Introduction
Photosystem II (PSII) is a multisubunit complex embedded in the thylakoid membranes of higher plants, algae and cyanobacteria. It uses light energy to catalyze a series of electron transfer reactions resulting in the splitting of water into molecular oxygen, protons and electrons. These reactions take place on an enormous scale, being responsible for the production of atmospheric oxygen and indirectly for almost all the biomass on the planet. Despite its importance, the catalytic properties of PSII have never been reproduced in any artificial system. Understanding its unique chemistry is not only important in its own right, but could have implications for the agricultural industry since PSII is a main site of damage during environmental stress. The aim of this article is to review our current knowledge of the three-dimensional structure of PSII in higher plants, an area of research which has developed rapidly over recent years. To aid this process we first outline the photochemical reactions that take place in this photosystem. We then summarize the main structural features of individual subunits, with particular focus on their likely transmembrane helical content, as well as on their cofactor and organizational characteristics. Electron microscopy of PSII will be reviewed in the following sections in order to relate the subunit and cofactor composition of PSII to its three-dimensional structure. Low resolution structural data on PSII, obtained from freeze-etch and freeze-fracture studies of thylakoid membranes will be reviewed initially. Such studies have provided information on the location, heterogeneity, as well as the overall size and shape of PSII and its antenna system in the thylakoid membrane at resolutions of 40-50Å. To obtain higher resolution information (~15-40Å), two other approaches have been used: single particle image averaging of detergent solubilised PSII complexes, and analysis of two-dimensional crystals. The former has yielded considerable information on the oligomeric state and subunit organization of PSII and its antenna systems while the latter offers the potential of an atomic resolution structure. Results obtained from both approaches are discussed in terms of the question of whether PSII exists as a monomer or dimer in vivo. Finally, the conclusions emerging from these studies are compared with biochemical and cross-linking data.
Electron Transport
A major role of many of the subunits of PSII, and its light harvesting antenna proteins, is to act as sophisticated and adaptable membrane embedded scaffolds which provide the ligands that bind and organize an excitonically linked network of pigments and other cofactors. Together, these cofactors trap, transfer and utilize solar energy, to drive the reactions of water splitting and plastoquinone (PQ) reduction. This section briefly outlines the light driven electron transfer events catalysed by PSII. The organization of the cofactors involved in these reactions will be elaborated upon in the next section.
Equation 1.1 summarises the overall reaction catalysed by PSII, while Fig. 1 illustrates the key photochemical processes involved.
2H2O + 2PQ |
------------> |
O2 + 2PQH2 |
The light energy used to drive the PSII reaction is captured by a large number of chlorophyll (Chl) a and b (averaging about 250 per reaction center) and carotenoid (b-carotene, lutein, neoxanthin and violoxanthin) molecules associated with light harvesting antenna proteins. Excitation energy is passed along an excitonically linked network of Chl molecules to P680 which is thought to consist of two associated Chl molecules, although the excitonic coupling is much weaker than in the "special pair" of the purple bacterial reaction center. The excited state, P680*, donates a single high energy unpaired electron to a molecule of pheophytin (Pheo), thereby forming the radical pair, P680+ Pheo- (Fig. 1). P680+ is the most oxidizing redox component of PSII and each time it is formed it accepts an electron from a specific amino acid residue (D1-Tyr161), and therefore is reduced to P680. Illumination of PSII allows the P680, P680*, P680+ cycle to be repeated and enables the sequential extraction of electrons from D1-Tyr161. As D1-Tyr161 donates an electron to P680+, it accepts another from water via a manganese (Mn) cluster. Joliot et al (112) and Kok et al (120) used short light flashes to induce a single turnover of P680 and showed that the Mn cluster passed through a series of oxidation states referred to as the S0-S4 cycle. Babcock (18) reviewed the evidence for the possibility that the S3-S4-S0 transition is the rate limiting step of this cycle. This was expanded by Plijter et al (198) who suggested that O2 release was the overall rate limiting step of the S-state cycle. After the extraction of four electrons from water by this process, an oxygen molecule is released. The Mn cluster therefore appears to act as a charge storage device during the water oxidation process.
