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1.
The light-harvesting accessory pigment-protein complex (LHC) with a chlorophyll (Chl) ab ratio of 1.2 was isolated by treating pea chloroplasts with Triton X-100. The LHC was used to investigate the action of ionic (sodium dodecyl sulfate) and non-ionic (Triton X-100) detergents. By optical methods (absorption and fluorescence spectra, measurements of fluorescence yield, ?, and lifetime, τ) two successive stages of the process were demonstrated, namely (1) interaction between detergent monomers and proteins and (2) solubilization of pigments into detergent micelles, which is facilitated by the presence of salts. The concentration ranges, characteristic of these stages, differ by 1.5–2 orders of magnitude for SDS, but slightly overlap for Triton X-100. At the second stage, certain changes occur in LHC absorption and fluorescence spectra. Several stable states of the LHC were established: (1) an aggregated state formed in the presence of 10 mM MgSO4 with τ ≈ 0.6 ns; (2) the dialyzed LHC with τ ≈ 0.9 ns; (3) the states of the LHC in detergent solution with τ ≈ 2.3, 2.9, 3.4 ns; (4) a 30 kilodalton monomer obtained by SDS-polyacrylamide gel electrophoresis with τ ≈ 4.1 ns. The fluorescence parameters of the LHC states were compared with those of Chl a in detergent micelles (for the micelles τ = 5.6–6.0 ns. The τ? ratio (the criterion for emission heterogeneity) for the LHC in the absence of a detergent was shown to be higher at least by a factor of 3.5 than that for Chl a in the presence of a detergent. Successive additions of the detergent to the LHC cause gradual decrease in the τ? ratio, and for the LHC monomer it reaches practically the same value as for Chl a in detergent micelles. The results are discussed on the basis of the data obtained previously. It is suggested that in vivo LHCs do not form such aggregates as in water solution without a detergent.  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.
Electrophoretic analysis by sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis showed that the light-harvesting chlorophyll ab-protein complex of barley thylakoids contains only one polypeptide of apparent molecular weight 26 000. The barley mutant, deficient in chlorophyll b and this light-harvesting complex, lacks this polypeptide.The addition of a nonionic detergent, Triton X-100, to the sodium dodecyl solubilization buffer prior to SDS polyacrylamide tube gel electrophoresis, allowed separation of a relatively stable complex, characterized as an oligomeric form of the light-harvesting complex. The oligomer also contained a polypeptide with an apparent molecular weight of 26 000. The absorption and fluorescence spectral properties of the oligomer are similar to those of the monomer. It is suggested that the oligomer of the light-harvesting chlorophyll ab-protein is closer to the in vivo form rather than the monomer.  相似文献   

4.
《BBA》1987,893(2):349-364
Steady-state and picosecond time-resolved fluorescence techniques in conjunction with circular dichroism have been used to study the light-harvesting chlorophyll-a/b protein complex (LHC) isolated from pea chloroplasts. In particular, the effect of changing the detergent / chlorophyll ratio on the state of the LHC has been investigated. Our results have been interpreted in light of the known protein geometry of the LHC in 2-dimensional crystals (Kühlbrandt, W. (1984) Nature 307, 478–479). The fluorescence lifetime data reveals 1 / e-lifetimes of 3.53 (±0.04) ns and 1.10 (±0.01) ns for a stable, efficiently energy-transferring state of the LHC. Subnanosecond lifetimes are observed under conditions leading to aggregation, while a long component of 5.50 (±0.16) ns corresponding to free Chl a is found when the detergent / chlorophyll ratio is high. The circular dichroism shows a major Chl-b exciton, a Chl-a / b exciton and a further ‘quenching’ Chl-b exciton. These have been attributed to: a C3 symmetric Chl-b interaction for which the intact C3 protein trimer geometry is a prerequisite; a dimeric Chl-a / b interaction, the presence of which is critically dependent on the detergent type; and a further Chl-b interaction which arises from the presence of aggregated trimers, respectively. We have found that the degree of heterogeneity with respect to the oligomeric state of the pigment-protein trimers is dependent upon the detergent / chlorophyll ratio used. Low detergent / chlorophyll ratios result in extensive aggregation of the trimers with a geometry similar to that found in 2-dimensional crystals of the LHC. Moderate detergent conditions yield predominantly non-aggregated trimers. Excess detergent conditions result in considerable chromophore heterogeneity and loss of the main Chl-b exciton consistent with protein denaturation through an initial break up of the trimer geometry. From these results we believe that in vitro the minimum stable functional unit corresponds to a C3 symmetric protein trimer.  相似文献   

