Photosynthesis Research (2005) 85: 3–13
Springer 2005
Regular paper
Structural differences in the inner part of Photosystem II between
higher plants and cyanobacteria
Claudia Büchel* & Werner Kühlbrandt
Max Planck Institute of Biophysics, Marie Curie Strasse 13–15, 60439 Frankfurt, Germany;
*Author for correspondence (e-mail; [email protected]; fax: +49-69-6303-3002)
Received 26 May 2004; accepted in revised form 13 September 2004
Key words: electron crystallography, electron density map, photosynthesis, P680, X-ray crystallography
Abstract
A detailed comparison of key components in the Photosystem II complexes of higher plants and cyanobacteria was carried out. While the two complexes are overall very similar, significant differences exist in the
relative orientation of individual components relative to one another. We compared a three-dimensional
map of the inner part of plant PS II at 8 Å resolution, and a 5.5 Å projection map of the same complex
determined by electron crystallography, to the recent 3.5–3.8 Å X-ray structures of cyanobacterial complexes. The largest differences were found in the rotational alignment of the cyt b559 subcomplex, and of the
CP47 core antenna with respect to the D1/D2 reaction centre. Within the D1/D2 proteins, there are clear
differences between plants and cyanobacteria at the stromal ends of membrane-spanning helices, even
though these proteins are highly homologous. Notwithstanding these differences in the protein scaffold, the
distances between the critical photosynthetic pigment cofactors seem to be precisely conserved. The different protein arrangements in the two complexes may reflect an adaptation to the two very different
antenna systems, membrane-extrinsic phycobilisomes for cyanobacteria, and membrane-embedded chlorophyll a/b proteins in plants.
Abbreviations: chl – chlorophyll; cyt – cytochrome; PS – photosystem; RC – reaction centre
Introduction
Oxygenic photosynthesis in plants and cyanobacteria depends on Photosystem II (PS II), the only
enzyme in nature able to oxidize water. PS II is an
assembly of more than 15 membrane proteins and
three extrinsic proteins (for review see Hankamer
et al. 2001a), which form a dimeric complex in the
thylakoid membrane. The two central proteins, D1
and D2, bind the pigment cofactors that carry out
light-induced charge separation and primary electron transport. These pigments include six chlorophylls (Chl), two pheophytins and two
plastoquinones, QA and QB (for review see Debus
1992), related by approximate twofold symmetry.
The two central chlorophylls PD1 and PD2 are
together referred to as P680 and form the special
pair. The two chlorophylls next to the special pair
are referred to as ChlD1 and ChlD2. Two Chl z
molecules, one each bound by helix B of the D1
and D2 proteins, do not participate in charge
separation but are required for photoprotection of
PSII by secondary electron transfer (for review see
Stewart and Brudvig 1998). The actual watersplitting reaction is carried out by a cluster of four
Mn atoms close to the special pair. The electrons
abstracted from water re-reduce the central Chls
via a Tyr residue on the D1 protein upon light-
4
induced charge separation. The two subunits of cyt
b559 and PsbI are tightly bound to the D1 and D2
proteins. Therefore, the complex of D1, D2,
cytb559, and PsbI is often referred to as the reaction centre (RC) (Nanba and Satoh 1986).
In the PS II core complex the reaction centre
proteins are surrounded by the inner antenna
proteins, CP47 and CP43, each with six
membrane-spanning a-helices binding Chl a and
b-carotene, and several small one-helix membrane
proteins. The sequences of most PS II subunits in
plants and cyanobacteria are highly homologous,
with up to 89% identity for the D2 protein. Major
differences exist in the three extrinsic proteins
shielding the Mn cluster on the lumenal side of the
complex, two of which are different in higher
plants and cyanobacteria.
The first structure of plant PS II (Rhee et al.
