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1.
Fractions enriched in either Photosystem I or Photosystem II activity have been isolated from the blue-green alga, Synechococcus cedrorum after digitonin treatment. Sedimentation of this homogenate on a 10–30% sucrose gradient yielded three green bands: the upper band was enriched in Photosystem II, the lowest band was enriched in Photosystem I, while the middle band contained both activities. Large quantities of both particles were isolated by zonal centrifugation, and the material was then further purified by chromatography on DEAE-cellulose.The resulting Photosystem II particles carried out light-induced electron transport from semicarbizide to ferricyanide of over 2000 μmol/mg Chlorophyll per h (which was sensitive to 3-(3,4-dichlorophenyl)-1,1-dimethylurea), and was nearly devoid of Photosystem I activity. This particle contains β-carotene, very little phycocyanin, has a chlorophyll absorption maximum at 675 nm, and a liquid N2 fluorescence maximum at 685 nm. The purest Photosystem II particles have a chlorophyll to cytochrome b-559 ratio of 50 : 1. The Photosystem I particle is highly enriched in P-700, with a chlorophyll to P-700 ratio of 40 : 1. The physical structure of the two Photosystem particles has also been studied by gel electrophoresis and electron microscopy. These results indicate that the size and protein composition of the two particles are distinctly different.  相似文献   

2.
The functional role of a chlorophyll ab complex associated with Photosystem I (PS I) has been studied. The rate constant for P-700 photooxidation, KP-700, which under light-limiting conditions is directly proportional to the size of the functional light-harvesting antenna, has been measured in two PS I preparations, one of which contains the chlorophyll ab complex and the other lacking the complex. KP-700 for the former preparation is half of that of the preparation which has the chlorophyll ab complex present. This difference reflects a decrease in the functional light-harvesting antenna in the PS I complex devoid of the chlorophyll ab complex. Experiments involving reconstitution of the chlorophyll ab complex with the antenna-depleted PS I preparation indicate a substantial recovery of the KP-700 rate. These results demonstrate that the chlorophyll ab complex functions as a light-harvesting antenna in PS I.  相似文献   

3.
An O2-evolving Photosystem II subchloroplast preparation was obtained from spinach chloroplasts, using low concentrations of digitonin and Triton X-100. The preparation showed an O2 evolution activity equivalent to 20% of the uncoupled rate of fresh broken chloroplasts, but had no significant Photosystem-I-dependent O2 uptake activity. The preparation showed a chlorophyll ab ratio of 1.9 and a P-700chlorophyll ratio of 12400. Absorption spectra at room temperature and fluorescence emission spectra of chlorophyll at 77 K suggested a significant decrease in Photosystem I antenna chlorophylls in the O2-evolving Photosystem II preparation.  相似文献   

4.
J. Barber  G.F.W. Searle  C.J. Tredwell 《BBA》1978,501(2):174-182
The MgCl2-induced chlorophyll fluorescence yield changes in broken chloroplasts, suspended in a cation-free medium, treated with 3,-(3′,4′-dichlorophenyl)-1,1-dimethylurea and pre-illuminated, has been investigated on a picosecond time scale. Chloroplasts in the low fluorescing state showed a fluorescence decay law of the form exp ?At12, where A was found to be 0.052 ps?12, and may be attributed to the rate of spillover from Photosystem II to Photosystem I. Addition of 10 mM MgCl2 produced a 50% increase in the steady-state fluorescence quantum yield and caused a marked decrease in the decay rate. The fluorescence decay law was found to be predominantly exponential with a 1/e lifetime of 1.6 ns. These results support the hypothesis that cation-induced changes in the fluorescence yield of chlorophyll are related to the variations in the rate of energy transfer from Photosystem II to Photosystem I, rather than to changes in the partitioning of absorbed quanta between the two systems.  相似文献   

