 1 only; the relatively large half width of the band, as compared to the emission bands at 683 and 696 nm, suggests that it is possibly due to overlapping emission bands of different pigments. 相似文献
A specific effort was made to measure the difference spectrum for the photooxidation of P680 under conditions (chloroplasts frozen to −196 °C in the presence of ferricyanide) where a stable, Photosystem II mediated EPR signal, attributed to P680+ has been reported. The difference spectra, however, did not show that P680+ was stable at −196 °C under any conditions tested. Absorbance measurements induced by saturating flashes at −196 °C (in the presence or absence of ferricyanide) indicated that all of the P680+ formed by the flash was reduced in the dark either by a secondary electron donor or by a backreaction with the primary electron acceptor. We conclude that P680+ is not stable in the dark at −196 °C: if the normal secondary donor at −196 °C is oxidized by ferricyanide prior to freezing, P680+ will oxidize other substances. 相似文献
A sulfite reductase was purified from spinach leaves. Broken chloroplasts and sulfite reductase reduced sulfite to sulfide in the light when ferredoxin was added. NADP+ was not required for this reduction.
The results suggest that in chloroplasts a sulfate activated by ATP (phosphoadenosine phosphosulfate) is reduced to sulfite by a sulfhydryl compound and that sulfite is reduced to sulfide by a ferredoxin-dependent sulfite reductase. 相似文献