The electrons, which are accepted by P680+, are passed down the electron transport chain. In this way, Pheo accepts electrons from P680 and passes them on to a plastoquinone molecule (QA), tightly associated with the reaction center. QA- passes its electron on to a second plastoquinone molecule, associated with the QB site of PSII. This electron transfer is aided by the presence of a non-heme iron located between QA and QB. Each plastoquinone associated with the QB site is able to accept two electrons derived from water and two protons from the stroma before being released into the lipid matrix in the form of reduced plastoquinone (PQH2).
THE SUBUNIT AND COFACTOR COMPOSITION OF PSII
In higher plants and green algae well over 20 subunits are associated with PSII in vivo and have been named after the genes encoding them (PsbA-PsbY, Lhcb1-Lhcb6). The location and organization of the genes that encode these proteins has been reviewed in detail (64, 84, 111). Here, the PSII subunits are listed in Table 1, in a way that reflects the PSII particle type that they are associated with; the smallest being the isolated reaction center (RC) and the largest, PSII with its complete antenna. The cofactors bound by each subunit are given, together with subunit function, common name and the number of a-helices that the subunit is predicted to contain. The structure, cofactor composition and functions of many of the PSII subunits, have now been studied in some detail and are also summarized below.
The PSII Reaction Center D1/D2 Proteins
At the heart of PSII is the photochemically active reaction center (RC), which consists of the D1 and D2 proteins (24, 173, 243). These proteins are highly conserved between higher plant species and both consist of 353 amino acids prior to processing (64, 211). The mature D1 and D2 subunits consist of N-acetyl-Thr2-Ala344 and N-acetyl-Thr2-Gly353, respectively, and are calculated to have molecular masses of 38 kDa and 39.4 kDa. In higher plants the N-terminal residues of both proteins are reported to be reversibly phosphorylated (150, 241, 177, 211). Both subunits are predicted to contain 5 membrane spanning a- helices (reviewed in 23, 150, 211) with their N-termini exposed to the stromal surface of the membrane (150). The D1 and D2 proteins also have approximately 15% sequence homology with the L and M subunits of the purple bacterial RCs of Rhodopseudomonas viridis and Rhodobacter sphaeroides (reviewed in 23, 148, 211). Although this level of homology is low, the conserved amino acids correspond largely to the binding sites of the photochemically active cofactors (23, 148, 211). Thus the structure of the purple bacterial reaction center has proved useful in the localization of cofactors associated with the D1 and D2 proteins. In PSII, these cofactors include a 4 atom Mn cluster, P680, Pheo, QA, QB, non-heme iron, accessory Chls and b-carotene (b-Car).
THE Mn CLUSTER AND D1-TYR161 The 4 atom Mn cluster (see Fig. 1) is located on the lumenal surface of the D1 and D2 proteins (for reviews see 39, 91, 208, 214). It has been proposed to consist of either a trimeric Mn cluster and an associated mononuclear Mn center (90, 74, 193) or of two distinct binuclear Mn centers (85) or a single tetranuclear cluster (for review see 39). Britt (39) recently reviewed the data obtained from X-ray absorption fine structure (EXAFS) spectroscopy, electron paramagnetic resonance (EPR) spectroscopy, X-ray absorption near edge structure (XANES) spectroscopy, UV and IR difference spectroscopy, electron-nuclear double resonance (ENDOR) and electron spin echo envelope modulation (ESEEM) studies. The combined data obtained by these approaches appear to be consistent with the conclusion that the 4 atom Mn cluster is composed of a pair of di-m-oxobridged dinuclear Mn units linked together via an oxo-bridge and a pair of amino acids providing carboxalato bridges (39). In the model of Britt (39), it is proposed that D1-His190 forms a ligand with the Mn cluster and D1-Tyr161 is thought to be positioned close to it. This conclusion is consistent with the following findings. Gilchrist et al (77) calculated D1-Tyr161 to be located about 4.5Å from the Mn cluster. Furthermore, D1-Tyr161 can act as a strong proton donor (192) and its re-reduction occurs on the same time scale as the release of oxygen from the Mn cluster during the S3-S4-S0 transition (18). Recently Tommos et al (249) and Hoganson et al (101) have also concluded that there must be a close association of D1-Tyr161 with the Mn cluster. These results are consistent with the H+ abstraction model (249) in which Tyr161 is placed close to, but not binding, the Mn cluster. A number of other D1 and D2 residues have been proposed as potential ligands for the Mn cluster based on mutagenesis experiments. These residues include the highly conserved D1-Asp170 (178, 34), D1-Ala344 (177) and D2-Glu69 (258). Histidine residues D1-His190, His332 and His377 are also potential ligands for the Mn cluster (42, 43, 57, 177, 179).