5.
C.J. Arntzen  C.L. Ditto 《BBA》1976,449(2):259-274
When isolated chloroplasts from mature pea (Pisum sativum) leaves were treated with digitonin under “low salt” conditions, the membranes were extensively solubilized into small subunits (as evidenced by analysis with small pore ultrafilters). From this solubilized preparation, a photochemically inactive chlorophyll · protein complex (chlorophyll ab ratio, 1.3) was isolated. We suggest that the detergent-derived membrane fragment from mature membranes is a structural complex within the membrane which contains the light-harvesting chlorophyll ab protein and which acts as a light-harvesting antenna primarily for Photosystem II.Cations dramatically alter the structural interaction of the light-harvesting complex with the photochemically active system II complex. This interaction has been measured by determining the amount of protein-bound chlorophyll b and Photosystem II activity which can be released into dispersed subunits by digitonin treatment of chloroplast lamellae. When cations are present to cause interaction between the Photosystem II complex and the light-harvesting pigment · protein, the combined complexes pellet as a “heavy” membranous fraction during differential centrifugation of detergent treated lamellae. In the absence of cations, the two complexes dissociate and can be isolated in a “light” submembrane preparation from which the light-harvesting complex can be purified by sucrose gradient centrifugation.Cation effects on excitation energy distribution between Photosystems I and II have been monitored by following Photosystem II fluorescence changes under chloroplast incubation conditions identical to those used for detergent treatment (with the exception of chlorophyll concentration differences and omission of detergents). The cation dependency of the pigment · protein complex and Photosystem II reaction center interactions measured by detergent fractionation, and regulation of excitation energy distribution as measured by fluorescence changes, were identical. We conclude that changes in substructural organization of intact membranes, involving cation induced changes in the interaction of intramembranous subunits, are the primary factors regulating the distribution of excitation energy between Photosystems II and I.  相似文献   

6.
J. Haveman  P. Mathis 《BBA》1976,440(2):346-355
A comparative study is made, at 15 °C, of flash-induced absorption changes around 820 nm (attributed to the primary donors of Photosystems I and II) and 705 nm (Photosystem I only), in normal chloroplasts and in chloroplasts where O2 evolution was inhibited by low pH or by Tris-treatment.At pH 7.5, with untreated chloroplasts, the absorption changes around 820 nm are shown to be due to P-700 alone. Any contribution of the primary donor of Photosystem II should be in times shorter than 60 μs.When chloroplasts are inhibited at the donor side of Photosystem II by low pH, an additional absorption change at 820 nm appears with an amplitude which, at pH 4.0, is slightly higher than the signal due to oxidized P-700. This additional signal is attributed to the primary donor of Photosystem II. It decays (t12 about 180 μs) mainly by back reaction with the primary acceptor and partly by reduction by another electron donor. Acid-washed chloroplasts resuspended at pH 7.5 still present the signal due to Photosystem II (t12 about 120 μs). This shows that the acid inhibition of the first secondary donor of Photosystem II is irreversible.In Tris-treated chloroplasts, absorption changes at 820 nm due to the primary donor of Photosystem II are also observed, but to a lesser extent and only after some charge accumulation at the donor side. They decay with a half-time of 120 μs.  相似文献   

7.
Three chlorophyll-protein complexes of a Chroomonas species (Cryptophyceae) have been separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis. The two bands at 100 and 42 kDa are Complex I (CP I) and Complex IV (CP IV), the ubiquitous chlorophyll a-proteins associated with Photosystems I and II, respectively. The third 55 kDa band, which had two peptide subunits (24 and 20 kDa), contained both chlorophyll a and chlorophyll c2 in a molar ratio of 1.4 chlorophyll a to 1 chlorophyll c2 (chlorophyll achlorophyll c2 ratio in whole cells = 4). A chlorophyll ac2 fraction with similar spectral and electrophoretic properties was isolated by digitonin-sucrose density gradient centrifugation. This fraction had no photochemical activity and contained only a single carotenoid species with absorbance maxima in methanol at 424, 448 and 476 nm. Efficient energy transfer from chlorophyll c2 to chlorophyll a occurred in the complex.  相似文献   

