photosystems, Photosystem I (PSI) and I1 (PSII), which act together in a ... (ii) A thorough analysis of the structural changes which occur when isolated thylakoid ...
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600th MEETING, OXFORD
The Structure and Function of Light-Transducing Membranes Colloquium organized on behalf of the Membrane Group and the British Photobiology Society by P. J. Quinn and D. J. Chapman and edited by P. J. Quinn (London)
Interactions between Photosystem I and I1 membrane organization has arisen from two different approaches. (i) Improved isolation and characterization of various types of thylakoid membrane fragments using phase-separation techniques (Andersson, 1978; Anderson, 1981) (ii) A thorough analysis of the structural changes which occur when isolated thylakoid membranes are subjected to different ionic environments (Barber & Chow, 1979; Barber, 1980a). It is the latter approach which I will mainly focus on in the present communication, although reference to fragmentation studies will be made. When thylakoid membranes of higherplant chloroplasts are isolated and suspended in a medium containing low levels of univalent cations (1-IOmM) they become fully unstacked and show a low yield of chlorophyll fluorescence even when the PSII reaction centre is closed by the addition of the herbicide DCMU [3-(3,4-dichlorophenyl)-I , 1dimethylureal. Increasing the levels of cations in the medium
J. BARBER Department of Pure and Applied Biology, Imperial College, London S . W.7, U.K. 0,-evolving photosynthetic organisms contain two different photosystems, Photosystem I (PSI) and I1 (PSII), which act together in a collaborative manner to extract electrons and protons from water and raise them to a redox potential capable of reducing CO,. The early work of Wessels (1964) and of Boardman & Anderson (1964) suggested that these two different photosystems, consisting of a photochemical reaction centre and associated light-harvesting pigment antennae, existed as separate intrinsic protein entities within the chloroplast thylakoid membrane. More recently, a much clearer picture has emerged of the relationship between PSII and PSI, which raises important questions about the mechanism of both energy and electron transfer between them. The new picture of thylakoid
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High fluorescence
Good energy transfer
/ ' d
Low fluorescence
Lateral pigment-protein diffusion
4
/ I
Salt
I I
Time
Fig. 1 . A diagrammatic representation o, how alterinz the electrolyte composition of medium so as to change the screening of surface electrical charges brings about lateral diflusion of integral pigment-protein complexes in the chloroplast thylakoid membrane The protein complex of PSII, including the light-harvesting chlorophyll a/chlorophyll b protein (shown as an unshaded particle, a), is postulated to carry a low net electrical charge on its exposed surface and therefore preferentially aggregates in regions where close membrane interaction can occur (strong van de Waals interactions between adjacent complexes and between adjacent membrane surfaces in the absence of significant short-range coulombic repulsion). The pigment-protein complexes of PSI are shown as black circles (0)and postulated to carry net negative charge on their exposed external surfaces. Because of this they aggregate less readily on addition of salt and are excluded from those membrane regions where the coulombic repulsion is suficient not to favour membrane appression. Under low-salt conditions, coulombic repulsion is large so as to prevent aggregation and membrane appression so that the PSH and PSI proteins are randomized in the plane of the membrane. The change from the randomized to the aggregated condition is reversible and can be readily monitored by detecting changes in the yield of chlorophyll fluoresence which reflects alterations in the degree of energy transfer from the PSII to PSI complexes as shown (Barber, 1980a). VOl.