P680 AND ACCESSORY CHLOROPHYLLS The sequence homology between the L and M subunits of the purple bacterial reaction center with the D1 and D2 subunits of PSII, led to the suggestion that P680, might be a chlorophyll dimer like its purple bacterial counterpart (reviewed in 23, 148, 211, 212). Biophysical characterizations appear to support this view (180, 212) but indicate that its structure and functional properties differ considerably from the special pair of BChl in the purple bacterial reaction center (see 212). For example, P680+ has a far higher oxidizing potential (approx. 1V) enabling it to drive the water splitting reaction. Furthermore, the constituent Chl molecules of P680 are also more weakly excitonically coupled than the bacterial special pair suggesting that P680 triplet and P680+ states are localized on single Chls but not necessarily the same ones attributed to P680* (see 59, 256). ###Earlier it was thought that based on the above mentioned sequence homology studies and biophysical characterizations, one of the Chls of P680 was H-bonded via two keto groups to D1-Thr286, and either Ser291 or Thr292, in such a way that it is orientated at a 30o angle to the membrane plane (180, 212). However, now it has been suggested that there is no H-bonding with the two keto-groups, but may be an H-bond with the ester group of Chl1 on the D1 protein via Thr286. The Ser291 and Thr292 also do not H-bond to the cation state of P680 or the triplet state of P680 (pers. comm. Dr. Mary Sarcina) ### The Mg ion within its porphyrin ring may be liganded via D1-His198 (212). One of the keto groups of a second P680 Chl may be bound by D2-Ser283, while its Mg ion is proposed to interact with D2-His198. This cofactor binding model suggests that the second P680 Chl is held at an angle closer to the perpendicular of the membrane plane and would account for the weak excitonic coupling between the two P680 Chls. In its isolated form the PSII reaction center is associated with 6 Chl molecules (80, 119, 280). Based on spectroscopic studies, it has been suggested that 4 of the Chls and the 2 Pheo interact excitonically leading to the suggestion that P680 acts as a chlorin multimer rather than a dimer (59). The other two outer Chls may be located at the periphery of the complex and involved in the transfer of excitation energy from CP43 and CP47 to P680. D1-His118 and D2-His118, which are not found in corresponding positions in the L and M subunits, may function as ligands for these two "extra" accessory Chls (121).
PHEOPHYTIN The atomic map of the purple bacterial reaction center shows that together the L and M subunits bind two bacteriopheophytin molecules (see 10, 47, 48, 149) but only one of these is photochemically active (117, 261). Extrapolation from the structure of the purple bacterial RCs, led to the proposal that the RC of PSII might also bind two molecules of Pheo. Analysis of PSII RC preparations and D1-D2 complexes confirmed this hypothesis (173, 243). The structural similarity of the cofactor organization in the RCs of purple bacteria and PSII was emphasized further by the finding that one of these Pheo molecules was photo-reducible (173, 197) while the other was not (158). D1-Glu130 is proposed to bind the photochemically active Pheo via its 9-keto group (139, 162, 169). Indeed, such liganding is similar to that observed between L-Glu104 and bacteriopheophytin in the Rhodopseudomonas viridis reaction centers (149). Proof of the involvement of D1-Glu130 comes from recent mutational studies using the cyanobacterium Synechocystis 6803 where spectral shifts in pheophytin absorption were induced by modifying the equivalent residue (78). Svensson et al (235) suggest that D1-Tyr126 may correspond to the Rhodopseudomonas viridis L-Trp100, which also forms a hydrogen bond with bacteriopheophytin. Taking the analogy a step further, D1-Tyr147 and D1-Ala150 are proposed to correspond to L-Pro124 and L-Phe121 which are in contact with the bacteriopheophytin ring plane in purple bacteria (151, 235).
