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Annu. Rev. Biophys. Biomol. Struct. 2001. 30:307–28
c 2001 by Annual Reviews. All rights reserved
Copyright °
PHOTOSYSTEM II: The Solid Structural Era
Kyong-Hi Rhee
Medical Research Council, Laboratory of Molecular Biology, Hills Road, Cambridge,
CB2 2QH, United Kingdom; e-mail: khrhee@mrc-lmb.cam.ac.uk
Key Words reaction center, P680, cytochrome b559, evolution, electron
crystallography
■ Abstract Understanding the precise role of photosystem II as an element of oxygenic photosynthesis requires knowledge of the molecular structure of this membrane
protein complex. The past few years have been particularly exciting because the structural era of the plant photosystem II has begun. Although the atomic structure has yet to
be determined, the map obtained at 6 Å resolution by electron crystallography allows
assignment of the key reaction center subunits with their associated pigment molecules.
In the following, we first review the structural details that have recently emerged and
then discuss the primary and secondary photochemical reaction pathways. Finally, in
an attempt to establish the evolutionary link between the oxygenic and the anoxygenic
photosynthesis, a framework structure common to all photosynthetic reaction centers
has been defined, and the implications have been described.
CONTENTS
PERSPECTIVES AND OVERVIEW . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
STRUCTURE DETERMINATION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Electron Cryo-Microscopy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Structure at 6 Å Resolution . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
SUBUNIT ASSIGNMENT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
D1/D2 Heterodimer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
CP47 and CP43 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Cytochrome b559 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
IMPLICATIONS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
An Emerging Framework Structure Among Reaction Centers . . . . . . . . . . . . . . .
Electron-Transfer Pathways in Photosystem II . . . . . . . . . . . . . . . . . . . . . . . . . .
PROSPECTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
307
310
310
310
311
311
313
314
315
315
317
319
PERSPECTIVES AND OVERVIEW
Life without oxygen? Without oxygen, life certainly could not have evolved to
its contemporary level of complexity. Studies on photosystem II (PSII) shed light
on how this integral membrane protein complex utilizes solar energy to liberate
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molecular oxygen from water. Water is a very stable molecule. Thus to oxidize
water, the PSII reaction center (RC) must catalyze an extremely strong oxidizing
condition. P680, a photoactive chlorophyll a complex in PSII, carries out this
unique activity driven by the light-induced charge separation (for an overview in
photosynthesis research, see Govindjee, 45a).
Photosystem II is located in the thylakoid membrane and consists of more than
20 protein subunits (Table 1) composed of the reaction center core, the oxygenevolving complex (OEC), and the peripheral light-harvesting antenna assembly.
Biochemical and biophysical studies over the past several decades helped elucidate the nature of this enzyme: (a) Purification of a functional PSII complex has
been successfully developed (10a, 87), and various reconstitution protocols have
been devised. (b) This opened the door to a direct spectroscopic analysis of the
isolated membrane-free PSII complex and consequently has stimulated structural
studies of this enzyme. However, the large size, structural complexity, and the heterogeneity of the PSII complex (61) have hindered the high-resolution structure
determination until recently. Previous efforts using crystallography and electron
microscopy (16, 17, 30, 49, 51, 57, 77, 78, 79, 80, 82, 86, 88, 107, 115, 124, 125) provided a glimpse of the shape of the various PSII assemblies, although limited to
relatively low-resolution.
Electron crystallography of two-dimensional (2D) crystals combined with electron cryo-microscopy (cryo EM) is an established method for determining the
structure of proteins. This technique, advantageous for membrane proteins, has
been used to investigate the structure of an ∼160 kDa plant photosystem II reaction
center complex. The map resolved at 6 Å resolution (99, 101) provided the first
solid three-dimensional (3D) structure where the location of the D1, D2, CP47,
and cytochrome b559 (Cyt b559) α/β subunits and the likely position of their
associated cofactors were assigned.
Solely from a structural point of view, the time is ripe for a new paradigm:
Photosynthetic RCs were thought to be divided into two types, the quinone type
RC and the Fe/S type RC (reviewed in 14, 26). Purple bacteria, green filamentous
bacteria, and PSII belong to the former group, whereas green sulphur bacteria,
heliobacteria, and PSI belong to the latter. Although this classification originally
derives from the nature of the primary electron acceptor, it has often been interpreted as a structural classification. In recent years, there has been considerable progress in unveiling structures of PSI and PSII, which allows structural
comparison of both types of reaction centers. Indeed, it now seems apparent
that there is a structural universality among the photosynthetic reaction centers
and that all reaction centers share a common evolutionary ancestor (90, 101,
110).