8.
J.A. Van Best  P. Mathis 《BBA》1978,503(1):178-188
Absorption changes (ΔA) at 820 nm, following laser flash excitation of spinach chloroplasts and Chlorella cells, were studied in order to obtain information on the reduction time of the photooxidized primary donor of Photosystem II at physiological temperatures.In the microsecond time range the difference spectrum of ΔA between 750 and 900 nm represents a peak at 820 nm, attributable to a radical-cation of chlorophyll a. In untreated dark-adapted material the signal can be attributed solely to P+?700; it decays in a polyphasic manner with half-times of 17 μs, 210 μs and over 1 ms. The oxidized primary donor of Photosystem II (P+II) is not detected with a time resolution of 3 μs. After treatment with 3–10 mM hydroxylamine, which inhibits the donor side of Photosystem II, P+II is observed and decays biphasically (a major phase with t12 = 20–40 μs, and a minor phase with t12 ? 200 μs), probably by reduction by an accessory electron donor.In the nanosecond range, which was made accessible by a new fast-response flash photometer operating at 820 nm, it was found the P+II is reduced with a half-time of 25–45 ns in untreated dark-adapted chloroplasts. It is assumed that the normal secondary electron donor is responsible for this fast reduction.  相似文献   

9.
Thylakoid membrane protein phosphorylation affects photochemical reactions of Photosystem II. Incubation of thylakoids in the light with ATP leads to: (1) an increase in the amplitude of three components (4–6, 25–45 and 280–300 μs) of delayed light emission after a single flash without any change in their kinetics; (2) a reduction of the flash-dependent binary oscillations of chlorophyll a fluorescence yield associated with electron transfer from the primary quinone acceptor, Q, to the secondary quinone acceptor, B; (3) an increase in the B?B ratio resulting from an increase in stability of the semiquinone anion during dark adaptation; and (4) no change in the redox state of the plastoquinone pool as determined by flash-induced photooxidation of the Photosystem I reaction center, P-700. All the above observations are reversible upon dephosphorylation of the thylakoid membranes. These data are explained by a protein phosphorylation-induced stabilization of the bound semiquinone anion, B?. It is proposed that this increased stability may be due to an alteration in the accessibility of an endogenous reductant to B, or to an increase in dissipative cycling of charge around Photosystem II.  相似文献   

10.
The wavelength-resolved fluorescence emission kinetics of the accessory pigments and chlorophyll a in Porphyridium cruentum have been studied by picosecond laser spectroscopy. Direct excitation of the pigment B-phycoerythrin with a 530 nm, 6 ps pulse produced fluorescence emission from all of the pigments as a result of energy transfer between the pigments to the reaction centre of Photosystem II. The emission from B-phycoerythrin at 576 nm follows a nonexponential decay law with a mean fluorescence lifetime of 70 ps, whereas the fluorescence from R-phycocyanin (640 nm), allophycocyanin (660 nm) and chlorophyll a (685 nm) all appeared to follow an exponential decay law with lifetimes of 90 ps, 118 ps and 175 ps respectively. Upon closure of the Photosystem II reaction centres with 3-(3,4-dichlorophenyl)-1,1-dimethylurea and preillumination the chlorophyll a decay became non-exponential, having a long component with an apparent lifetime of 840 ps. The fluorescence from the latter three pigments all showed finite risetimes to the maximum emission intensity of 12 ps for R-phycocyanin, 24 ps for allophycocyanin and 50 ps for chlorophyll a.A kinetic analysis of these results indicates that energy transfer between the pigments is at least 99% efficient and is governed by an exp ?At12 transfer function. The apparent exponential behaviour of the fluorescence decay functions of the latter three pigments is shown to be a direct result of the energy transfer kinetics, as are the observed risetimes in the fluorescence emissions.  相似文献   