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induces a re-organization of the membranes into their normal stacked and unstacked regions (Barber & Chow, 1979; Barber, 1980a). Concomitant with this conformational change is an 0 increase in the level of chlorophyll fluorescence, which is I usually thought to reflect a decrease in the energy transfer I ; Granal from the light-harvesting chlorophylls of PSII to those of PSI (Williams, 1977). The properties of the cation-induced changes in stacking and chlorophyll fluorescence have been studied in great detail and it has been concluded that the two phenomena are closely related and reflect changes in the balance between electrostatic and electrodynamic forces which exist between I protein complexes and between adjacent membrane surfaces (Barber, 1980a; Chow et al., 1980). It has been possible to use the classical electrostatic theory of Gouy & Chapman and new theories of Van der Waals forces for macroscopic systems to give a semi-quantitative analysis of the forces involved (Barber, 1980b; Sculley et al., 1980). The picture which has emerged from the various studies is shown in Fig. 1, where it can be seen that the addition of cations to the medium results in a change I from a randomized mixing of PSII and PSI pigment-protein complexes to discrete domains of PSII and PSI. The formation of domains implies that the surface properties of the PSI and PSII regions will be different, assuming that intrinsic protein complexes involved have exposed segments with different compositions. Because the PSII complexes aggregate in the appressed-membrane region, it follows that these proteins probably have a surface charge density sufficiently low to allow close membrane-membrane interaction (Barber, 1980b; Sculley et ab, 1980; Rubin ef al., 1981). If this argument is correct, it seems likely that the regions enriched in PSI are highly charged so that close membrane interaction is not normally possible, owing to coulombic repulsion. Freeze-fracture and other studies indicate that the organizational changes suggested in Fig. 1 do occur on adding cations (Wang & Packer, 1973). The model shown in Fig. 1 should be contrasted with other possible ways bf inducing membrane stacking, namely by electrostatic neutralization of surface electrical charges. Such a neutralization can al., 1980). The decrease in membrane fluidity was found to be occur, owing to cation binding or protonation giving rise to associated with an inhibition of the cation-induced fluorescence extensive stacking without the segregation of PSII and PSI rise and membrane stacking. Fractionation of thylakoids into protein complexes into separate domains (Barber, 1980a; stroinal (unstacked) and granal (stacked) fragments has allowed Barber et al., 1980). a closer examination of the heterogeneity of fluidity (Ford et al., The concept presented in Fig. 1 emphasizes the usefulness of 1982). As Fig. 2 shows, it seems that the stromal membranes are the chlorophyll fluorescence signal to monitor the dynamic more fluid than the granal membranes. This apparent difference changes which occur in thylakoid membrane organization on in the physical state of the two membrane regions was confirmed addition of cations. Thus the kinetics of the fluorescence rise by electron paramagnetic resonance studies using the spin should give information about the lateral diffusion of pigmentprobes, 5-doxyldecane and 12-doxylstearate (Ford et al, 1982). protein complexes within the lipid matrix of the membrane. By The higher fluidity of the stromal lamella compared with the using the coagulation theory of Schmoluchowski and the granal membranes is consistent with their lower protein-to-lipid twodimensional-analysis method of Adam & Delbruck, we ratio (1.22f0.11 for stromal and 1.84f0.16 for granal), have been able to estimate lateral diffusion coefficients for the although no significant difference in the degree of fatty acid PSII protein complex (Rubin et al., 1981). Values obtained unsaturation could be detected for the two membrane regions. to 3 . 