Emphasis here lies on the biological implications; in particular, I focus
on the structure of the primary electron donor (P680) and the structure of cytochrome b559, which are characteristic in oxygenic photosynthetic reaction
centers.
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TABLE 1 Photosystem II subunits
Gene
Protein
Mr∗ (Ma∗∗)
Helices§
Function
Source
psb A
D1
38.8 (32)
5
Reaction center (RC)
PSBA SPIOL
psb B
CP47
56.3 (47)
6
Chl a-binding RC
antenna
PSBB SPIOL
psb C
CP43
51.8 (43)
6
Chl a-binding RC
antenna
PSBC SPIOL
psb D
D2
39.4 (32)
5
Reaction center
PSBD SPIOL
psb E
Cyt b559α
9.3 (9)
1
Photoprotection
PSBE SPIOL
psb F
Cyt b559β
4.5 (4)
1
Photoprotection
PSBF ORYSA
psb H
PsbH
7.8 (10)
1
Light-dependent
phosphorylation
PSBH ORYSA
psb I
PsbI
4.2 (4.8)
1
Unknown
SOPSBIA§§
psb J
PsbJ
4.2 (4)
0
Stabilization of
assembly
PSBJ MAIZE
psb K
PsbK
4.3 (3.5)
1
Unknown
CHSOPSBK§§
psb L
PsbL
4.5 (5)
0
Regulation of the
P680+ reduction
PSBL ORYSA
psb N
PsbN
4.7 (4.1)
1
Unknown
PSBN ORYSA
psb O
OEC1
27.0 (33)
0
Regulate oxygenevolution
PSBO WHEAT
psb P
OEC2
20.2 (23)
0
Regulate oxygenevolution
PSBP SPIOL
psb Q
OEC3
16.9 (17)
0
Regulate oxygenevolution
PSBQ CHLRE
psb R
PsbR
10.2 (10)
0 or 1
Unknown
PSBR SPIOL
psb S
PsbS
21.7 (22)
4
Regulation of lateral
location, light
harvesting
PSBS SPIOL
psb T
PsbTc
3.8
1
Protection of growth
PSBT SPIOL
psb W
PsbW
5.9 (6.1)
1
Control of the assembly
and accumulation
SORNAPIIP§§
psb X
PsbX
4.1
1
Unknown
(69a)
∗
Molecular weight calculated from the amino-acid sequence (kDa).
∗∗
Apparent molecular weight (kDa).
§Transmembrane helices.
§§Data and references originate from the GENEMBL databank.
Total Mr = ∼340 kDa.
Where available, the protein sequences from Spinacia oleracea, used for this analysis, were obtained from the protein data
bank “SWISSPROT” (references cited therein). For the cab gene products see the references: LHC-II (48), CP29 (97), CP26
(10), CP24 (38), and CP14 (59) (adapted from 99).
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STRUCTURE DETERMINATION
Electron Cryo-Microscopy
The use of electrons as an illuminating material for scattering analysis has several potential advantages. The central profit arises from the significantly greater
(ca. 100,000 times) cross-section of electrons than that of X-rays and the lower
amount (ca. 100 times) of radiation damage than for X-ray scattering (53). These
facts imply that the crystal size required for electron crystallography is much
smaller than that required for X-ray analysis. Secondly, no “phase problem” exists. Electron microscopic images maintain the phase information; thus, the phase
values are readily obtainable by numerical Fourier analysis of images. Thirdly,
charged electrons can easily be focused by magnetic lenses. Therefore, electron
microscopes can concomitantly record both diffraction patterns and images without any particular technical demands.
The image analysis of 2D projections for a reconstruction of a 3D structure
was first demonstrated with objects embedded in a thin film of heavy atom stains
(33, 58). Later, analysis with specimens of unstained biological macromolecules,
such as specimens prepared in glucose (127), in thin frozen, aqueous films (119),
or in a thin layer of vitreous ice (36), showed that, in principle, resolutions of
electron microscopic images can ultimately reach close to 3 Å resolution. To date,
four atomic resolution structures have been determined by cryo EM of 2D crystals:
bacteriorhodopsin (54; PDB code:1brd), light-harvesting complex II (73; no PDB
code deposited), tubulin (91; PDB code:1tub), and AQP (85b; PDB code:1fqy).
Several hundred medium resolution structures were obtained by electron cryomicroscopy, of which the following were determined using 2D crystals and are
approaching a near-atomic resolution: bovine rhodopsin (71), halorhodopsin (74),
PSII (99, 100), gap-junction (126), and NhaA (135). Various aspects of cryo EM
and electron crystallography have been reviewed (4, 7, 37, 44, 53).
