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© 2005 Nature Publishing Group
Towards complete cofactor arrangement in the
3.0 A
˚
resolution structure of photosystem II
Bernhard Loll
1
*†, Jan Kern
2
*, Wolfram Saenger
1
, Athina Zouni
2
& Jacek Biesiadka
1
Oxygenic photosynthesis in plants, algae and cyanobacteria is
initiated at photosystem II, a homodimeric multisubunit protein–
cofactor complex embedded in the thylakoid membrane
1
. Photo-
system II captures sunlight and powers the unique photo-induced
oxidation of water to atmospheric oxygen
1,2
. Crystallographic
investigations of cyanobacterial photosystem II have provided
several medium-resolution structures (3.8 to 3.2 A
˚
)
3–6
that explain
the general arrangement of the protein matrix and cofactors, but
do not give a full picture of the complex. Here we describe the most
complete cyanobacterial photosystem II structure obtained so far,
showing locations of and interactions between 20 protein subunits
and 77 cofactors per monomer. Assignment of 11
b
-carotenes
yields insights into electron and energy transfer and photo-
protection mechanisms in the reaction centre and antenna sub-
units. The high number of 14 integrally bound lipids reflects the
structural and functional importance of these molecules for
flexibility within and assembly of photosystem II. A lipophilic
pathway is proposed for the diffusion of secondary plastoquinone
that transfers redox equivalents from photosystem II to the
photosynthetic chain. The structure provides information
about the Mn
4
Ca cluster, where oxidation of water takes place.
Our study uncovers near-atomic details necessary to understand
the processes that convert light to chemical energy.
In photosystem II (PSII), excitation energy is transferred from the
antenna system to the reaction centre, where the primary electron
donor P680 formed by chlorophyll a (Chla) molecules is excited to
P680*, followed by release of an electron that travels along the
electron transfer chain by means of the pheophytin a Pheo
D1
to the
plastoquinone Q
A
, forming P680
þ†
Q
A
2
. After two steps of reduction
and protonation of the secondary plastoquinone Q
B
, the plasto-
quinol PQH
2
leaves the Q
B
site and is replaced by new plastoquinone
PQ (ref. 1). Oxidation of water at the Mn
4
Ca cluster occurs in four
steps (termed S states) in the so-called Kok cycle
2
; at each step the
water-oxidizing complex is oxidized to a higher oxidation state, and
after the fourth step molecular O
2
is released. The electrons are
transferred from the Mn
4
Ca cluster through redox-active Tyr
Z
to
P680
þ†
, which is reduced to P680 for another photosynthetic
cycle. Our structure of dimeric PSII from Thermosynechococcus
elongatus shows the location of 35 Chla,11
b
-carotene (Car), two
pheophytin (Pheo), two PQ, two haem, bicarbonate, 14 lipid and
three n-dodecyl-
b
-D-maltoside (
b
-DM) detergent molecules, the
Mn
4
Ca cluster, and one Fe
2þ
and one putative Ca
2þ
ion in each
monomer. The cofactor and protein arrangement is shown in Fig. 1
and Supplementary Fig. S1.
Various studies have indicated the influence of lipids on the
assembly and function of PSII
7,8
, but so far no structural information
about lipids bound to PSII is available. We found 14 lipids integrally
bound to PSII: four DGDG (digalactosyldiacylglycerol), six MGDG
(monogalactosyldiacylglycerol), three SQDG (sulphoquinovosyl-
diacylglycerol) and one PG (phosphatidyldiacylglycerol) molecule
(Fig. 1 and Supplementary Fig. S2). The composition of the lipids is
comparable to that of the thylakoid membrane
9
. Hydrophilic lipid
headgroups are close to the membrane surface, whereas hydrophobic
fatty acid chains are anchored between transmembrane
a
-helices
(TMHs) of different subunits. Negatively charged headgroups of
SQDG and PG are exclusively located at the cytoplasmic side
(Fig. 1b).