0 8 ~10-11cm2.s-' over the Recently, we have extended the steady-state DPH fluorescenceranged from 1.85 x temperature range 10-3OoC. The concept of lateral movements polarization studies by carrying out time-resolved fluorescence of pigment-protein complexes within the thylakoid membrane measurements (R.C. Ford & J. Barber, unpublished work). and the sensitivity of this movement to temperature focuses With intact freshly isolated thylakoid membranes, a biphasic attention on the fluidity of the lipid matrix. The thylakoid decay could be resolved of the form: membrane contains a high proportion of unsaturated fatty acid F = A le-t/rI + A,e-lIW (mainly a-linolenic acid), and we have investigated its fluidity by using the hydrophobic fluorescence probe DPH (1,6- with (s.E.M.), A2=0.78+0.06, r,= A,=0.22&0.06 diphenylhexa-1,3,5-triene). Steady-state D P H fluorescence 1.4 k0.06ns and r,=7.4 f0.3ns. When the same experiment measurements gave polarization values of 0.20-0.25 for freshly was conducted with separated stromal and g r a d lamellae~ isolated membranes, which is indicative of a relatively fluid lipid the decays were still biphasic, but best described by mean matrix (Ford & Barber, 1980). Introduction of cholesterol lifetimes rstroma, = 6.411s and rSrana, =4.8ns. hemisuccinate, using polyvinylpyrrolidone ( 1-ethenyl-2The results and concepts presented above lead to a picture of pyrrolidinone polymers) as the 'carrier', into the thylakoid the organization of the thylakoid membrane, as shown in Fig. 3. membrane increased the D P H polarization value to about 0.35 PSII and PSI pigment proteins, together with their reaction (Yamamoto et al., 1981). This increase in the rigidity of the centres, are mainly located in appressed (non-exposed) and membrane is in line with the action of sterols to decrease the non-appressed (exposed) membrane regions respectively. Direct fluidity of biological membranes. A similar stiffening of the evidence for this distribution of PSII and PSI comes from phase membrane occurred when they were allowed to age (Barber et separation of fragmented membranes (Anderson, 1978; Ander-
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600th MEETING. OXFORD
Exposed grana membrane (high a)
/ Appressed grane membrane (low 0)
0
Coupling factor (CF, + CF,)
LHC-PSII pigment protein
0 Cytochrome b,-f
PSI pigment protein
protein
Fig. 3. A model for the possib., distribution of functionally active protein components in and on the appressed and non-appressed thylakoid membranes of higher-plant chIoroplasts
son, 1981). The location of the coupling factor (ATP synthetase), consisting of its intrinsic (CF,) and extrinsic (CF,) components, in the non-appressed membranes is well established (Murakami & Kunieda, 1976). The cytochrome b6-f complex is an intrinsic membrane protein of the electron-transport chain which acts as a plastoquinol: plastocyanin oxidoreductase. Evidence has been presented which suggests that this complex is located both in the appressed- and non-appressed-membrane regions (Cox & Anderson, 1981). The static model shown in Fig. 3 is probably too simple, for it is possible that lateral movements of protein complexes occurs between the appressed- and non-appressed-membrane regions. For example, changes in vivo in energy transfer between PSII and PSI at the pigment level seems to involve phosphorylation of the exposed segment of the light-harvesting pigment-protein complex of PSII (LHC) regulated by a membrane-bound kinase under the control of the redox state of plastoquinone (Horton & Black, 1980; Allen et al., 1981). It seems quite feasible that the phosphorylation would alter the surface-charge properties in the appressed-membrane region so as to induce coulombic repulsion favouring lateral displacement of some of the PSII complexes into the non-appressed membranes in such a way as to enhance PSII-to-PSI energy transfer (Barber, 1982). Dephosphorylation would reverse this process. Conclusive evidence for this type of structural regulation has yet to be presented, but there are findings in its favour, including changes in a- and p-centres before and after phosphorylation, where a-centres represent PSII complexes in domains and p-centres represent single PSII complexes, possibly in the non-appressed membranes (Butler. 1980: Kyle et al., 1982). Clearly the spatial separation of PSII from PSI to different membrane regions raises questions about the intercommunication of the two photosystems at the electrontransport level. It is generally accepted that a pool of plastoquinone acts as the electron/proton carrier between PSII and PSI and that it is this step in the overall electron/proton transfer from water to NADP which is rate-limiting, being 20ms under optimal conditions. Accepting that plastoquinone/plastoquinol diffusion would need to occur over about 500 nm, then a VOl.