Structure at 6 Å Resolution
The 2D crystals used for structure determination of the monomeric PSII RC complex were obtained in the dark, by detergent removal (86, 102). This method is
one of several methods of 2D crystallization of membrane proteins that have been
developed over the years (62, 68, 98, 103). Biochemical evidence indicated that
the crystals contain the D1, D2, CP47, Cyt b559 α- and β-subunits, and several
small polypeptides including PsbI, PsbK, PsbL, PsbTc, and PsbW (138). This
monomeric complex appears to be active by its excitation spectrum and in electron transfer (12). The projection structure of the unstained, frozen 2D crystals
(101, 102) indicated that the rectangular unit cell has the dimension of α = 168.3 Å
and β = 155.2 Å with the 2-sided plane group of p22121 symmetry.
A three-dimensional map to a resolution of 8Å in the membrane plane has
been published (101). A 6 Å resolution structure has also been reconstructed by
analyzing 45 images (99), and has subsequently been improved by merging 79
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TABLE 2 Electron crystallographic data used for the 3D structure
determination of plant PSII (100)
Two-dimensional crystals
2-sided plane group
Unit cell parameters
Thickness (Å)
p22121
α = 168.3 Å, β = 155.2 Å, γ = 90◦
70–80
Phase determination from images
No. of merged lattices
79
Maximum tilt angle
Resolution limit for merging
No. of reflections merged
No. of independent phases
Overall weighted phase residual
in resolution range (Å):
200.0–14.0
14.0–10.0
10.0–8.2
8.2–7.0
7.0–6.0
Resolution in membrane plane
Resolution in membrane normal
0–20◦ :
20–30◦ :
30–40◦ :
40–50◦ :
50–67◦ :
67◦
6.0 Å
59456
7193
29.9◦
22 lattices
16 lattices
10 lattices
19 lattices
12 lattices
23.2◦
30.5◦
38.2◦
41.8◦
48.7◦
6.0 Å
11.4 Å
images recorded at tilt angles ranging from 0◦ to 67◦ (Table 2). In Figure 1, all
7193 independent structure factors, with an overall phase residual of 29.9◦ , are
plotted in the azimuthal projection. The distribution of the tilt axis angles and the
tilt angles of each crystal lattice must be considered in order to build up reciprocal
Fourier space evenly, so that it can be effectively sampled in all directions (112).
These are represented in Figure 1 by the point-spread function of the raw data. The
algorithms for the image processing of 2D crystals (54, 55) and the MRC-LMB
image processing programs (28) were used.
SUBUNIT ASSIGNMENT
D1/D2 Heterodimer
The D1 protein was first identified as a major translation product of greening
chloroplasts (13, 47). Later, the primary sequence (140) showed that the D1 protein
is homologous to the D2 protein (56, 104). Deisenhofer et al (29) suggested on the
basis of the analogy to the L and M subunits of the Rhodopseudomonas viridis, that
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Figure 1 Azimuthal projection of raw data and point-spread function (inset). The spot
size correlates with the reliability (IQ value) of the phase (55). The larger the dot, the greater
the reliability of the individual amplitude and phase. The point-spread function calculated
with a cut-off of 6 Å resolution shows that the vertical elongation factor is 1.9 mainly due
to the missing cone (K-H Rhee, unpublished data).
the D1 and D2 proteins each contain five transmembrane α-helices. Soon after this
proposal, experimental evidence confirmed this five transmembrane α-helix model
(109). The redox-active components necessary for the primary photochemical
reactions appeared to be associated with this heterodimer (87). These cofactors
are (from the oxidizing side to the reducing side) a tetramanganese cluster with
a calcium- and a chloride-ion, two tyrosine residues as referred to YZ and YD,
four to six chlorophyll a molecules, two pheophytins, and two plastoquinones.
Compared to these, cofactors, such as non-heme iron and bicarbonate anions
(46), do not function as a direct electron carrier but are thought to be crucial
for this process. Two β-carotene molecules are likely to be present in the RC and
probably involved in the secondary photochemical reactions (see the section on
secondary cyclic pathway in “Electron-Transfer Pathways in Photosystem II”). The
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debate over the stoichiometry of the cofactors, especially the chlorophyll a content,
brought to light the fact that PSII RC probably contains more chlorophylls than
does the bacterial RC. The experimentally determined number varied from 4 to 6
per RC, depending largely on membrane preparation (9, 24, 41, 45, 67, 87). A free
radical signal observed by EPR under low temperature illumination (77K) of darkadapted PSII RC, in which Cyt b559 had been oxidized, suggested the presence
of an additional cationic chlorophyll radical in PSII RC (32). Some groups have
speculated that the binding ligands of these chlorophylls are the histidines-118 of
both D1 and D2 polypeptides, and that these histidines are located in the second
transmembrane α-helices of both subunits (58a, 69, 109a).