1997, 1998) was determined at 8 Å resolution by
electron crystallography of two-dimensional crystals consisting of D1, D2, CP47, the two subunits
of cyt b559 and three single-helix subunits. This PS
II subcomplex is referred to as CP47RC. The 8 Å
map resolved all membrane-spanning a-helices
and the chlorophyll cofactors in CP47RC, and
revealed a close structural similarity between the
D1 and D2 proteins and the L and M subunits of
the bacterial reaction centre. Another electroncrystallographic structure of the PS II core at
lower resolution revealed the symmetrical
arrangement of the two inner antenna proteins,
CP47 and CP43, on each side of the D1-D2 heterodimer (Hankamer et al. 1999).
Recently the structures of PS II core complexes
from two different species of the cyanobacterium
Thermosynechococcus were determined by X-ray
crystallography at, respectively, 3.8 Å (Zouni et al.
2001), 3.7 Å (Kamiya and Shen 2003) and 3.5 Å
(Ferreira et al. 2004) resolution. All structures show
the cofactors bound to the RC, the Chls of CP47 and
CP43, the manganese cluster and the alpha-carbon
backbones of all a-helices. Kamiya and Shen (2003)
used the location of bulky side chains to model some
of the transmembrane helices more precisely,
whereas Ferreira et al. (2004) were able to trace
almost all residues. Overall, the three models are
almost identical with respect to the location and
orientation of the a-helices and the arrangement of
cofactors. Significant differences exist in the location of two b-carotene molecules close to the RC. In
addition, slight variations in numbers and location
of Chl a molecules in CP47 and CP43 exist and
Ferreira et al. (2004) were able to assign some
further densities in these inner antenna proteins to
b-carotene. The most striking difference is the lack
of a helix close to psbE in the model of Ferreira et al.
(2004) which was described in both other models.
Additionally, Ferreira et al. (2004) for the first time
showed the arrangement of the second plastoquinone acceptor, QB, and the arrangement of a Ca2+
together with Mn in the water splitting complex.
Other differences concern the assignment of the
single-helix subunits. Because the model of Ferreira
et al. (2004) is the most complete, being based on a
tracing of all side chains, we here attribute the single-helix subunits according to their model.
Although the polypeptides of pro- and
eukaryotic PS II are highly homologous, there are
some important structural differences. These are
most apparent in the overall shape of the PS II
core dimer (da Fonseca et al. 2002). In cyanobacteria four helices are found between cyt b559
and CP43 whereas in spinach only two helices were
detected in this part of the complex. On the other
hand, one additional helix is found on the opposite
side of CP43 in spinach, i.e. between CP43 and D1
(Hankamer et al. 2001b). Other differences were
evident in a 3D map of the spinach PS II core
when compared to the Thermosynechococcus PS II
(Hankamer et al. 2001b). In this comparison the
two photosystems are roughly aligned to fit the D1
and D2 proteins. The most obvious differences
were the location and orientation of CP47 and
CP43. However, the limited resolution precluded a
more detailed comparison. Here we compare the
three models of cyanobacterial PS II (Zouni et al.
2001; Ferreira et al. 2004; Kamiya and Shen 2003)
with the 8 Å 3D map (Rhee et al. 1998) and a
5.5 Å projection map of spinach CP47RC crystals
to provide an accurate structural model of plant
PS II.
Materials and methods
Maps were displayed and coordinates were
adjusted using the program O (Jones et al. 1991).
Models of PS II from Thermosynechococcus
elongatus, pdb accession numbers 1FE1 (Zouni
et al. 2001) and 1S5L (Ferreira et al. 2004), and
from Thermosynechococcus vulcanus (Kamiya and
Shen 2003), pdb accession number 1IZL, were
5
compared to the 8 Å 3D map of spinach CP47RC
(Rhee et al. 1998). Subunits not present in
CP47RC were removed from the cyanobacterial
models and all membrane-extrinsic loops were
omitted since they are not visible in the spinach
map. An accurate model of plant PS II based on
the structure of cyanobacterial PS II core using the
T. elongatus coordinates (Ferreira et al. 2004) was
generated as follows. The magnification of the 8 Å
3D map from spinach was increased by 2.8% to
match the size of the cyanobacterial complex.