11.
A.W.D. Larkum  Jan M. Anderson 《BBA》1982,679(3):410-421
A Photosystem II reaction centre protein complex was extracted from spinach chloroplasts using digitonin. This complex showed (i) high rates of dichloroindophenol and ferricyanide reduction in the presence of suitable donors, (ii) low-temperature fluorescence at 685 nm with a variable shoulder at 695 nm which increased as the complex aggregated due to depletion of digitonin and (iii) four major polypeptides of 47, 39, 31 and 6 kDa on dissociating polyacrylamide gels. The Photosystem II protein complex, together woth the P-700-chlorophylla protein complex and light-harvesting chlorophyll ab-protein complex (LHCP) also isolated using digitonin, were reconstituted with lipids from spinach chloroplasts to form proteoliposomes. The low-temperature (77 K) fluorescence properties of the various proteoliposomes were analysed. The F685F695 ratios of the Photosystem II reaction centre protein complex-liposomes decreased as the lipid to protein ratios were increased. The F681F697 ratios of LHCP-liposomes were found to behave similarly. Light excitation of chlorophyll b at 475 nm stimulated emission from both the Photosystem II protein complex (F685 and F695) and the P-700-chlorophyll a-protein complex (F735) when LHCP was reconstituted with either of these complexes, demonstrating energy transfer between LHCP and PS I or II complexes in liposomes. No evidence was found for energy transfer from the PS II complex to the P-700-chlorophyll a-protein complex reconstituted in the same proteoliposome preparation. Proteoliposome preparations containing all three chlorophyll-protein complexes showed fluorescence emission at 685, 700 and 735 nm.  相似文献   

12.
Ta-Yan Leong  Jan M. Anderson 《BBA》1983,723(3):391-399
The hypothesis that chloroplasts having different light-saturated rates of photosynthesis will have different proportions of the intrinsic thylakoid complexes engaged in light-harvesting and electron transport (Anderson, J.M. (1982) Mol. Cell. Biochem. 46, 161–172) has been tested. Peas were grown in light regimes which varied in light intensity, quality and time of irradiance, and ranged from sunlight through red to blue-enriched light of very low radiation. The electron-transport capacity at saturating light of Photosystem I and Photosystem II of chloroplasts isolated from light-adapted peas was 2-fold and 5–6-fold lower, respectively, in the lowest radiation compared to sunlight. There was a marked increase in the amount of total chlorophyll associated with the main chlorophyll ab-proteins (LHCP1, LHCP2 and LHCP3) and a 2-fold decrease in the core reaction centre complex of Photosystem II (CP a) as the radiation decreased; the LHCP1–3CP a ratio changed from 3.5 to 9.0. The amount of chlorophyll associated with Photosystem I varied from 34% in sunlight to 27% in the lowest radiation, but the antenna size of Photosystem I was not markedly different; there was a 2-fold decrease in the amount of cytochrome f on a chlorophyll basis, which partly accounted for the decreased electron-transport capacity of Photosystem I. Since the increases or decreases in the levels of each of the components correlated with decreasing radiation, it is clear that the light-adaptation of both light-harvesting and electron-transport components is indeed closely co-ordinated.  相似文献   

13.
Phosphorylation in vitro of the light-harvesting chlorophyll ab protein complex associated with Photosystem II (LHCII) resulted in the lateral migration of a subpopulation of LHCII from the grana to the stroma lamellae. This movement was characterized by a decrease in the chlorophyll ab ratio and an increase in the 77 K fluorescence emission at 681 nm in the stroma lamellae following phosphorylation. Polyacrylamide gel electrophoresis indicated that the principal phosphoproteins under these conditions were polypeptides of 26–27 kDa. These polypeptides increased in relative amount in the stroma lamellae and decreased in the grana during phosphorylation. Pulse/chase experiments confirmed that the polypeptides were labelled in the grana and moved to the stroma lamellae in the subsequent chase period. A fraction at the phospho-LHCII, however, was unable to move and remained associated with the grana fraction. LHCII which moved out into the stroma lamellae effectively sensitized Photosystem I (PS I), since the ability to excite fluorescence emission at 735 nm (at 77 K) by chlorophyll b was increased following phosphorylation. These data support the ‘mobile antenna’ hypothesis proposed by Kyle, Staehelin and Arntzen (Arch. Biochem. Biophys. (1983) 222, 527–541) which states that the alterations in the excitation-energy distribution induced by LHCII phosphorylation are, in part, due to the change in absorptive cross-section of PS II and PS I, resulting specifically from the movement of LHCII antennae chlorophylls from the PS-II-enriched grana to the PS-I-enriched stroma lamellae.  相似文献   