A belt of 11 lipids surrounds the reaction centre, separating it from
the antenna and smaller subunits (Fig. 2a). The remaining three
lipids and the detergent molecules are located at the monomer–
monomer interface. The unusually high lipid content in the PSII
complex provides a structural flexibility that might be required for
local mobility of subunits and promotes subunit–subunit recog-
nition. At high light intensities, Chla triplet states can be populated
and react with molecular oxygen to form singlet oxygen, leading to
oxidative damage of proteins. As subunit D1 is most prone to this
photodamage, it needs to be continuously replaced by newly syn-
thesized D1 (ref. 10). To facilitate this turnover of D1, a flexible
environment, such as that provided by the belt of lipids, might be
necessary. A similar ‘lubricant’ role may be fulfilled by lipid molecules
located at the dimerization interface, as observed in other multimeric
membrane-intrinsic complexes
11
.
The protein environment of Q
A
,Q
B
and non-haem-Fe
2þ
is
described in the Supplementary Discussion and Fig. S3. The
Q
B
-binding site (Fig. 2b) opens into a large cavity defined by
the TMHs of cytochrome b-559 (PsbE and PsbF), PsbJ, PsbK, TMH-d
and TMH-e of D1, and TMH-a of D2 (Fig. 2 and Supplementary
Fig. S4). The walls of the cavity are lipophilic as they are coated by
phytol chains of P
D2
, Chl
D2
, Pheo
D2
, Chla37, Chla44 and Chla46,
and by acyl chains of four lipids. In addition, Car11 and Car12 point
into the cavity. Fragmented electron density in the cavity is not
interpretable and could be due to two or three disordered lipophilic
molecules.
The cavity has two openings perpendicular to each other: the
larger one (,16 £ 16 A
˚
) opens towards the cytoplasmic side and the
smaller one (,10 £ 20 A
˚
), flanked by TMHs of cytochrome b-559
and PsbJ, faces the membrane interior (Fig. 2c). On the opposite side
of the membrane-facing opening, the Q
B
-binding pocket forms an
antechamber of the cavity that is filled by the quinone headgroup,
while its isoprenoid chain winds along the wall of the cavity. This
arrangement suggests that the cavity provides a flexible, lipophilic
environment for the diffusion of PQ/PQH
2
through the membrane-
facing opening, between the Q
B
-binding site in PSII and the plasto-
quinone pool of the thylakoid membrane (Fig. 2d). Similar lipophilic
LETTERS
1
Institut fu¨r Chemie und Biochemie/Kristallographie, Freie Universita
¨
t Berlin, Takustrasse 6, D-14195 Berlin, Germany.
2
Institut fu¨r Chemie/Max Volmer Laboratorium fu¨r
Biophysikalische Chemie, Technische Universita
¨
t Berlin, Straße des 17 Juni 135, D-10623 Berlin, Germany. †Present address: Max-Planck-Institut fu¨r Medizinische Forschung,
Abteilung fu¨r Biomolekulare Mechanismen, D-69120 Heidelberg, Germany.
*These authors contributed equally to this work.
Vol 438|15 December 2005|doi:10.1038/nature04224
1040
© 2005 Nature Publishing Group
pathways have been proposed for the photosynthetic cytochrome b
6
f
complex
12,13
and for the respiratory cytochrome bc
1
complex
14
. The
alternative exchange pathway for PQ through the larger cytoplasmic
opening is less favourable because hydrophobic PQ molecules would
have to pass through the cytosol before entering the membrane. It is
likely that PsbJ and cytochrome b-559, flanking the membrane-
facing opening, could regulate PQ diffusion. Indeed, PsbJ has been
shown to influence the electron transfer between Q
A
,Q
B
and the
Figure 2 | The plastoquinone diffusion pathway.
a, Lipid molecules located in PSII, viewed as in
Fig. 1. Lipid, detergent and quinone molecules are
shown in space-filling representation, and
a
-helices of protein subunits are shown as
cylinders. Subunits D1 and D2 and cytochrome
b-559 are coloured as in Fig. 1, the remaining
a
-helices are grey, and lipids are cyan. TMHs
forming the wall of the cavity are labelled.
b,Q
B
-binding pocket. The interacting residues of
D1 are labelled, arrows indicate or ientations of
TMH-d and TMH-e, and of the connecting
a
-helix
de. c, Electrochemical potential surface of PSII
(red, negative; blue, positive; white, neutral),
viewed along the membrane plane onto the cavity
opening that faces the membrane. The tail of Q
B
(violet) is nestled in the cavity. d, Schematic views
of Q
B
diffusion pathway in two orientations. Top,
same view as a. The positions of TMHs forming the
wall of the cavity (circles) and the membrane-
facing opening are indicated. Car molecules are
orange; lipid molecules are black. Bottom, PSII
embedded in the membrane. Positions and
approximate dimensions of the two openings of
the cavity, as well as the position of the Q
B
-binding
pocket and lipid molecules, are indicated.