Subunits in the homology model were fitted to the
map density manually by rotational and
translational movements as rigid bodies.
Individual a-helices were then moved into the 8 Å
map density. Torsion angles F and Y between the
Ca-and N-atoms or the Ca- and C-atoms in
transmembrane helices were adjusted only at residues where the sequence differed between Thermosynechococcus and spinach and where this
change was conserved in the plant kingdom compared to cyanobacteria. Torsion angles used for
particular side chains were in the allowed regions
of the Ramachandran plot (Kleytwegt et al. 1996;
Lovell et al. 2003). Finally, the positions of pigment cofactors were adjusted by translational
movements where necessary.
2D crystals of spinach CP47RC were produced
by a modified procedure based on the preparation
and crystallisation conditions of Rhee et al.
(1997). Briefly these changes included a higher
detergent concentration for the purification of
PSII enriched grana membranes, Na+ instead of
K+ and acetate instead of Cl) in the dialysis buffer
with the addition of propionate, and temperature
ramping (37–20 C) during dialysis. Two-dimensional crystals were examined by electron cryomicroscopy after flash-freezing on carbon-coated
electron microscopy grids in liquid ethane. Lowdose electron micrographs with an estimated dose
of 30 eÅ)2 were recorded in a JEOL 3000SFF
helium-cooled microscope at 300 kV acceleration
voltage at a calibrated magnification of 70,000· in
spot scan mode (Downing 1991). Images were
digitised on a Zeiss scanner at a resolution of 1 Å
per pixel at the specimen and analysed using the
MRC program suite (Crowther et al. 1996). Thirteen images were merged with overall weighted
phase residuals of 27 at 5.5 Å (90 ¼ random)
and a projection map was calculated. This 2D map
was compared to the 2D maps of the cyanobac-
terial and spinach models calculated using the
MRC programs. Quantitative comparison was
carried out using cross correlation coefficients of
the maps calculated in SPIDER (Frank et al.
1981).
Sequence comparisons of the D1 and D2 proteins were done with the program ClustalW on all
sequences available in SwissProt and Trembl for
PSII of higher plants and cyanobacteria. The
projection map of the cyanobacterial model was
calculated using the MRC and CCP4 program
suites. The structures were displayed with the
program Bobscript (Esnouf 1997).
Results
To identify the unique features of the inner part of
plant PSII, the structure of cyanobacterial PS II
was compared to the two electron crystallographic
maps of a subcomplex of spinach PS II, CP47RC.
Because the three cyanobacterial models showed
no major differences in this part of PS II, we mainly
considered the most detailed structure of Ferreira
et al. (2004). The few divergences from the models
of Zouni et al. (2001) and Kamiya and Shen (2003)
will be discussed later. The subunits not present in
the CP47RC complex and the extrinsic subunits,
which did not show up in the map of the plant
complex, are not considered in the comparison.
Figure 1A shows the relevant part of the model of
T. elongatus superimposed on the 8 Å map of the
spinach complex, viewed from the lumenal side.
It is immediately clear that overall, the plant
complex resembles the structure of the cyanobacterial PS II closely, but a more detailed examination revealed several interesting differences. For
the comparison, a homology model of the plant
complex was calculated, using the cyanobacterial
structure as a template. At first, each subunit was
fitted to the 3D map of plant PS II as a rigid body.
Then the helices within the subunits were fitted to
their map densities. In case of D1 and D2, Ca
torsion angles were adjusted at positions where the
polypeptide sequences differed between Thermosynechococcus and spinach. The resulting model of
plant PS II (Figure 1B) shows an excellent fit to
the 8 Å map.
The most apparent differences between the
cyanobacterial and the plant complex concerned
the location of cyt b559 in relation to D1 and D2.