14.
15.
W. Junge  H. Schaffernicht  N. Nelson 《BBA》1977,462(1):73-85
The mutual orientation of pigments in Photosystem I reaction centers from spinach is evaluated by polarized photochemistry. The photoinduced linear dichroism of the absorption changes of chlorophyll a1 at 701 nm is studied as function of the excitation wavelength. The Photosystem I reaction center particles contain about 100 and if depleted about 40 chlorophylls, respectively. To prevent their rapid Brownian rotation they were immobilized on DEAE-Sephadex.The excitation spectrum of the linear dichroism reveals a high degree of order between the long axis of β-carotene and the Qy transition moments of those chlorophyll a molecules absorbing at the red end of the spectrum. The latter are the most endangered ones for destructive oxidation via their triplet state. Hence, the location of β-carotene in close proximity to and in parallel with these chlorophylls seems to be most favourable for the protective role of β-carotene within the antennae system I. It is observed that the dichroic ratio of the absorption changes of chlorophyll a1 does not exceed a figure of 43, which characterizes a circularly degenerate system, even at far red excitation (724 nm). This will hit selectively those few chlorophyll a molecules with their peak absorption at about 700 nm (including the photooxidizable dimer). We conclude, if the dimer is the only species peaking at 700 nm then the two chlorophyll a within the dimer have their y-axes oriented perpendicular to each other. If there are some antennae in addition to the dimer, the y-axes of all chlorophyll-a peaking at 700 nm form a star which accounts for the circular degeneracy of absorption.  相似文献   

16.
(1) Five minor chlorophyll-protein complexes were isolated from thylakoid membranes of the green alga Acetabularia by SDS-polyacrylamide gel electrophoresis, after SDS or octylglucoside solubilization. None of them were related to CP I (Photosystem I reaction center core) or CP II (chlorophyll ab light-harvesting complex). (2) Two complexes (CPa-1 and CPa-2) contained only chlorophyll (Chl) a, with absorption maxima of 673 and 671 nm, and fluorescence emission maxima of 683 nm compared to 676 nm for CP II. The complexes had apparent molecular masses of 43–47 and 38–40 kDa, and contained a single polypeptide of 41 and 37 kDa, respectively. They each account for about 3% of the total chlorophyll. (3) Three complexes had identical spectra, with Chl ab ratios of 3–4 compared to 2 for thylakoid membranes, and a pronounced shoulder around 485 nm indicating enrichment in carotenoids. One of them was the complex ‘CP 29’ (Camm, E.L. and Green, B.R. (1980) Plant Physiol. 66, 428–432) and the other two were slightly different oligomeric forms of CP 29. They could be formed from CP 29 during reelectrophoresis; but about half the complex was isolated originally in an oligomeric form. Together they account for at least 7% of the total chlorophyll. Their function is unknown.  相似文献   

17.
The yield of P-700 photooxidation has been studied in isolated chloroplast membranes by measuring the extent of the flash-induced absorption increase at 820 nm (ΔA820) in the microsecond time range. The extent of ΔA820 induced by non-saturating laser flashes was increased by the following treatments. (1) Suspension of chloroplast membranes in Mg2+ free medium (plus 15 mM K+) which leads to unstacking of grana (as detected by a decrease in chlorophyll fluorescence). (2) Reduction of Q, the primary acceptor of Photosystem II, in the presence of 20 μM 3-(3,4 dichlorophenyl)-1,1-dimethylurea by a saturating xenon flash, fired 300 ms before the laser flash. (3) Phosphorylation of light harvesting chlorophyll ab-protein complex, which occurs in the presence of ATP after activation of protein kinase in the dark with NADPH and ferredoxin. We conclude that the Mg2+ concentration, the redox state of Q and the protein-phosphorylation all can control the photochemical efficiency of P-700 photooxidation in isolated chloroplasts, and we discuss these results in relation to control of excitation energy distribution between the two photosystems. We also discuss the significance of these results in relation to the regulation of photosynthetic electron transport in vivo.  相似文献   