Figure 1 | The PSII monomer from the cytoplasmic side. a, Overall view of
the PSII monomer. Transmembrane
a
-helices are represented as cylinders;
other protein elements are omitted for clarity. The main subunits are
highlighted as follows: reaction centre subunits D1 (yellow) and D2
(orange), antenna subunits CP43 (magenta) and CP47 (red), and the
a
- and
b
-chain of cytochrome b-559 (green and cyan, respectively). Low molecular
mass subunits are coloured grey. Unassigned TMHs are labelled X1–X3.
Cofactors are coloured green (Chla), yellow (Pheo), red (Car), blue (haem),
violet (quinone), black (lipids) and brown (detergent and unassigned alkyl
chains). The non-haem Fe
2þ
(blue) and putative Ca
2þ
(yellow) ions are
shown as spheres. Dashed line indicates the monomer–monomer interface.
b, Schematic representation of the view in a, with the same colour code.
Lipid and detergent molecules with headgroups pointing ‘downwards’ or
‘upwards’ are located at the lumenal or cytop lasmic side, respectively. Green
numbers in CP43 and CP47 indicate the positions of antenna Chla. The Q
B
diffusion cavity is indicated by a dotted line.
NATURE|Vol 438|15 December 2005 LETTERS
1041
© 2005 Nature Publishing Group
plastoquinone pool in tobacco
15
and, because the haem of cyto-
chrome b-559 is exposed to the cavity, an influence of the interior of
the cavity on the redox properties of cytochrome b-559 can also be
imagined.
The 11 carotenoid molecules were modelled as
b
-carotene in
all-trans configuration. Two carotenoids are bound to the D1 and
D2 subunits (Fig. 3), three are at CP43 and five at CP47 (Fig. 1).
Car12 is nestled between subunits PsbJ, PsbK, PsbZ and X1. The
carotenoids are distributed uniformly at the periphery of PSII except
for Car3, Car4, Car5 and Car6, which are clustered at the monomer–
monomer interface. This contrasts with the arrangement of seven
Car molecules in previous work
5
(Supplementary Discussion). In the
reaction centre, Car
D2
(Car11) is nearly parallel to the membrane
plane and located close to cytochrome b-559, as shown in previous
models
5,6
. Its counterpart Car
D1
(Car1) is oriented roughly perpen-
dicular to the membrane plane, the isoprenoid moiety being parallel
to TMH-a of D1 (Fig. 3a and Supplementary Fig. S5). The presence
of two differently oriented and therefore spectroscopically distinct
Car molecules has been shown for spinach PSII (ref. 16), where Car
D1
corresponds to Car
489
and Car
D2
to Car
507
.
Because Car
D1
does not bridge between Chlz
D1
and other cofactors
of the electron transfer chain (Fig. 3b), it is unlikely that Car
D1
participates in electron transfer reactions. Its position is rather
optimized for transfer and/or quenching of Chlz
D1
triplet states
and quenching of singlet oxygen that could be produced by
3
P680
(ref. 17). By contrast, the position of Car
D2
seems to be less efficient
for quenching of triplet states, but is in keeping with a role as a
‘molecular wire’ in putative secondary electron transfer when the
Mn
4
Ca cluster is not functional or is absent
17
. This idea is supported
by calculations of maximal electron transfer rates, showing that
secondary electron transfer reactions will predominantly occur on
the D2 side (Supplementary Table S4).