As represented in Figure 2, the crystal structure shows that in the central position
there are 10 transmembrane α-helices of the D1/D2 (yellow/orange) heterodimer
that are arranged in near twofold symmetry around a local twofold axis (101).
Along this axis six tetrapyrrole densities appear nearest to the position analogous
to that of the bacterial counterparts (101). These correspond to the photochemically
active P680 (two central green discs near helices E), the non-P680 chlorophylls
a (two nearly central green discs near helices D), and two pheophytins (brown
discs near helices D). Because the histidine residues that bind the accessory bacteriochlorophylls in the L and M subunits are not conserved in the sequences of
the D1 and D2 (81), the binding motif for the corresponding Chl a must be different from that of the bacterial counterparts. The exact function of the non-P680
chlorophylls remains under discussion.
In the periphery of the D1 and D2 heterodimer, two additional densities between
the helices A and B of both D1 and D2 proteins (Figure 2) were observed and
suggested to be the likely position of the redox-active ChlZ and ChlD (99). This
positioning agrees well with the spectroscopic measurement indicating the distance
of 39.5 Å ± 2.5 Å from the bacterial non-heme Fe(II) proposed by Koulougliotis
et al (69). The additional density on the D1 side has a corresponding distance
of ∼37 Å, and that on the D2 side has a distance of ∼40 Å (99). Notably, the
electron density on the D2 side is more reliable than that on the D1 side in the
D1/D2/CP47/Cytb559 complex (99). Because these densities are surrounded by
additional α-helices (blue cylinders in Figure 2), it is conceivable that the Chl a
content in PSII RC may vary, depending on the subunit composition sustained
during the preparation.
Both QA and QB were missing in the 2D crystals owing to the absence of the
CP43 subunit (108). Nevertheless, models for the QA binding site in the D2 subunit and for the QB binding site in the D1 protein have been suggested on the
basis of an analogy made with the X-ray structure of their bacterial homologs
(81, 105, 136, 137).
CP47 and CP43
Two chlorophyll a-protein complexes, which are designated in the current literature
as CP47 (= CPa-1) and CP43 (= CPa-2), are the RC antennae for PSII. The CP47
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and CP43 proteins were thought to be involved in the resonance energy transfer,
in which the absorbed energy is transferred to the reaction center through the
noncovalently bound Chl a. The protein sequences (56, 83) predicted that each
of the CP47 and CP43 proteins has six transmembrane α-helices (25, 132). In the
membrane domain, 12 histidine residues are thought to serve as axial ligands for
chlorophyll tetrapyroles, as is the case in the R. viridis reaction center (29) and in
light-harvesting proteins (73, 123). Biochemical properties of the CP47 and CP43
proteins have been reviewed elsewhere (19).
The CP47 protein is located closest to the D2 protein (85) as shown in Figure 2.
The characteristic arrangement of pairwise helices in CP47 shows a threefold
symmetry, of which two pairs share an internally repeating sequence homology;
the second transmembrane α-helix matches the fourth α-helix, and the third
α-helix matches the fifth transmembrane α-helix (99). This result suggests that
gene duplication may be common for the chlorophyll-binding proteins. For
instance, internal sequence repetition has also been observed in PsbS protein
(64, 133). It is interesting to note that no similar sequence repetition has been
found in the CP43 protein (99).
In the interior of CP47, fourteen chlorophylls are accommodated within 17 Å
of each other (99, 101). This allows them to play the role of light-harvesting and
excitation energy transfer (130). CP47 binds less chlorophyll than does the corresponding antenna part of the PsaA and PsaB proteins of photosystem I (PSI).
Unlike anoxygenic photosynthetic apparatus, the inner-antenna chlorophylls are
clustered rather than encircle RC.
The location of CP43 has been inferred by comparing the projection map of
a RC core dimer (49) with the structure of a monomeric D1/D2/CP47/Cytb559
complex (102). The model for CP43 (gray cylinders) in Figure 2 was built up
using the twofold rotational symmetry of the CP47 around the local twofold axis.
Cytochrome b559
Since the photooxidation of cytochrome b559 was first demonstrated (66), it has
been assumed that Cyt b559 is closely associated with the oxidizing side of PSII.
Soon after the determination of the primary sequence of both Cyt b559 α- and
β-subunits (55a), it became clear that, like other b-type cytochromes, the axial
ligands of the heme iron of Cyt b559 are two histidines (6). These are likely to be
His-23 in both α- and β-subunits (plants numbering, 94). Cyt b559 has long been
thought to be essential for the assembly of the functional RC (87, 95, 96). The
puzzling nature of the physical properties of Cyt b559 (see below) and the lack
of homology in purple bacterial RC led to an intense investigation of Cyt b559.
However, the exact regulative mechanism is not yet understood.