5.
The time-resolved chlorophyll fluorescence emission of higher plant chloroplasts monitors the primary processes of photosynthesis and reflects photosynthetic membrane organization. In the present study we compare measurements of the chlorophyll fluorescence decay kinetics of the chlorophyll-b-less chlorina-f2 barley mutant and wild-type barley to investigate the effect of alterations in thylakoid membrane composition on chlorophyll fluorescence. Our analysis characterizes the fluorescence decay of chlorina-f2 barley chloroplasts by three exponential components with lifetimes of approx. 100 ps, 400 ps and 2 ns. The majority of the chlorophyll fluorescence originates in the two faster decay components. Although photo-induced and cation-induced effects on fluorescence yields are evident, the fluorescence lifetimes are independent of the state of the Photosystem-II reaction centers and the degree of grana stacking. Wild-type barley chloroplasts also exhibit three kinetic fluorescence components, but they are distinguished from those of the chlorina-f2 chloroplasts by a slow decay component which displays cation- and photo-induced yield and lifetime changes. A comparison is presented of the kinetic analysis of the chlorina-f2 barley fluorescence to the decay kinetics previously measured for intermittent-light-grown peas (Karukstis, K. and Sauer, K. (1983) Biochim. Biophys. Acta 725, 384–393). We propose that similarities in the fluorescence decay kinetics of both species are a consequence of analogous rearrangements of the thylakoid membrane organization due to the deficiencies present in the light-harvesting chlorophyll ab complex.  相似文献   

6.
Beverley R. Green  Edith L. Camm 《BBA》1982,681(2):256-262
Reelectrophoresis of the oligomer form (CP II1) of the chlorophyll ab light-harvesting complex (LHC) from the green alga Acetabularia yields two green bands which run at the position typical of the monomer (CP II). The upper green band (CP II1) is enriched in the 27 kDa polypeptide of the LHC, while the lower is enriched in the 26 kDa polypeptide. The fact that both bands have both chlorophyll (Chl) a and b, and in the same ratio, implies that the LHC is made up of two Chl ab proteins. Neither of these bands can be attributed to the Chl ab complex ‘CP 29’ (Camm, E.L. and Green, B.R. (1980) Plant Physiol. 66, 428–432). Resolution of CP II1 and CP II2 of spinach can be obtained if sucrose gradient fractions of an octylglucoside extract are subjected to SDS-polyacrylamide gel electrophoresis. CP II1 and CP II2 are interpreted as being fundamental subunits of the light-harvesting complex as it is defined on SDS-polyacrylamide gels.  相似文献   

7.
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   

8.
Jan M. Anderson 《BBA》1983,724(3):370-380
Eight chlorophyll-protein complexes were isolated from thylakoid membranes of a Codium species, a marine green alga, by mild SDS-polyacrylamide gel electrophoresis. CP 1a1, CP 1a2, CP 1a3 and CP 1a4 were partially dissociated Photosystem (PS) I complexes, which in addition to the core reaction centre complex, CP 1, possessed PS I light-harvesting complexes containing chlorophyll (Chl) a, Chl b and siphonaxanthin. LHCP1 and LHCP3 are orange-brown green chlorophyll ab-proteins (Chl aChl b ratios of 0.66) that contain siphonaxanthin and its esterified form, siphonein. CP a and CP 1, the core reaction centre complexes of PS II and PS I, respectively, had similar spectral properties to those isolated from other algae or higher plants. These P-680- or P-700-Chl a-proteins are universally distributed among algae and terrestrial plants; they appear to be highly conserved and have undergone little evolutionary adaptation. Siphonaxanthin and siphonein which are present in the Codium light-harvesting complexes of PS II and PS I are responsible for enhanced absorption in the green region (518 and 538 nm). Efficient energy transfer from both xanthophylls and Chl b to only Chl a in Codium light-harvesting complexes, which have identical fluorescence emission spectra at 77 K to those of the lutein-Chl ab-proteins (Chl aChl b ratios of 1.2) of most green algae and all higher plants, proved that the molecular arrangement of these light-harvesting pigments was maintained in the isolated Codium complexes. The siphonaxanthin-Chl ab-proteins allow enhanced absorption of blue-green and green light, the predominant light available in deep ocean waters or shaded subtidal marine habitats. Since there is a variable distribution of lutein, siphonaxanthin and siphonein in marine green algae and siphonaxanthin is found in very ancient algae, these novel siphonein-siphonaxanthin-Chl ab-proteins may be ancient light-harvesting complexes which were evolved in deep water algae.  相似文献   

9.
(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   

10.
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   

11.
The fluorescence properties of chlorophyll a and b monomolecular films at the air-water interface were measured by a high sensitivity fluorophotometer using the photon-counting method. The fluorescence intensity of chlorophyll molecules in monomolecular films in the absence of any diluents did not decrease simply with the mean distance of chlorophyll molecules. Over the range of the mean distances from 27 to 21 Å, three fluorescence components (peaks at 685, 695 and 715 nm) of chlorophyll a were observed. In the case of chlorophyll b, two fluorescence components (peaks at 667 and 685 nm) were observed over the range of the mean distances from 34 to 24 Å. When the mean distance was 18 Å, the short wavelength component of chlorophyll b disappeared, and only the long wavelength component was observed.  相似文献   