To make this subunit fit the map, it was rotated as
6
Figure 1. Comparison of the 8 Å 3D map of spinach CP47RC (Rhee et al. 1998) (chicken wire) with the coordinates of
cyanobacterial PS II (A) (Ferreira et al. 2004) and the fitted homology model of spinach PS II (B) viewed from the lumenal side
of the membrane. For clarity, only the backbones of a-helices and tetrapyrrole pigment cofactors are displayed. The extra density
not included in the fit correspond to additional monomers in the electron density map.
a rigid body counter-clockwise by 10 and shifted by 2.5 Å each in x- and y-direction
(Figures 1B and C, all changes are relative to PS II
oriented as shown in Figure 1A). This moves the
cyt b559 complex 4 Å away from its position in
the cyanobacterial PS II. However, the two helices
of PsbE and F have the same orientation relative
to one another, and to the membrane plane. The
position of the heme group of cyt b559 differs
between the three cyanobacterial models published
7
previously. The orientation shown by Ferreira
et al. (2004) did not fit the electron densities
obtained from spinach crystals. However, no evidence was found for a change in the position and
orientation of the heme group relative to the
helices of PsbE and PsbF when compared to the
structure published by Kamiya and Shen (2003).
The inner antenna protein CP47 was fitted
likewise as a rigid body including all chlorophylls
to the map of the plant complex. A clockwise
rotation of 6.5 and a shift by 1.5 Å in x- and
y-direction resulted in an excellent fit of the six
helices and most of the chlorophylls. There was no
density for one of the chlorophylls, Chl32 (Ferreira et al. 2004), indicating that it is absent in the
plant complex. The same was true for Chl43
(Kamiya and Shen 2003), which was only found in
T. vulcanus. Both are thus not included in our
model of plant PS II. Chl25 and Chl37 (Ferreira
et al. 2004) were shifted in x-direction by 2 and
2.5 Å, respectively, which resulted in a good fit.
Adjustments were also required for the singlespan PS II subunits. PsbI was rotated clockwise by
15. PsbX was shifted by 3 Å towards psbH and
D2, respectively. The helix of psbH was less tilted
by 10 in spinach compared to T. elongatus and
thus psbX and psbH are closer together in the
plant PS II, especially on the stromal side.
Although the overall fit of the central subunits
D1 and D2 to the map (Figure 1A) is convincing,
a closer look revealed small but significant differences in the position of the two subunits relative to one another, and in the position and
orientation of individual helices. This became
clear when the two molecules were aligned as one
rigid group, which gave a reasonable fit for either
the D1 or the D2 protein, but not for both. Only
if the D1 protein was separately rotated clockwise
by 4 and shifted by 1 Å in x- and y-direction,
the helices coincided with the map densities. A
further improvement of the fit resulted from
moving single helices or adjusting their curvatures
and tilt angles.
Membrane-spanning a-helices are well defined
in the 8 Å electron microscopy map, and differences down to 1 Å in their position or a few
degrees in tilt angle are detected reliably. As shown
in Table 1, only a few residues in these helices are
different in the sequences from Thermosynechococcus and spinach. However, most of these
changes are conserved throughout cyanobacteria
and higher plants, respectively. For a better fit,
adjustments of the F and Y torsion angles at Ca
atoms were made for residues with non-conservative changes, taking care to avoid side-chain clashes. All residues marked grey in Table 1 were
tested to find out whether such a change in torsion
angle would improve the model. Changes were
only accepted when angles were lying in the
allowed regions for a-helices in a Ramachandran
plot. This adjustment led to a considerably better
fit at all residues in the case of D2. For three residues of the D1 protein the fit was not improved by
changing the Ca-torsion angles (Ala-143 and Thr154 in helix C; Met-199 in helix D).