The structural assignment of Cyt b559 in PSII (99) is shown in Figure 2. Here,
Cyt b559 is located near the helix A of the D2 protein. It is most likely that
there is only one Cyt b559 per reaction center in plant PSII (21, 99 and references
therein). However, we cannot exclude the possibility that the stoichiometry of
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Figure 3 Schematic diagram of the Cyt b559
α/β heterodimer in plant PSII RC. Cylinders represent the transmembrane α-helices and the disc
represents the heme group. The putative boundaries of the thylakoid membrane are indicated
by the parallel lines (adapted from 99).
Cyt b559 in the cyanobacterial PSII may differ from one (78a, 79a and references
therein). The 2D crystal structure shows that the α- and β-subunits of Cyt b559
form a heterodimer with the bound heme, which is placed approximately 11 Å
from the stromal surface with respect to the estimated membrane thickness of
36 Å (Figure 3; 99). This agrees with the topography previously deduced from
antibody assays and proteolysis (117, 118, 128).
Some earlier deletion mutagenesis studies have shown an intimate structurefunction relationship between Cyt b559 and the D2 protein (89, 94). The crosslinking results of Barbato et al (8) are consistent with our structure in which Cyt
b559 is closer to the DE loop of D1 than to the DE loop of D2 on the stromal side
(see Figure 2). Importantly, the heme is positioned near the QB binding site. It is
thus possible that reduced QB may function as the direct reductant to Cyt b559,
and that there is a probable secondary electron transfer pathway from the catalytic
core, in the context of the intact PSII reaction center (121). A model for electron
donation from QB− to Cyt b559 has been deduced from the spectroscopic analysis
under various inhibitor conditions (22). This is discussed further in the section on
secondary cyclic pathway in “Electron-Transfer Pathways in Photosystem II.”
IMPLICATIONS
An Emerging Framework Structure Among Reaction Centers
Canonical Heterodimer Figure 4 (top;) shows a comparison of the transmembrane α-helices from the D1/D2 proteins (yellow/orange cylinders) with those
of the L/M subunits from R.viridis (dark/light blue ribbons) (99). The overall folding is very similar, except for the lumenal end of the helices 3 and 30
(see below). Similarly, the structure of PsaA protein of the PSI reaction center has been superimposed with the structure of the corresponding PSII subunits
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(Figure 4, middle and bottom). The structure of the RC with five C-terminal αhelices of PsaA, drawn in pink ribbons in Figure 4 (middle), shows that the position and orientation of the helices near the local twofold axis match with the
corresponding helices of PSII, whereas the inward radial displacement increases
away from the twofold axis by up to 12.0 Å. The elegance in this comparison was
the finding of the structural similarity in the region outside the reaction center.
The CP47 protein has a structure surprisingly similar to that of the N-terminal six
α-helices of PsaA (Figure 4, bottom; 101), which are known as the RC antenna
region of the PSI. A minor realignment was required for the best fit with respect
to the PsaA protein (102). It is obvious that if D1 and D2 proteins were covalently
fused to the CP43 and CP47 respectively, then the offspring would have, to a large
extent, the same structure as the PSI RC (in Figure 5, middle and bottom).
This comparison demonstrates that there is a common framework structure
among all kinds of photosynthetic reaction centers (Figure 5). Reaction centers
are composed of two homologous proteins that form a heterodimeric core in which
a near twofold symmetry is maintained. Along the local twofold axis, a pair of
central (bacterio) chlorophylls, a pair of accessory (bacterio) chlorophylls, and a
pair of primary electron acceptors of either pheophytins (PSII, bRC) or chlorophylls a (PSI) are positioned. They are anchored by a heterodimeric protein core
that provides the hydrophobic environment. The protein folding and the S-shaped
packing seems to be energetically favored, so that the overall scaffolding has been
stable over several billion years of evolution. However, the exact configuration of
cofactors seems to be diverse (3, 29, 70, 99), but still within certain functional criteria, as spectroscopic studies and theoretical quantum field analyses have pointed
out (84 and references therein). It is therefore probable that there are some canonical geometries of the redox cofactors that together form a resonance hybrid within
the context of the protein. Perhaps we can think of this framework as a functional
unity, at least in the context of the proteomic evolution.
Evolutionary Footprints On the basis of our knowledge of the structure of reaction centers, we attempt to understand how oxygenic photosynthesis evolved.
Some characteristic structures of the PSII that distinguish this oxygenic reaction
center from its anoxygenic bacterial counterparts are starting to emerge.