12.
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   

13.
K. Csatorday  E. Lehoczki  L. Szalay 《BBA》1975,376(2):268-273
The process of energy transfer was studied in the chlorophyll a-containing detergent micelle, serving as a possible model of the photosynthetic unit. Chlorophyll a was added to aqueous solutions of the detergent Triton X-100 and incorporated into the micelles. The energy transfer process was studied by investigating the concentration depolarization of fluorescence of chlorophyll a. On the basis of the experimental depolarization curves as well as the value of the Förster parameter R0 = 56 A? calculated from the overlap of absorption and fluorescence spectra it was concluded that energy transfer between chlorophyll a molecules in this model follows the Förstertype mechanism of inductive resonance. Furthermore it was found that the local concentration of chlorophyll a in the micelles is higher by 1–3 orders of magnitude than its overall concentration in the solution and by choosing the appropriate ratio between the concentration of chlorophyll a and the detergent it is possible to reach the in vivo chlorophyll concentration of 0.1 M within the micelles. Thus the chlorophyll-detergent micelle model may be applied as a model of the separate package-type photosynthetic unit.  相似文献   

14.
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   

15.
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   

16.
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   

17.
The present study examined the protein associations and energy transfer characteristics of chlorophyll c and fucoxanthin which are the major light-harvesting pigments in the brown and diatomaceous algae. It was demonstrated that sodium dodecyl sulfate (SDS)-solubilized photosynthetic membranes of these species when subjected to SDS polyacrylamide gel electrophoresis yielded three spectrally distinct pigment-protein complexes. The slowest migrating zone was identical to complex I, the SDS-altered form of the P-700 chlorophyll a-protein. The zone of intermediate mobility contained chlorophyll c and chlorophyll a in a molar ratio of 2 : 1, possessed no fucoxanthin, and showed efficient energy transfer from chlorophyll c to chlorophyll a. The fastest migrating pigment-protein zone contained fucoxanthin and chlorophyll a, possessed no chlorophyll c, and showed efficient energy transfer from fucoxanthin to chlorophyll a. It is demonstrated that the chlorophyll ac-protein and the chlorophyll afucoxanthin-protein complexes are common to the brown algae and diatoms examined, and likely share similar roles in the photosynthetic units of these species.  相似文献   

18.
This report concerns the large circular dichroic (CD) signal of intact chloroplasts of higher plants. The CD spectra of chloroplasts are compared with the aggregated form of the light-harvesting chlorophyll ab complex at 25°C and ?250°C. The light-harvesting chlorophyll aggregate has a CD of magnitude equal to or greater than chloroplasts, but of opposite sign, and it is not related to the CD of the unaggregated form, and hence its arrangement is an artefact compared to the arrangement in the chloroplast. We suggest that this preparation, which has pseudo-lamellar structure, is a clear example of a large CD signal being generated by macromolecular association. The asymmetry of organization in the chloroplast has an opposite sense to that of the aggregate, but affects only chlorophyll a, not chlorophyll b.  相似文献   

19.
We have observed the development and decay of triplet excitons formed in the ‘antenna’ chlorophyll ab protein complex by high-intensity laser excitation. The carotenoid triplet (3Car) appeared 5 ns after excitation in the protein isolation, commonly termed CP-II; the risetime in a larger antenna particle, called LHC (light-harvesting complex) was 12 ns. The quantum yield of 3Car in CP-II decreased 11-fold as intensity was increased from 1016 to 2 · 1017 photons/cm2 per pulse. The effect is attributed to exciton annihilation during the initial period of triplet formation. Above 5 · 1016 photons/cm2 per s, the 3Car lifetime decreases substantially from its low intensity value of 8.7 μs. A comparison of the transient absorption spectrum of CP-II with those of chlorophyll and carotenoid in vitro indicates that ‘trapped’ chlorophyll triplets formed at high intensities. We present a simple model of destructive interaction between 3Car and chlorophyll triplets which is compatible with the observed increased rate of 3Car decay. Indirect evidence suggests similar effects occur in LHC.  相似文献   

20.
O. Machold  A. Meister 《BBA》1979,546(3):472-480
Thylakoids of Vicia faba chloroplasts disaggregated by sodium dodecyl sulfate were separated by means of different electrophoretic systems. Under the conditions of a high resolving gel system the chlorophyll containing zone previously termed chlorophyll-protein complex II or light-harvesting chlorophyll ab-protein was found to be inhomogeneous. It represents a mixture of two distinct chlorophyll-proteins characterized by different spectral properties and different apoproteins. One chlorophyll-protein exhibits a chlorophyll ab ratio of 0.9 and is associated with polypeptides of 24 000 and 23 000 daltons. The 24 000 dalton band is proved to bind chlorophyll and has a light-harvesting function. The function of the 23 000 dalton band is unknown. The second chlorophyll-protein has a chlorophyll ab ratio of 2.1 and an additional absorption maximum in the position of 637 nm. It is associated with only one polypeptide which has an apparent molecular weight of 23 000. The two 23 000 dalton polypeptides occurring in both complexes are not identical.  相似文献   

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