A comparison of the models of the spinach
and cyanobacterial D1 and D2 protein is shown
in Figure 2A (lumenal view) and Figure 2B (side
view). The model of the cyanobacterial complex
did not fit to the electron density around helix A
of the D2 protein, which was therefore shifted
by 3 Å towards psbF. Deviations were also
found around helix B, especially on the stromal
side of the complex. In the spinach D2 protein,
Gly-121 is replaced by Ala-120. Thus, we changed the torsion angle around the Ca-atom of
Ala-120 in order change the curvature of helix B
as indicated by the map. In addition, helix B was
shifted by 2 Å in y-direction. Helix C of D2,
after a minor change in the angles around the
Ca-atom of Gly-148, was also shifted in by 1 Å
y-direction. Thus, not only the location of helix
A but also the helix positions of B and C in
the D2 protein are different in plant PS II
(Figure 2A).
The divergence between the cyanobacterial
model and the spinach densities was less pronounced in helices D of both D1 and D2.
However, a minor change in the angles around the
Ca-atoms of spinach D2 Ala-207 and D1 Ser-211
improved the fit. The same was true for D1 Val122 in helix B and D1 Val-46 in helix A. In addition, this helix was shifted by 1 Å in y-direction.
Thus, the distance between the first two helices of
D1 was smaller in the plant complex.
We then compared our resulting D1 and D2
structures to all models of cyanobacterial PS II. In
summary, the D1 and D2 proteins are oriented
differently with respect to each other in plant PS
II. In the D1 protein, helices B and C are closer to
helix A. Especially the shift in helix A of the D2
protein and the changes in tilt of both helices B
8
Table 1. Sequence comparison of the a-helices of the D1 and D2 proteins
D1 - protein
helix
A
T. vulcanus:
T. elongatus:
36
B
S. oleracea:
T. vulcanus:
T. elongatus
35
109
C
S. oleracea:
T. vulcanus:
T. elongatus:
108
141
D
S. oleracea:
T. vulcanus:
T. elongatus
140
193
E
S. oleracea:
T. vulcanus:
T. elongatus:
192
270
S. oleracea:
269
IMIPTLLAATICFVIAFIAAPP
IMIPTLLAATICFVIAFIAAPP
:******:** *:********
LMIPTLLTATSVFIIAFIAAPP
GGPYQLIIFHFLLGASCYMGR
GGPYQLIIFHFLLGASCYMGR
****:**::*****.:*****
GGPYELIVLHFLLGVACYMGR
PWICVAYSAPLASAFAVFLIYPIG
PWICVAYSAPLASAFAVFLIYPIG
***.******:*:* *********
PWIAVAYSAPVAAATAVFLIYPIG
LMHPFHQLGVAGVFGGALFCAMHGSLVTSSL
LMHPFHQLGVAGVFGGALFCAMHGSLVTSSL
****** *********:**.***********
LMHPFHMLGVAGVFGGSLFSAMHGSLVTSSL
SLHFFLAAWRVVGVWFAALG
SLHFFLAAWPVVGVWFTALG
********* ***:**:***
SLHFFLAAWPVVGIWFTALG
D2 - protein
helix
A
T. elongatus:
35
B
S. oleracea:
T. elongatus:
35
109
C
S. oleracea:
T. elongatus:
109
141
D
S. oleracea:
T. elongatus:
141
191
E
S. oleracea:
T. elongatus:
191
266
S. oleracea:
266
ILLFPCAYLALGGWLTGTTFV
:*******:*****:******
LLLFPCAYFALGGWFTGTTFV
GLWTFIALHGAFGLIGFMLR
***:*:******.*******
GLWAFVALHGAFALIGFMLR
YNAIAFSAPIAVFVSVFLIYPLG
*******.***************
YNAIAFSGPIAVFVSVFLIYPLG
WTLNPFHMMGVAGVLGGALLCAIHGAT
****************.**********
WTLNPFHMMGVAGVLGAALLCAIHGAT
WLHFFMLFVPVTGLWMSAIG
******************:*
WLHFFMLFVPVTGLWMSALG
In case of the D2 protein no sequence of T. vulcanus is a available and therefore, only the sequence of T. elongatus is shown. Underlined
residues are not conserved in either higher plants or cyanobacteria. Residues shaded in grey were tested for refinement of the fit by
changing the torsion angles around the Ca-atoms. Only using those shaded in dark grey led to improvement of the model.