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typical two-dimensional diffusion coefficient for membrane lipids of cmz. s-I would be in line with a rate-limiting step in the region of 20ms. Reduction of the fluidity of the membrane by lowering the temperature or by the incorporation of cholesterol hemisuccinate decreases the rate of electron transport from PSII to PSI in the way expected (Yamamoto et al., 1981). Also the artificial introduction of additional lipid into the membrane brings about an increase in the spatial separation of the functional proteins and decreases the concentration of the plastoquinone pool, resulting in a slowing down of electron transfer from PSII to PSI (P. Millner, D. J. Chapman & J. Barber, unpublished work). Even though the role of plastoquinone as a long-range diffusing carrier seems logical, there is the problem that some cytochrome b6-f complexes may be located close to PSII in the appressed membranes. In these cases the transfer of electrons from reduced cytochrome f to oxidized P700 would require the lateral diffusion of plastocyanin. Such diffusion of this extrinsic protein along the inner surface of the membrane is feasible, but it seems unlikely that this lateral diffusion would not be greatly affected by changes in the fluidity of the lipid matrix. Allen, J. F.. Bennett, J., Steinback, K. E. & Arntzen. C. J. (1981) Nature (London) 291. 1-5 Anderson, J. M. (1981)FEBSLett. 124, 1-10 Anderson, B. (1978)Ph.D. Thesis, University of Lund Barber, J. (19800)FEES Left. 118, 1-10 Barber, J. (1980b)Biochem. Biophvs. Acta 594. 253-308 Barber, J. (1982)Annu. Rev, Plant Physiol. 33,261-295 Barber. J. & Chow, W. S. (1979)FEES Lett. 105.5-lO Barber, J., Chow, W. S., ScoufRaire. C. & Lannoye, R. (1980) Biochim. Biophys. Acta 591,92-103
Boardman. N. K. & Anderson, J. M. (1964)Narure (London) 203. 166- I67
Butler. W.L. (1980)Proc. Natl. Acad. Sci.U.S.A. 77.4697-4701 Chow, W. S.. Thorne, S. W.. Duniec, J. T.. Sculley. M. J. & Boardman. N.K. (1980)Arch. Biochem. Biophvs. 201.347-355 Cox, R. P. & Anderson, B. (1981)Biochem. Biophvs. Res. Commun. 103, 1336-1642
Ford, R. C. & Barber, J. (1980)Phofobiochem. Photobiophys. 1, 263210
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Ford, R. C., Chapman, D. J., Barber, J., Pedersen, J. Z. & Cox, R. P. (1982)Biochim.Biophys. Actn in the press Horton, P. & Black, M.T. (1980)FEBS Len. 119,141-144 Kyle, D. J., Haworth, P. & Arntzen, C. J. (1982) Biochim. Biophys. Acta in the press Murakami, S . & Kunieda, R. (1976)Cell Struct. Function 1,389-392 Rubin, B. T.,Barber, J., Paillotin, G., Chow, W. S. & Yarnarnoto, Y. (198I) Biochim. Biophys.Acta 63869-74 Rubin, B. T., Chow, W. S. & Barber, J. (1981)Biochim. Biophys. Acta 634, 174- 190
Sculley, M.J., Duniec, J. T., Thorne, S. W., Chow, W. S. & Boardman, N. K. (1980)Arch. Biochem. Biophys. 201,339-346 Wang, A. Y. I. & Packer, L. (1973) Biochim. Biophys. Actn 305, 488-492 Wessels, J. S. C. (1964)Biochim.Biophys. Acta 19,64&642 Williams, W. P. (1977)in Primary Processes in Photosynthesis, ool. 2: Topics in Photosynthesis (Barber, J., ed.), pp. 94-147, Elsevier, Amsterdam Yamamoto, Y., Ford, R. C. & Barber, J. (1981) Plant Physiol. 67, 1069- I072
The structure of the bacterial photosynthetic unit RICHARD J. COGDELL.,* JANE VALENTINE,* J. GORDON LINDSAY? and KARIN SCHMIDT$ Departments of *Botany and tliochemistry, University of Glasgo w, Glasgo w GI 2 8QQ, Scotland, U.K., and $Institute of Microbiology, University of Gottingen, Gottingen, Federal Republic of Germany