18.
O2 uptake in spinach thylakoids was composed of ferredoxin-dependent and -independent components. The ferredoxin-independent component was largely 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) insensitive (60%). Light-dependent O2 uptake was stimulated 7-fold by 70 μM ferredoxin and both uptake and evolution (with O2 as the only electron acceptor) responded almost linearly to ferredoxin up to 40 μM. NADP+ reduction, however, was saturated by less than 20 μM ferredoxin. The affinity of O2 uptake for for O2 was highly dependent on ferredoxin concentration, with K12(O2) of less than 20 μM at 2 μM ferredoxin but greater than 60 μM O2 with 25 μM ferredoxin. O2 uptake could be suppressed up to 80% with saturating NADP+ and it approximated a competitive inhibitor of O2 uptake with a Ki of 8–15 μM. Electron transport in these thylakoids supported high rates of photophosphorylation with NADP+ (600 μmol ATP/mg Chl per h) or O2 (280 μmol/mg Chl per h) as electron acceptors, with ATP2e ratios of 1.15–1.55. Variation in ATP2e ratios with ferredoxin concentration and effects of antimycin A indicate that cyclic electron flow may also be occurring in this thylakoid system. Results are discussed with regard to photoreduction of O2 as a potential source of ATP in vivo.  相似文献   

19.
In intact, uncoupled type B chloroplasts from spinach, added ATP causes a slow light-induced decline (t12 ≈ 3 min) of chlorophyll a fluorescence at room temperature. Fluorescence spectra were recorded after fast cooling to 77 K and normalized with fluorescein as an internal standard. Related to the fluorescence quenching at room temperature, an increase in Photosystem (PS) I fluorescence (F735) and a decrease in PS II fluorescence (F695) were observed in the low-temperature spectra. The change in the F735F695 ratio was abolished by the presence of methyl viologen. Fluorescence induction at 77 K of chloroplasts frozen in the quenched state showed lowered variable (Fv) and initial (F0) fluorescence at 690 nm and an increase in F0 at 735 nm. The results are interpreted as indicating an ATP-dependent change of the initial distribution of excitation energy in favor of PS I, which is controlled by the redox state of the electron-transport chain and, according to current theories, is caused by phosphorylation of the light-harvesting complex.  相似文献   

20.
G. Kulandaivelu  H. Senger 《BBA》1976,430(1):94-104
The kinetics (region of seconds) of the light-induced 520 nm absorbance change and its dark reversal have been studied in detail in the wild type and in some pigment and photosynthetic mutants of Scenedesmus obliquus. The following 5 lines of evidence led us to conclude that the signal is entirely due to the photosystem I reaction modified by electron flow from Photosystem II.Gradual blocking of the electron transport with 3(3,4-dichlorophenyl)-1,1-dimethylurea resulted in diminution and ultimate elimination of the biphasic nature of the signal without reducing the extent of the absorbance change or of the dark kinetics. On the contrary, blocking electron flow at the oxidizing side of plastoquinone with 2, 5-dibromo-3-methyl-6-isoprophyl-p-benzoquinone or inactivating the plastocyanin with KCN, prolonged the dark reversal of the absorbance change apart from abolishing the biphasic nature of the signal.Action spectra clearly indicate that the main signal (I) is due to electron flow in Photosystem I and that its modification (Signal II) is due to the action of Photosystem II.Signal I is pH independent, whereas Signal II demonstrates a strong pH dependence, parallel to the O2-evolving capacity of the cells.Chloroplast particles isolated from the wild type Scenedesmus cells demonstrated in the absence of any added artificial electron donor or acceptor and also under non-phosphorylation conditions the 520 nm absorbance change with approximately the same magnitude as whole cells. The dark kinetics of the particles were comparatively slower. Removal of plastocyanin and other electron carriers by washing with Triton X-100 slowed down the kinetics of the dark reversal reaction to a greater extent. A similar positive absorbance change at 520 nm and slow dark reversal was also observed in the Photosystem I particles prepared by the Triton method.Mutant C-6E, which contains neither carotenoids nor chlorophyll b and lacks Photosystem II activity, demonstrates a normal signal I of the 520 nm absorbance change. This latter result contradicts the postulate that carotenoids are the possible cause of the 520 nm absorbance change.  相似文献   

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