and helices D differ significantly from all three
cyanobacterial structures. Due to the lower resolution perpendicular to the membrane plane electron crystallography might underestimate helix
tilts. This is unlikely for both helices B because
they are extremely well resolved in the electron
density map of spinach. In addition, systematic
underestimation of tilt angles can be ruled out as
9
Figure 2. Direct comparison of the spinach model (red) with the cyanobacterial structure (green). (A) Top view from the lumenal side
of the membrane. (B) Side view of the comparison of the locations of the D1 and D2 proteins with their cofactors P, Chl, pheophytin
(Pheo) and Chl z in spinach and cyanobacteria.
some of the helices are even more tilted in our
model compared to the cyanobacterial PS II, e.g.
helix D of the D2 protein. In case of an underestimation of tilt angle one would expect similar
deviations on both the lumenal and stromal end of
a helix. As shown in Figure 2B, the deviations of
the two models are generally more pronounced on
the stromal side of the PS II complex than on the
10
lumenal side. Thus, the changes in tilt of helices D
are most likely real as well.
Densities for the tetrapyrrole groups of Chls,
pheophytins and hemes are visible in the 8 Å map,
although they are less well defined than the membrane-spanning helices, especially when their planes
are oriented approximately parallel to the membrane. In the two cyanobacterial structures the
centre-to-centre distances of reaction centre Chls
and pheophytins are virtually identical. We fitted
the central four Chls and two pheophytins as a rigid
group by matching the model to the densities as far
as possible, keeping the distances to the cofactor
binding side chains constant. This proved possible,
despite the structural differences in the D1 and D2
proteins, which include the binding sites for PD1 and
PD2. The two Chl z molecules at the periphery of D1
and D2 required a separate fit to match the densities.
Because their chlorine rings are oriented almost
perpendicular to the membrane plane, the densities
were strong enough for a reliable adjustment. Both
Chl z molecules were moved slightly outwards. As a
result, distances were 25.4 Å between Chl zD2 and
ChlD2 and 25.0 Å between Chl zD1 and ChlD1,
slightly longer than in the two cyanobacterial
models (24 and 24.5–25 Å, respectively). The centre-to-centre distance between the Mg2+ of Chl zD2
and the central iron of cyt b559, was 28 Å in spinach,
compared to 26.4–27.4 Å in cyanobacteria. Surprisingly, this distance is almost the same in both PS
II complexes, even though the cyt b559 complex had
moved by 4 Å. Carotenes would not be visible at
8 Å resolution, and therefore we did not include the
two b-carotenes identified by Kamiya and Shen
(2003) or Ferreira et al. (2004) in our model.
A 5.5 Å projection map (Figure 3) of plant PS
II was obtained from 2D crystals that were more
highly ordered than those of Rhee et al. (1997,
1998). A corresponding 5.5 Å projection map was
calculated from the coordinates of the T. elongatus
complex (Figure 3A). The structural differences
described above were equally evident, in particular
the different positions of cyt b559 and CP47.
Figure 3B shows a comparison of the 5.5 Å projection map and a projection calculated from the
coordinates of our final model of spinach
CP47RC. The improved fit and the agreement of
the spinach model and map is also evident at this
higher resolution and thus further validates the
new model. The maps show essentially the same
features except for the highly tilted helices PsbF
Figure 3. Comparison of projection maps of PS II at 5.5 Å
resolution. In A the projection map of spinach PS II is shown in
red, the projection map of the cyanobacterial model calculated
at the same resolution in green. In B the projection map of
spinach PS II is shown in red, the calculated projection map of
the fitted homology model of spinach PS II in blue.
and helices A of D1 and D2. To quantify the
improvement gained by fitting we calculated the
cross correlation coefficients between the projection map and the maps derived from the cyanobacterial and spinach models. At a resolution of
5.5 Å the overall cross correlation coefficient
between the 2D map of the cyanobacterial model
and the projection map of plant PS II was only
0.64. Cross correlation of the projection map with
the 2D map of our new spinach model yielded
0.70, thus further validating our fitting.