Most of the Car molecules located in the antenna subunits are in
van der Waals contact with Chla molecules, thereby allowing transfer
of excitation energy from Car to Chla, as well as protection of PSII
from destructive triplet states of Chla by means of rapid triplet–
triplet transfer. However, not all Chla molecules are located close to a
Car molecule. As coupled Chla molecules of the antenna might act as
excitation energy sinks, with a longer lifetime of the excited state and
hence higher probability of triplet formation, sufficient protection
from triplet states could be achieved if these groups are connected to
at least one Car molecule. Strongest coupled Chla groups in CP47
(Chla11, Chla12, Chla13 and Chla14, Chla17, Chla25, Chla26,
Chla27) have a Car molecule at van der Waals contact, whereas
corresponding groups in CP43 (Chla34, Chla46 and Chla45, Chla47)
are more distant from any Car, indicating lower triplet quenching
efficiency. A cluster of four Car molecules (Car3, Car4, Car5 and
Car6) is located at the monomer–monomer interface, close to a
coupled group of five Chla molecules in CP47 (Fig. 1 and Sup-
plementary Fig. S6). Three of the clustered Car molecules are at van
der Waals distance to each other, forming a coupled Car multimer,
which could broaden the absorption spectrum of the antenna system.
The shape of the electron density of the Mn
4
Ca cluster can be best
approximated by four Mn ions arranged as a ‘hook’ (Fig. 4a)
resembling a Y-shaped structure
3,6
. The Mn ions are numbered
Mn1 to Mn4, starting from the highest electron density at the bend
of the ‘hook’. The Mn–Mn distances could not be derived directly at
3.0 A
˚
resolution and thus were crystallographically refined with the
aid of restraints obtained from EXAFS studies (Supplementary
Methods). Mn1–Mn2 and Mn2–Mn3 are 2.7 A
˚
apart and probably
connected by di-
m
-oxo bridges, whereas Mn1–Mn3 and Mn3–Mn4
are at 3.3 A
˚
, suggesting mono-
m
-oxo bridges (Fig. 4b). This arrange-
ment is compatible with the ‘3 þ 1’ models suggested for the Mn
cations
18
. The position of Ca
2þ
between the Mn and Tyr
Z
is supported
by spherical anomalous difference electron density (Supplementary
Fig. S7). Ca
2þ
forming the vertex of a trigonal pyramid is equidistant
(,3.4 A
˚
) to Mn1, Mn2 and Mn3.
The interpretation of the Mn
4
Ca structure and coordination must
account for likely radiation damage of the cluster during X-ray data
collection. EXAFS studies of PSII crystals and solutions indicated
that Mn
3þ
and Mn
4þ
ions of the Mn
4
Ca cluster are rapidly reduced
by X-ray generated radicals to Mn
2þ
(ref. 19). This reduction is
probably associated with structural changes caused by the disruption
of
m
-oxo bridges and Mn–ligand interactions and may lead to
disorder, as indicated by uneven distribution of electron density in
the cluster (Supplementary Fig. S7).
The two short and two long Mn–Mn interactions and the three
equal Mn–Ca
2þ
interactions differ from the numbers of such
interactions derived from EXAFS studies
20
. This discrepancy could
be caused by the still low resolution of the diffraction data and by
possible radiation damage. In particular, the positions of Ca
2þ
and
Mn4 are less well defined in the electron density, leading to higher
coordinate errors for these atoms. The proposed coordination of Mn
and Ca
2þ
is shown in Fig. 4b. The residues of the second coordination
Figure 3 | Redox-active cofactors and electron transfer chain. a, View
along the membrane plane. The cofactors of the electron transfer chain
(P
D1
/P
D2
, Chl
D1
/Chl
D2
, Pheo
D1
/Pheo
D2
,Q
A
/Q
B
) are related by the pseudo-
C2 axis (arrow). Fe
2þ
(blue), Mn (red) and Ca
2þ
(yellow) ions are shown as
spheres. b, Schematic representation of the view in a. Selected distances
(in angstroms) are drawn between cofactor centres (black lines) and edges of
p
systems (red dotted lines).
LETTERS NATURE|Vol 438|15 December 2005
1042
© 2005 Nature Publishing Group
sphere (Fig. 4b, grey), even though they are not close enough to
coordinate the Mn
4
Ca cluster directly in the oxidation states present
in the crystals, might be suitable for interaction through water
molecules or may provide ligation in different redox states of the
Mn
4
Ca cluster.