NON-HEME IRON BINDING SITE The histidines which form ligands to the non-heme iron in the purple bacterial reaction center (L-190 and 230 and M-217 and 264) seem to be conserved in the D1 and D2 proteins (D1-His215 and -His272, D2-His215 and -His269) (see 23, 148, 211, 236). The fifth ligand in the bacterial system (M-Glu232) is not so obvious in the D2 protein and may be replaced by a ligation to bicarbonate (see 56). Under phosphorylating conditions, the phosphate group on D1-Thr2, which is exposed to the stromal side of the membrane, may also interact with this non-heme iron, thereby exerting an effect on QA and QB electron transport (150). However, green algae, such as Chlamydomonas and cyanobacteria, do not have a phosphorylated form of the D1 protein and therefore the above suggestion can not be true generally.
QA BINDING SITE Models of the QA binding site based on the sequence homology between the purple bacterial M subunit and the D2 protein suggest that the following amino acids are QA ligands (corresponding M subunit amino acid of Rp. viridis are given in brackets); D2-Thr218 (M-Thr220), D2-Phe253 (M-Phe249), D2-Trp254 (M-Trp250) (24, 56, 148, 207, 250). These place QA close to the stromal surface of the membrane.
QB BINDING SITE Mutagenesis studies have suggested that D1-Tyr237 (not conserved between L subunit and D1), D1-Tyr254 (L-Tyr215), D1-Phe255 (L-Phe217) and D1-Gly256 (not conserved between L subunit and D1) are involved in binding QB (58, 99, 148, 203). Ser264 is conserved in both the D1 protein and the L subunit (L-Ser223) and was recently argued to be involved in QB protonation (131, 185). L-Glu212, also implicated in the protonation of QB in purple bacteria, is not conserved in the D1 protein (see 146, 224). The identification of the above amino acids places the QB binding site near the stroma surface of the membrane, a conclusion substantiated by the finding that thylakoids, subjected to a mild trypsin digestion, lost their QB binding activity without affecting electron transport between the oxygen evolution apparatus and QA (201, 262, 263).
LOCATION OF b-CAROTENE Little is known about the location of the two molecules of b-Car associated with the PSII reaction center (24, 173). As b-Car does not directly quench the P680 triplet state these components do not appear to be in van der Waals contact (246). b-Car is, however, involved in the quenching of singlet oxygen and so fulfills a protective role (246). By analogy with the orientation of two molecules of lutein in the subunits of light harvesting complex II (LHCII), it is likely that the b-Cars of the RC lie in hydrophobic grooves of the membrane spanning a-helices of D1 and D2.
