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334 BIOCHEMICAL SOCIETY TRANSACTIONS 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 - a, 1982 600th MEETING, OXFORD 335 I ’ complex found in Chromatium vinosum (Thornber, 1970). The position of the long-wavelength-absorption maximum in both the B800-830 and the type 2 B800-850 complexes is somewhat variable. It seems to depend upon the salt and detergent concentrations in the solution. The 830nm band varies between 815 and 830nm, whereas the 850nm band varies between 840 and 850nm. This type of variability is not shown by either the B880 or type 1 B80Ck850 complexes. Monomeric BcM a in organic solvents such as 7:2 (v/v) acetone/methanol absorbs at 772nm (Clayton, 1963). In the light-harvesting complexes the Bchl. a is non-covalently bound to the protein and is easily removed by extraction with organic solvents or by denaturing the protein. When the Bchl. a is bound within the complex, its absorption in the n.i.r. is strongly redshifted compared with free Bchl. a, and the position of the 800 900 800 850 Wavelength (nrn) absorption bands of the complexes are a sensitive indication of the integrity of the complex. It is therefore reassuring that the Fig. 2. The n.i.r. absorption spectra of the direrent pigmentabsorption maxima of the isolated complexes exactly correspond protein complexes isolated from Rps. acidophila to those seen in the absorption spectrum of the intact membrane (compare Fig. 2 with Fig. 1). In each case the complexes were suspended in 20rn~-Tris/HCl In those species of photosynthetic bacteria where light(pH 8.0)/0.1% LDAO (v/v). The circles indicate where the harvesting pigment-protein complexes have been studied in recording spectrophotometer stopped and the spectra were detail (Cogdell & Thornber, 1980), the pigments are usually continued point by point in another machine. (a) Complexes found associated with rather small, hydrophobic polypeptides in from strain 7750 grown at 2000k: 0 , type 1 B800-850; 0, the 5000-14OOO molecular-weight range. We have attempted to RC-B880. (b) Complexes from strain 7050 grown at 4 0 0 1 ~ :0, resolve the polypeptide composition of the Rps. acidophila type 2 B800-850; this spectrum was raised to make it more pigment-protein complexes using electrophoresis on sodium clearly visible; 0, B800-830. dodecyl sulphate/polyacrylamide gradient gels. The type 1 B800-850 complex shows two clearly resolved polypeptides in the 5000-9000 molecular-weight region. The other complexes further purified by a second passage over a DE52 cellulose only yield a diffuse band in this region of the gel and although in column. Each of the different pigment-protein complexes has a each case they are clearly distinct, further work is required to try different carotenoid composition. This is very convenient, since and achieve a clearer picture of their polypeptide composition. it allows them to be easily distinguished on the basis of colour. Only the RC-880 complex shows any polypeptides with a Fig. 2 shows the absorption spectra of the isolated, purified molecular weight in excess of 12000. The B 8 0 6 8 3 0 and both pigment-protein complexes. The first pigmented complex which the types of B80Ck850 complex appear to be quite free of is eluted from the column is the B880 light-harvesting complex. contaminating polypeptide and show remarkably high Bchl. This fraction also contains reaction centres and appears to be protein ratios [usually in the range of 2&24% (Bchl. a rather similar to ‘fraction A’ which Thornber (1970) obtained expressed as a percentage, w/w, of the protein present)]. from Chromatium vinosum. When cells of Rps. acidophila strain We need to examine the pigment content of these complexes 7750 grown at 2000lx are used in this preparation, the in an attempt to determine the pigment-protein composition of B800-850 antenna complex is eluted after the B880 complex the minimal functional unit of each type of complex. from the column. Spectrally this complex (Fig. 2) is very similar to the B800-850 light-harvesting complexes which have been This work was supported by a grant from the Science and isolated from Rps. sphaeroides (Clayton & Clayton, 1972) and Engineering Research Council and we would also like to thank Mrs. from Rps. capsuluta (Feick & Drews, 1978). It is a charac- Irene Durant for her expert technical assistance. teristic of this type of B800-850 that the absorbance at 850nm is usually between 1.5 and 2 times larger than the absorbance at Clayton, R. K. (1963) in Bacterial Photosynthesis (Gest, H., San Pietro, A. & Vernon, L. P., eds.), pp. 495-500, Antioch Press, 800nm. In order to distinguish this B800-850 complex from the Yellow Springs, OH B800-850 complex isolated from cells of strain 7050 grown at Clayton, R. K. & Clayton, 8. J. (1972) Biochim. Biophys. Acta 283, 400lx, we have called it ‘type 1 B800-850’. Fractionation of the 492-504 ‘400lx’ 7050 cells yields B800-830 and type 2 B 8 0 6 8 5 0 Cogdell, R. J. & Thornber, J. P. (1980) FEBS Letr. 122, 1-8 complexes (Fig. 2) as well as the analagous B880-reactionFeick, R. & Drews, G. (1978) Biochirn. Biophys. Acto 501,499-5 13 centre fraction. Absorbances at 800 and 850nm in the type 2 Pfennig, N. (1969) J. Bacteriol. 99, 597-602 B 8 W 8 5 0 complex are nearly equal. The type 2 B800-850 Schmidt, K. (I97 1) Arch. Mikrobiol. 77, 23 1-236 antenna complex is therefore rather similar to the B800-850 Thornber, J. P. (1970) Biochemistry 9,2688-2698 1.o Protein-lipid interactions in the photosynthetic membrane W. PATRICK WILLIAMS*, ARINDAM SEN? and PETER J. QUINN? Departments of *Biophysics and ?Biochemistry, Chelsea College, University of London, London SW3 6LX, U.K. The molecular organization of chloroplast membranes is generally described in terms of the fluid mosaic model. On this basis, the central matrix of the membranes consists of a simple bilayer of polar lipids to which various globular proteins are VOl. 10 attached or inserted. The most important, and well characterized, of the peripheral membrane proteins that are attached to the bilayer surface are carboxydismutase and the CFJ components of the CFo-CF, ATPase complex (Howell & Moudrianakis, 1967a,b; Miller & Staehelin, 1976) and the $ Abbreviations: CF, coupling factor; Chl., chlorophyll; LHCP, chlorophyll alb light-harvesting protein; RCP, reaction-centre complex.