A prominent feature on the oxidizing side of the PSII RC that altered during the
evolution is found in the region of the helices 3 and 30 of the D1 and D2 proteins
(Figure 4, top left). Each L and M subunit has a kink, brought about by their
proline residues, whereas the D1 and D2 subunits both appear as straight helices,
oriented towards the lumen. Primary sequence analysis indicates that the location
of the corresponding prolines in D1 and D2 (Figure 6), with respect to the V-157
and F-158 (which are conserved in the L subunit as their counterparts V-133 and
F-134), is likely to be shifted by an insertion of two residues to the C-terminus.
It is interesting that the redox-active tyrosines, YZ-161 and YD-161, the electron
carriers between P680 and the oxygen evolving complex appear as the inserted
residues in this integral membrane domain (99). This implies that the positions of
the YZ and YD, as one helix turn above the kinks (99), point to the helices 5 and 50
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Figure 6 Shifted prolines by the insertion of the YZ-161 and YD-161 in D1 and D2,
respectively. The amino acid sequences of interest are given with the residual number of
the D1, D2, L, and M proteins (adapted from 99).
of the D1 and D2 proteins, respectively. The 6 Å resolution structure retains the
twofold symmetry in this region. In the section “Electron-Transfer Pathways in
Photosystem II,” two notable features evolved only in PSII RC are discussed.
Electron-Transfer Pathways in Photosystem II
Primary ‘Uphill’ Reactions The two central chlorophylls in closest proximity to
the lumenal surface and to the local twofold axis within the D1/D2 heterodimer are
of particular interest. These are the positions equivalent to the bacterial “special
pair” and are believed to function as the photochemically active P680. The photooxidation of P680 creates an unusual redox potential of Em = ∼1.1 V (63b, 65),
which is high enough to oxidize water (+0.8V), whereas other primary electron
donors, such as P870 (+0.45 V, purple nonsulphur bacteria), P700 (+0.49 V, photosystem I), or P840 (+0.25 V, green sulphur bacteria) cannot accomplish this
function (99 and references therein). By accumulating four positive charges, the
photooxidized P680+ successively extracts four electrons from the manganesewater cluster (63a, 67a), thereby releasing molecular oxygen into the atmosphere.
The mechanism of water oxidation on this terminal-electron-donor side has not
yet been fully established.
The crystal structure obtained by electron crystallography shows that the centerto-center distance of the central chlorophyll pair may be ∼11 Å, which is significantly larger than the corresponding distance of 7.6 Å of the purple bacterial
special pair (101) as shown in Figure 7. This more distant separation may explain
the weaker exciton coupling observed in PSII RC (18, 120). A similar distance of
11.5 Å has also been suggested on the basis of the magnetic resonance spectrum
of P680 in its triplet state (23). As a result of the distant association of the P680
dimer, it is likely that the interaction between P680 and the adjacent chlorophylls
analogous to the accessory bacteriochlorophylls is tighter than that in purple bacteria. Therefore, we may not rule out the possibility that the P680 dimer is weakly
coupled to several neighboring pigments. In the model of a P680 multimer (40),
the exciton state is delocalized over the reaction center chlorophylls a including the
pheophytin electron acceptor. The biphasic nature of the charge separation process
in PSII has also been experimentally demonstrated by Greenfield et al (48a).
Concerning the physical as well as the kinetic properties, the subtle differences
between the individual tetrapyrrole components have yet to be determined. The
inability to separate the character of chlorophylls, for which the optical spectra
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strongly overlap, has made the determination difficult. However, several observations led to the proposal of an asymmetric nature of P680 (reviewed by 34, 39, 92,
129, 131), although some homology-based modeling approaches favor the symmetric arrangement of the P680 dimer (105, 116, 136, 137). The structural features
of the central chlorophyll pair give rise to at least at 6 Å resolution, a likely distribution of the asymmetric electron densities over the P680 dimer (99). To dissect
the quantum thermodynamic details of all protein-pigment and pigment-pigment
interactions in intact PSII RC, requires theoretical as well as experimental proof.
Secondary Cyclic Pathway The ability of Cyt b559 as an electron carrier in photosynthesis is brought about by the heme, whose iron atom undergoes oxidationreduction. In PSII, Cyt b559 is closely associated with the first transmembrane
α-helix of the D2 protein (Figure 2). The heme group of Cyt b559 is faced
to the stromal side and in proximity to the QB binding site of the D1 protein
(99). This positioning implies that Cyt b559 is likely to mediate secondary
electron-transfer in PSII RC either by oxidizing plastoquinol (134) or by QB (22).
The reduction of Cyt b559 by QB− is more probable because plastoquinol and
QA− do not seem to be the direct reductant to Cyt b559, as demonstrated by the
dark reduction response of Cyt b559 under the DCMU-treatment (22).