Structure and Function of Antenna Subunits Lhcb1-6
PSII is closely associated with six chlorophyll a/b binding proteins which form the light harvesting antenna of PSII (Lhcb1-6) and are encoded by the nuclear cab genes. The structure, cofactor organization and function of these subunits have been reviewed in detail (81, 82, 83, 84, 103, 111, 191). Pigment composition values can be found in (111) and primary sources are referenced there. Of these six chlorophyll a/b binding proteins:
Lhcb1-3 are the components of LHCII and have apparent molecular masses of 25, 27 and 28 kDa. It is the function of the light harvesting outer antenna system to collect sunlight and deliver the derived excitation energy to PSII in a controlled manner. Sequence homology studies have shown that the proteins Lhcb1-6 all have three highly conserved transmembrane helices and bind varying amounts of Chl a, Chl b, b-Car and xanthophylls (83, 84, 111). Four highly conserved b-turn regions have also been detected (84). Lhcb1, 2, 3 and 4 also have a highly conserved sequence of six amino acids which may act as a phospholipid binding site, suggested to be involved in Lhcb protein trimerization (100, 191). The structure of a trimeric form of LHCII from pea has been determined to high-resolution by electron crystallography (123, 124). Based on the high degree of sequence homology between the 6 Lhcb proteins it is likely that their atomic structures will also be similar to that of the LHCII monomer . Within each Lhcb protein monomer of LHCII, the two longest transmembrane helices are surrounded by chlorophyll a and b molecules, and two molecules of lutein. The chlorophylls are arranged in two layers, towards the lumenal and stromal membrane surfaces and the main ligands are Glu, Asu, Glu, His and Gly. The close association of the luteins with the excitonically linked chlorophylls, prevents singlet oxygen formation and the damage induced by it (124). The luteins also play a structural role in that they form a cross-brace in the centre of the complex, providing a direct and strong link between the peptides loops at both surfaces (124).
Lhcb4-6 are often referred to as CP24, CP26 and CP29, respectively and have been shown to be more closely associated with PSII than Lhcb1-3 as they remain bound to the core complex under conditions that result in LHCII dissociation (195). Several pieces of evidence point to Lhcb4-6 being involved in the transfer of excitation energy from LHCII to the PSII reaction centre via CP43 and CP47. The first is that the LHCII components bind 50-65% of the chlorophyll while the Lhc4-6 proteins together only bind about 20% of this pigment (46, 82, 195). The larger number of chlorophylls associated with the LHCII subunits would suggest that it is these polypeptides which act as the predominant light harvesting components of the antenna system. Thus, although it is likely that the Lhcb4-6 proteins can harvest light energy as well, their major role is thought to be one of facilitating and controlling excitation energy transfer from LHCII to PSII. The finding that the absorption spectra of Lhcb4-6 are blue shifted with respect to those of the PSII core components is consistent with this hypothesis (26, 29, 30). Uncontrolled excitation energy transfer could, however result in an excessively high rate of P680 turnover and cause acceptor or donor side photoinhibition and subsequent D1 damage. It is therefore pertinent to note that the Lhcb proteins bind 78% of the total violoxanthin associated with the PSII antenna system. Strong illumination converts violoxanthin to zeaxanthin, which in turn is able to dissipate excess excitation energy (53). This protective mechanism is important in that it shows that the function of Lhcb proteins is not only to transfer excitation energy to the reaction center but also to control its rate of transfer to PSII.
Structure and Function of Cytochrome b559
Cytochrome b559 (cyt b559) is a heme protein closely associated with the reaction center. It consists of - (PsbE) and - (PsbF) subunits which, in their unprocessed forms, are 83 and 39 amino acids in length. Both are predicted to contain a single transmembrane a-helix and a stromally exposed N-terminus (237). Their N-terminal methionines are removed after translation (107, 108, 270). Recent mass spectrometric analyses have confirmed that the mature - and -subunits isolated from peas contain 82 and 38 amino acids and have molecular masses of 9284 and 4395 Da, respectively (218). The N-terminus of the a-subunit is unmodified but that of the b-subunit is acetylated (218). Both subunits are present in PSII in a ratio of 1:1 (273). The heme group has been shown to be liganded to the complex, via two histidine