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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.