complexes are arranged within the membrane to form the functional photosynthetic unit. Rps. acidophila is a purple non-sulphur photosynthetic bacterium and it takes its name from the fact that the optimal pH for its growth is 5.2 (Pfennig, 1969). The major lightabsorbing pigments in Rps. acidophila are bacteriochlorophyll a Bchl. a) and carotenoids of the ‘normal spirilloxanthin series’ In most species of photosynthetic bacteria, the components (Schmidt, 1971). Two strains of Rps. acidophila, 7050 and required for the ‘light reactions’ are localized in and on the 7750, were used in the present study. These two strains differ in intracytoplasmic membranes. The pigment-protein complexes both their response to growth at different light intensities and that make up the photosynthetic unit (the light-harvesting their content of pigment-protein complexes. complexes and the photochemical reaction centres) account for Fig. 1 shows the absorption spectrum of whole cells of Rps. most of the protein in these membranes. It is therefore important acidophila strain 7050 grown semi-aerobically and photofor a detailed understanding of the structure of the photo- synthetically at two different light intensities. The absorption in synthetic membrane to characterize these pigment-protein the near infrared (nir.) is due to the Bchl. a, and the different complexes. maxima seen reflect the presence of a variety of different Here we present an account of our attempts, so far, to resolve pigment-protein complexes. The semi-aerobic cells have few, if the photosynthetic unit of Rhodopseudomonas acidophila into any, intracytoplasmic membranes, a low specific Bchl. a content its constituent pigment-protein complexes, and to use this (typically < l p g of Bcht a/mg of protein), and only show one information to provide the background for studies on how these major absorption band in the n.i.r. at 885nm. This is due to the presence of B88O. The photosynthetically grown cells show a much higher Bchl. a content (for the cells shown in Fig. 1, 53pg of Bchl. a/mg of protein at 4OOIx and 29pg of Bchl. almg of protein at 2000lx). This increase in Bchl. a content is associated with the development of an elaborate system of intracytoplasmic lamellae, together with an increase in both the number and 0.6 types of pigment-protein complexes present. The ‘4001~’cells show additional maxima/shoulders in the n.i.r. at -800, 830 and 850nm, whereas the ‘2000 Ix’cells are dominated by absorptions at 800 and 850nm. The absorption spectrum of strain 7750 (results not shown) is not so variable as that of strain 7050. The absorption spectrum of semi-aerobic cells of strain 7750 is identical with that of semi-aerobic cells of strain 7050, whereas at both the light intensities used in Fig. 1 the absorption spectrum of cells of strain 7750 resembles that of the strain-7050 cells grown at 20001~.It is clear from the results presented below that these additional absorption bands present in the photosynthetically grown cells reflect the presence of the following extra light-harvesting complexes, B800-830 and two types of B 8 W 8 5 0 . The isolation and purification of these different pigmentprotein complexes is comparatively straightforward in Rps. acidophila. The cells are disrupted by passage through a French pressure cell at -1.51 x 107kg/m2(-10ton/in*) and the broken membranes isolated by centrifugation. The membranes are 1 1 I I resuspended in 20m~-Tris/HCl,pH 8.0, to give an absorbance 900 800 at 850nm of 50cm-I. This solution is then made 1% (v/v) with Wavelength (nm) the zwitterionic detergent NN-dimethyldodecylamine N-oxide (LDAO). The unsolubilized material is removed by a low-speed Fig. 1. The n.i.r. absorption spectra of whole cells of Rps. centrifugation at 12OOOg for 15min. The solubilized fraction is acidophila strain 7050 grown under direrent conditions then diluted 5-fold with 20m~-Tris/HCl,pH 8.0, and loaded on -, Cells grown anaerobically at 20001~; . . cells grown to a column of DE52 cellulose, equilibrated with 2 0 m ~ semi-aerobically; ----, cells grown anaerobically at 4001~. Tris/HCI, pH 8.0. The various complexes are eluted by a NaCl Different amounts of cells were used in each case, so that the gradient of & 3 0 0 m ~ made up in 20m~-Tris/HCI(pH8.0)/ spectra appeared on the same absorbance scale. 0.2% LDAO. The different complexes are collected, diluted and
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