11
Discussion
We present here a detailed comparison between
spinach and cyanobacterial PS II, based on the
electron-crystallographically determined structure
of spinach CP47RC and the X-ray models of a
cyanobacterial PS II core complex. Several previous
studies have highlighted the overall similarity of the
PS II core complexes in cyanobacteria and plants
(Zouni et al. 2001; Hankamer et al. 2001b; Barber
and Nield 2002; da Fonseca et al. 2002; Kamiya and
Shen 2003; Ferreira et al. 2004). However, the
structures of the two complexes cannot be the same
because their subunit composition is different
(Hankamer et al. 2001b). We show here that
important differences exist even in the highly conserved, main subunits that are common to both.
The work on spinach was carried out using a
subcomplex of PS II, CP47RC. We therefore need
to consider carefully whether the different orientation reflects the true structure in vivo or an
artefactual rearrangement due to crystal contacts
or the exposure to detergent. Our conclusion that
the observed differences are real is supported by
several lines of evidence:
(i) Whereas 3D crystals of PS II contain high
levels of detergent and consist of detergent-stabilised complexes, this is not the case in 2D crystals. In
there, the detergent is replaced by lipids and the
complexes are reconstituted into a native-like lipid
environment. The lipid to protein contacts of some
subunits indeed replace the in vivo protein to protein
contacts and this might influence their conformation. However, almost no changes were found in
those helices that are completely surrounded by
other proteins in vivo and are at the periphery of the
CP47RC subcomplex, e.g. helix A of D1. On the
contrary, some of the helices retaining their in vivo
neighbours showed significant changes, e.g. helix B
of D2. Thus, we can rule out detergent effects as a
cause of the observed differences.
(ii) The most striking difference between the
structures of spinach and cyanobacterial PS II is
manifest in the rotational alignment of the
subunits relative to one another. This is most
obvious for cyt b559, which is located at the
periphery of the CP47RC subcomplex and the PS
II core. In this case crystal contacts might be
responsible for the observed positional difference.
However, in the 10 Å map of the complete spinach
PS II core complex as shown by Hankamer et al.
(2001b), cyt b559 and CP47 have the same apparent
orientation and thus our crystallisation protocol
cannot be responsible for their orientation.
Moreover, the CP47RC can be easily superimposed on the spinach core model, thus demonstrating that the loss of subunits did not result in
major changes in the arrangement of helices. In
addition, the differences found between cyanobacterial core complexes and spinach CP47RC are
most evident on the stromal side of the complex
and thus unlikely to be due to the loss of extrinsic
subunits.
(iii) As a consequence of the orientation of cyt
b559 and the position of Chl z in the two assemblies,
one might expect significant differences in the distances from the cyt b559 heme to Chl z and the
central D1/D2 cofactors. This would have measurable effects on the rate of electron or energy
transfer, which are strongly distance-dependent.
However, in the two forms of PS II the distances
between these cofactors are the same within 1.5 Å.
The fact that these distances are essentially identical
despite the differences in the protein scaffold again
argues strongly against an artefactual arrangement
of subunits in the CP47RC complex. Evidently, the
cofactor-binding polypeptides of both complexes
are arranged in ways to conserve these functionally
important distances almost exactly, as required for
the finely-tuned action of PS II. In support of our
finding, there is no spectroscopic evidence to indicate any such difference in the distance or orientation of the key cofactors in PS II.
(iv) Spinach CP47RC complexes do not contain the water splitting complex, but were recently
shown to be able to evolve oxygen upon photoactivation (Büchel et al. 1999). Only 18% of the
RCs could be activated in these experiments, but
the quantum yield per active RC was comparable
to PS II in grana membranes. This proves that the
CP47RC subcomplexes are able to evolve oxygen
when reconstituted with Mn and Ca. These findings concerned dimers as well as monomers in
detergent solution, a much harsher environment
than the membrane lipids in the 2D crystals analysed here. These results further support the closeto-native structure of plant CP47RC. Thus we
conclude that the differences in position and orientation of subunits in higher plants and cyanobacteria are real.