Although the limited resolution does not allow a definite decision
about rotamers of ligating side chains, five carboxylates are in
positions that could act as bidentate ligands bridging different
cations. The combination of bidentate ligands and mono- or di-
m
-
oxo bridges could enhance the stability of the cation arrangement
and facilitate rearrangement of
m
-oxo bridges during the S-state
cycle. Mutational studies of ligating residues
21,22
found that, except
for Glu 333 and Asp 342 of D1, replacement of a ligating group with a
non-ligating group still leads to a (partially) functional cluster. Our
structure shows that in most cases a second ligating group is present,
which could hold the cations in place even when the exchanged
residue cannot coordinate them.
Glu 189 and Ala 344 in D1 are identified as possible ligands for
Ca
2þ
, with the former also ligating Mn1 and the latter Mn2, in
contrast to previous findings
5
. Taking into account the positional
error of Ca
2þ
and results from Fourier transform infrared (FTIR)
spectroscopy indicating that Ala 344 of D1 should ligate Mn
(ref. 23) but not Ca
2þ
(ref. 24), it is likely that Ca
2þ
is coordinated
only by Glu 189 of D1. Mn4, which is coordinated by Asp 170 and
Glu 333 of D1 and represents the first Mn ion bound to the high-
affinity binding site during assembly of the cluster
22,25
, seems to be
more distinct from the other three Mn ions. Our electron density
suggests that Mn4 is most prone to radiation damage and subsequent
disorder.
Our structure of the Mn
4
Ca cluster differs considerably from the
postulated cubane-like model
5
because the Mn–Mn distances in
the pyramid formed by three Mn and Ca
2þ
are not equal and the
pyramid is connected asymmetrically to Mn4 (Fig. 4c; for a detailed
comparison of both structures, see Supplementary Discussion and
Fig. S8). Even though the same coordinating residues have been
proposed
5
, none of them was modelled as bridging two metal ions,
leading to a lower saturation of the metal coordination spheres.
Consequently, the restraints for possible
m
-oxo-bridging are altered,
resulting in a different architecture of the whole oxygen-evolving
complex and providing new implications for the mechanism of water
oxidation.
Combining the structure with results from FTIR studies
23,26,27
and
assuming an oxidation state distribution of Mn(
III)
2
(IV)
2
for the S
1
state of the Kok cycle
28,29
, we can suggest localization of oxidation
states on individual metal ions of the cluster. Mn1 and Mn3 can
attain either oxidation state
III or oxidation state IV in the S
1
state.
Mn4, which is ligated by Asp 170, is not oxidized during S
0
–S
1
–S
2
–S
3
transitions
26
and is therefore probably present as Mn(IV), whereas
Mn2, which is ligated by the carboxy-terminal carboxylate of Ala 344,
changes from Mn(
III) to Mn(IV) in the S
1
–S
2
transition
23
and can be
considered as the site of first oxidation in the catalytic cycle of water
splitting.
METHODS
Dimeric PSII from T. elongatus was purified, characterized and crystallized as
described
30
. X-ray diffraction data were collected at the European Synchrotron
Radiation Facility (ESRF, Grenoble, France). The crystal structure of PSII was
determined to 3.0 A
˚
resolution and refined to R and R
free
factors of 0.24 and 0.29,
respectively (Supplementary Methods and Tables S1 and S2).
Received 1 July; accepted 13 September 2005.
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and CP43 (magenta) are indicated; Mn is red and Ca
2þ
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2F
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Supplementary Information is linked to the online version of the paper at
www.nature.com/nature.
Acknowledgements We thank D. DiFiore and C. Lu¨neberg for technical
assistance; K.-D. Irrgang, H. Lokstein, J. Messinger, F. Mu¨h, T. Renger,
E. Schlodder and J. Yano for discussions; and R. Clarke, G. Renger, K. Sauer and
V. Yachandra for critically reading the manuscript. Beam time and support at
the ESRF, SLS, BESSY and DESY are gratefully acknowledged. We thank
Deutsche Forschungsgemeinschaft and Fonds der Chemischen Industrie for
support.
Author Information Atomic coordinates have been deposited in the Protein
Data Bank under the accession number 2AXT. Reprints and permissions
information is available at npg.nature.com/reprintsandpermissions. The authors
declare no competing financial interests. Correspondence and requests for
materials should be addressed to W.S. (saenger@chemie.fu-berlin.de) and A.Z.
(zouni@phosis1.chem.tu-berlin.de).
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