When PSII is exposed to an excess of photons the highly reactive P680+ becomes harmful, while the oxidizable peripheral chlorophylls which have a relatively low potential are harmless. Suggestions were made whether the reduced Cyt
b559 donates the electron to P680+ directly (52) or via the redox-active peripheral ChlZ (121). The peripheral chlorophyll in the D2 subunit (ChlD2 in Figure 8),
referred to as ChlD in the literature, is located with a center-to-center distance
of ∼27 Å from the heme (author’s unpublished observation); thus the electron
donation from the cytochrome b559 to this ChlD2 is plausible. Only recently has
site-directed mutation analysis shown that this ChlD2 might be involved in the RC
photochemistry (63).
The involvement of β-carotene in the secondary electron-transfer cycle has been
proposed on the basis of the spectroscopic identification (50). PSII RC contains
two β-carotene molecules (45, 67) that adopted probably a 15-cis configuration
(11) and are possibly coupled excitonically (76), of which one β-carotene seems
to be located on the D2 side interacting with an indole nitrogen of a tryptophan
residue, such as D2-112 and D2-168 (31). There are homologue partners of these
residues in the M subunit of the bacterial RC where the carotenoid is associated.
In purple bacteria, β-carotene has the function of protecting the reaction center
from photoinhibition by quenching triplet states of bacteriochlorophyll (reviewed
in 42). Such triplet quenching by β-carotene is unlikely in PSII RC (39), and the
β-carotene may alternatively function as an electron carrier in PSII.
Taken together, the results support the existence of a secondary cyclic electrontransfer pathway in PSII RC via Cyt b559. There remains, however, current discussion about whether the β-carotene is reduced by Cyt b559 or by ChlD2 (Figure 8).
Nevertheless, the cyclic electron-transfer reactions seem to be essential to protect
the oxidizing side of the reaction center from photoinhibition (22, 121).
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319
Figure 8 Model of the secondary electron-transfer pathway in PSII RC. Under the photo
excess conditions, an electron from QB− is transferred to the heme and then recycled to
the P680 via β-carotene (white solid arrows). The paths indicated by dotted arrows are
currently under investigation. The location of QA, QB and the non-heme iron is assumed
on the basis of the structure of the R. viridis RC.
Of special interest is the finding that Cyt b559 exists in transformable potential
forms. The midpoint potential of Cyt b559 varies with a range of unusually high
(+375 mV) to low (+50 mV) potential (reviewed in 27). Several other physiologically relevant intermediate potential forms of Cyt b559 have also been reported
(60, 122). The large decrease in potential that results from the removal of the 17and 23-kDa extrinsic polypeptides has been interpreted as being due mainly to an
increased solvent accessibility of the heme group (122). A model calculation of the
dielectric field of an α-helix in membranes shows that the redox properties of Cyt
b559 may be affected by the membrane electrostatic environment (72). However,
bearing in mind more recent results (1a, 60), it is possible that the association of the
17- and 23-kDa proteins has an indirect role in keeping Cyt b559 in high potential
form. The 3D structure of the Cyt b559 α- and β-subunits shows that both transmembrane α-helices are highly tilted with respect to the membrane normal and the
lumenal end of both α-helices closely comes together near the membrane surface
(Figure 3). As for the existence of the different forms of Cyt b559, its regulatory
mechanism has remained unclear. The physical properties of the heme and its photochemical behavior are comprehensively reviewed by Stewart & Brudvig (114).
PROSPECTS
It has become more important than ever to have a high-resolution structure with
which exact functional mechanisms of the PSII reaction center can be explained. A
three-dimensional single crystal of PSII has been obtained from the thermophilic
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cyanobacterium Synechococcus elongatus that diffracts X-rays to a resolution of
3.8 Å (139). Crystals belong to the space group P212121, with the unit cell dimension of a = 134 Å, b = 227 Å, and c = 310 Å. The native crystals are still active
in the water oxidation reaction. Other forms of the monomeric PSII RC complex
from both spinach and pea have also been crystallized in three dimensions using
a mixture of a variety of detergents (1). These crystals contain at least 8 subunits (D1, D2, CP47, CP43, 33 kDa subunit, Cyt b559 large- and small-subunits,
and 4.8kDa subunit) and diffract to 6.5Å resolution. They belong to a hexagonal space group with unit cell parameters of a = 495 Å, b = 495 Å, c = 115 Å,
α = β = 90◦ , and γ = 120◦ .
Electron crystallography combined with electron cryo-microscopy is becoming an increasingly powerful method as an alternative to X-ray crystallography.
Both 2D crystal and single particle approaches may help understand the structurefunction relationship of various PSII complexes. A number of subcomplexes have
shown a tendency to crystallize as two-dimensional arrays. A well-resolved 3D
structure determined by single particle cryo-EM analysis, for which no crystal is
required, would allow the subunits that are absent in both 2D and 3D crystals to
be assigned.