We now examine the inner subunits D1 and
D2. These are slightly more apart in spinach than
12
in cyanobacteria, with a clockwise rotation of D2
relative to D1. However, they do not participate in
crystal contacts, due to their central position in the
complex. We therefore conclude that the differences seen in these subunits, including the subtle
internal helix rearrangements, are also real.
Considering an evolutionary distance of more
than 1 billion years, the overall sequence homology
of PS II subunits from higher plants and
cyanobacteria is astonishingly high, with more than
85% identical residues in D1/D2, which bind the
most important cofactors. Therefore, it may seem
surprising that a few amino acid exchanges should
result in structural differences visible in an 8 Å map.
We notice that these differences are more pronounced on the stromal side of the complex, whereas
the critical cofactors bind near the lumenal side.
The stromal surface of PS II is more or less flat.
There are no extrinsic subunits on this side of the
membrane, and the loops are mainly short. In the
grana stacks of plant chloroplasts, this surface is
tightly appressed against the opposite thylakoid
membrane, where it makes extensive contact with
the stromal surface of PS II or the equally flat
stromal surface of LHCII (Kühlbrandt et al.
1994). In cyanobacteria the thylakoid membranes
do not stack. Instead, huge antenna complexes, the
phycobilisomes, are attached on this side, directly
on top of PS II. This would explain why the
structural differences in the PS II complexes of
plants and cyanobacteria are more pronounced on
the stromal side.
The phycobilisomes consist of three to five
allophycocyanin rods parallel to the membrane, to
which the peripheral rods are connected. Two of
the allophycocyanin rods are in direct contact with
the membrane. They consist of four disc-shaped
hexameric units of three a- and b-subunits each. In
one of the inner two discs, an a-subunit is replaced
by a linker protein (LCM), which connects the
phycobilisome to the PS II core. Biochemical,
mutational and single particle analyses (Bald et al.
1996; Barber et al. 2003) have been used to pinpoint the region where the linker protein interacts
with PS II. This region includes part of the D2 and
D1 proteins, as well as the low-density area
between CP43 and cyt b559, where cyanobacteria
and spinach vary in the number of single-helix
subunits. This region is a good candidate for the
phycobilisome binding site (da Fonseca et al.
2002). Interestingly, in this area of the spinach
complex there are even fewer contacts available
for protein–protein interactions, due to the more
remote location of cyt b559. In addition, helices C
and D in D2, and helices C, D and E in D1, all end
in this region. On the stromal side especially helix
C of D2, but also both D helices are slightly displaced in cyanobacteria compared to spinach. One
explanation for the observed structural differences
in the part of PS II that is thought to bind the
phycobilisome in cyanobacteria might therefore be
the missing interaction with the phycobilisome in
spinach PS II.
Whereas the subunit composition of the inner
part of PS II is conserved, this is not the case
for the surrounding polypeptides. In green
plants, the PS II core is more or less surrounded
on all sides by chlorophyll a/b-binding LHCs,
with which it forms various supercomplexes. In
the best-defined of these supercomplexes the core
antenna proteins CP47 and CP43 as well as
some of the single-helix subunits appear to be
located at the interface between the core and the
LHCs (Boekema et al. 1999; Nield et al. 2000).
Any constraints on the PS II core structure
imposed by the interaction with LHC would not
apply to the cyanobacterial core complex, which
does not bind LHCs. These lateral constraints
might account for the different rotational alignment of the CP47 core antenna in the two
complexes and the localisation of cyt b559 closer
to CP47.
Acknowledgements
This work was supported by the DFG (BU 812/3-1).
C. B. gratefully acknowledges a Heisenberg Fellowship (BU 812/2-1, 2-2).
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