Finally, a sound understanding of all the elements involved in the energy conversion and the water oxidation in PSII will be successful only if structural, biochemical, and spectroscopic studies collaborate.
ACKNOWLEDGMENTS
The European Molecular Biology Organisation (EMBO) and Max Perutz Fund at
the MRC Laboratory of Molecular Biology helped me to cover the travel costs
for my attendance at the Gordon Research Conference on Photosynthesis in June
2000. I am most grateful to the colleagues who encouraged me to write this article
and with whom the constructive discussions were made. It gives me pleasure to
express my thanks to Drs. Richard Henderson and Govindjee for the comments
on the manuscript.
Visit the Annual Reviews home page at www.AnnualReviews.org
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Figure 2 Location of the PS II reaction center subunits. The map shows the central position of the D1 (yellow) and D2 (orange) heterodimer, and the adjacent CP43 (gray) and
CP47 (red) proteins, respectively. The Cyt b559 α/β subunits (purple) are located in the
outermost of the D2 subunit from the local twofold symmetry axis. The likely positions of
the chlorophylls a (green), the pheophytins (brown), and the heme (white) are represented
by discs. The remaining five, presumably nonpigment binding α-helices resolved in the
monomeric D1/D2/CP47/Cytb559 complex, are color-coded in blue (adapted from 99).
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Figure 4 A comparison of the plant PSII RC with the bacterial RC from R. viridis (top)
and with the PSI RC from Synechococcus elongatus (middle). A comparison of the RC
antenna α-helices of PSI and PSII (bottom). (a) A view from the lumenal side of the
thylakoid membrane. The cylinders represent the D1/D2 (yellow/orange) α-helices. The
transmembrane α-helices of the L/M (dark/light blue) subunits of R. viridis are drawn as
ribbons. The numbers indicate the order of the transmembrane α-helices in the L and M
subunits. (b) A 90◦ rotation of those shown in (a) (lumenal surface below). (c) A view from
the lumenal side. The C-terminal five transmembrane α-helices of the PSI PsaA/B proteins
(pink) are represented as ribbons. (d ) A 90◦ rotation of those shown in (c) (lumenal surface
below). (e) A view from the lumenal side. The red columns represent the six α-helices of
CP47. Capitals are arbitrary labeling. The N-terminal six α-helices of the PSI PsaA (pink)
are drawn as ribbons. ( f ) A 90◦ rotation of those shown in (e) (lumenal surface below).
Coordinates were taken from the Brookhaven Protein Databank (1PRC, 2PPS) (adapted
from 99).
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Figure 5 Nature’s choice for the photochemistry in the reaction centers. Top: Bacterial
RC (bRC). The L and M subunits (orange) create a charge separation using the cofactors
(green and red) attached to the transmembrane α-helices that are arranged with a pseudotwo-fold symmetry. The outer light harvesting antenna proteins, LH2 (light green), transfer
the light energy to the LH1 (light red) and then to the reaction center which it encircles.
Cytochrome (Cyt) donates electrons to the reaction center. Center: Photosystem II. The
D1 and D2 proteins (orange) are the evolutionary descendants of the L and M subunits
of the purple bRC. The overall arrangement of the cofactors (green) necessary for the
primary charge separation appears in a similar order, but the distance between the central
pair is significantly larger than that of the bacterial special pair. The RC antenna proteins,
CP43 and CP47 (light red), are located adjacent to either side of the D1 and D2 proteins,
reflecting an extended heterodimeric organization. Cyt b559 (purple) is positioned at the
D2 side only. LHC-II (light green) collects light energy, which is eventually transferred
to the reaction center. The photooxidation of P680 enables the manganese-cluster (Mn) to
catalyze water oxidation. Below: Photosystem I. The homologous PsaA and PsaB proteins
(orange) resemble the PSII-like heterodimer. Each of PsaA and PsaB consists of a reaction
center equivalent to D1 or D2, fused with an antenna equivalent to CP43 or CP47. Electrons
flow “uphill” from plastocyanin (PC) to iron-sulphur cluster (Fe/S) through a cascade of
redox reactions (adapted from 99).
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Figure 7 Active constituents of the electron-transfer pathway in the R. viridis RC and
a comparison with the PSII. The components are arranged symmetrically and include the
special pair of chlorophylls (P), two bacteriochlorophylls (BCha), two bacteriopheophytins
(BP), and the terminal quinones (QA and QB). This geometry is similar in the PSII RC (black
contours), where the manganese (Mn) cluster accumulates the four oxidizing equivalents
required to produce one molecule of dioxygen. Figure reprinted with permission (41a,
copyright 1998, Macmillan Magazines Limited).