residues (19). Since the - and -subunits of cyt b559 each only contain a single histidine residue, in their respective membrane spanning domains, the heme must cross-link two polypeptides into a dimeric complex (45, 273). If only one cyt b559 dimer was present per reaction center, as has been reported by Kobayashi et al (119), Gounaris et al (80) and Barbato et al (21), then the cytochrome complex could only be an - and -heterodimer. However, several reports of a stoichiometry of two hemes per reaction center PSII core complex (51, 130, 168, 277) have been published. If two hemes are indeed present, then the existence of and cyt b559 homodimers has to be considered. Herrmann et al (97) showed that ion exchange chromatography was unable to resolve two distinct fractions corresponding to and , even though these two homodimers differ by 10 charge units, lending support to the ab heterodimer theory. Furthermore, the bi-cistronic nature of the mRNA of psbE and psbF also supports the heterodimer theory (reviewed in 64). Perhaps the most convincing evidence for the -cyt b559 heterodimer unit comes from the 14C labelling experiments of Alizadeh et al (9) which showed the presence of one copy of each subunit per reaction center. Site directed mutants of cyt--His22 and cyt--His22 as well as psbE and psbF deletion mutants of Synechocystis sp PCC 6803, all fail to accumulate the D1 and D2 proteins (187, 188). Similarly, deletion of psbE in Chlamydomonas led to a PSII-free mutant (163). This emphasizes the importance of the and subunits of cyt b559 and their His residues in particular, in maintaining the structural integrity of PSII. Truncation of the C-terminus of the a-subunit in Synechocystis PCC 6803 by 31 amino acids, also results in a 80-90% reduction in PSII accumulation but does not prevent cyt b559 assembly (238). Cytochrome is reported to exist in a high or low potential state. Many different functions have been postulated for this cytochrome and the most favored is its possible protective role against photoinduced damage of the reaction center. For example, Nedbal et al (174) and Barber and De Las Rivas (25) suggested that the low potential form of cyt b559 can accept electrons from Pheo, thus preventing the double reduction of QA and the consequential recombination of the P680+ Pheo- radical pair. As a result, cyt b559 may provide some protection against 3P680 and singlet oxygen formation. It has been suggested that the high potential form of cyt b559 is able to protect PSII against donor side photoinhibition by donating electrons to P680+. This would prevent the irreversible oxidation of carotenoids and Chls, as well as amino acids, closely associated with it (25, 247).
Structure and Cofactors of CP47 and CP43
The two largest PSII subunits, CP47 (PsbB) and CP43 (PsbC) are closely associated with the reaction center and form an antenna within the core complex. Their biochemical characterization was recently reviewed (35, 37) and only the essential points relating to primary and secondary structure and cofactor organization are presented here.
CHLOROPHYLL BINDING PROTEIN, CP47 The mature PsbB (CP47) protein of spinach is reported to consist of 508 amino acids, 72% of which are identical between spinach and tobacco. Most of the remaining residues are conservatively replaced (see 35). The calculated molecular mass of the mature protein of spinach is 56.278 kDa (164). Hydropathy plots suggested that CP47 contained either seven (164) or six (260) transmembrane helices. The latter theory is now favored as the additional helix seems to be both too short and too hydrophilic to span the membrane: it is now considered to be part of a 19 kDa lumenally exposed loop (loop E). The N- and C-termini are thought to be located on the stromal side of the membrane (264). One of the functions of CP47 is to transfer excitation energy from the Chl a/b binding proteins to the PSII reaction center, in the case of higher plants and green algae, and from phycobilisomes in the case of cyanobacteria and red algae (26, 29, 30, 205). CP47 binds a number of cofactors including Chl a, b-Car and Lut, but does not bind Pheo or Chl b (8, 28). CP47 has been estimated to contain 10-25 molecules of Chl a (8, 21, 55, 244). This discrepancy is extremely large and clearly requires clarification. Our own results indicate that CP47 binds approximately 15 Chl a molecules (281). Information on the pigment organization within CP47 has been obtained from mutagenesis studies (219) and by comparison with atomic maps of other pigment binding proteins (48, 124, 282). It has been noted that histidines act as ligands for bacteriochlorophyll in purple bacterial reaction centers (48, 282), while Kühlbrandt et al (124) identified glutamate, glutamine and glycine, as well as histidine, as Chl ligands in LHCII. Thus, any of the conserved amino acids of these types located in the putative transmembrane regions of CP47 may be potential Chl ligands. CP47 contains 14 conserved histidine residues of which 12 are located within the transmembrane helices and arranged in two layers near the stromal and lumenal surfaces of the membrane. As all of the Chl a molecules associated with LHCII are bound by amino acids in similar positions, these histidine residues are prime candidates for Chl ligands and many have been studied by site directed mutagenesis (219). In all cases, mutants with altered His residues showed a reduction in the light harvesting capability of their PSII complexes, with multiple mutations resulting in cumulative effects. This suggests that all of the histidine residues may provide ligands for the Mg of the bound Chl molecules. CD spectra showed that some of the Chls in CP47 are excitonically linked to one another (8), which is to be expected if CP47 transfers excitation energy from the outer antenna system to the PSII reaction center. Replacing His469 in helix VI with glutamine, resulted in a drastic reduction in PSII levels, suggesting that this ligand is structurally important (62). CP47 has a 77K fluorescence emission band peaking at 695 nm (122, 171, 189, 213, 255) which was greatly reduced in the His469 Glu mutant (62). The existence of a CP47 specific 695 nm fluorescence emission band is indicative of an excitonic trap within this subunit, which would be expected to be shallow enough to allow subsequent excitation energy transfer to the reaction center of PSII. The 695 nm fluorescence emission band probably originates from a monomeric Chl whose fluorescence dipoles are orientated perpendicular to the plane of the membrane (245, 255). It is interesting to speculate that a Chl anticipated to be associated with His 469, might be excitonically linked to the accessory Chls of the PSII reaction center. Loop E of CP47 has been subjected to site directed mutagenesis, crosslinking studies and immunolabelling (37). These experiments indicate that residues Lys304-Lys321, Pro360-Ser391 as well as Lys389-Lys419 are closely associated with the 33 kDa extrinsic subunit (61, 68, 79, 86). The crosslinking experiments of Odom and Bricker (181) suggest that the domain His364-Asp440 forms a salt bridge with the N-terminal domain of the 33 kDa subunit while site directed mutagenesis of Arg384-Arg385 causes a 50% loss in oxygen evolution (200). Mutating Arg448 to Gly448 or Ser448 in Synechocystis sp PCC 6803 prevents the organism from growing photoautotrophically under Cl- limiting conditions. The involvement of CP47 in excitation energy transfer and its interactions with the 33 kDa subunit of the OEC explains why deletion of the psbB gene inhibits both the assembly and stability of PSII (257, 259).
CHLOROPHYLL BINDING PROTEIN, CP43 The mature PsbC (CP43) protein of spinach is reported to consist of 473 amino acids, 67% of which are identical in spinach and tobacco with the remainder being conservatively replaced (35). The mature protein of spinach is calculated to have a molecular mass of 50.092 kDa (11, 150) and has an N-terminus, N-acetyl-Thr15, which in higher plants and green algae can be phosphorylated (150). Both its N- and C-termini are reported to be exposed to the stroma (41, 150, 215). Hydropathy plots and immunological studies indicate that CP43 contains six transmembrane a-helices (41, 215) and a large lumenally exposed loop (Loop E). This protein is reported to contain 12 conserved histidine residues of which 10 appear to be located in the transmembrane helices. Their positions close to the stromal and lumenal surfaces of the membrane are of striking similarity to those of CP47. This suggests that the histidines within the transmembrane helices may act as Chl ligands, but little mutagenesis data are available to confirm this (35). CP43 binds a number of cofactors including Chl a, -Car and Lut, but does not appear to bind Pheo or Chl b (8, 28). CP43 has been estimated to bind 9-25 molecules of Chl a (4, 8, 21, 55). As in the case of CP47 this discrepancy clearly requires clarification. Our own results indicate that CP43 is associated with approximately 15 Chl a molecules (88, 281), suggesting that ligands other than His residues are also required to bind them. Although biochemical studies indicate that CP43 is less tightly associated with the reaction center than CP47 (51, 73), it is structurally important since PSII reaction centers only accumulate at low levels in CP43-less mutants (40, 54, 204). Those PSII complexes that did accumulate were not functional in oxygen evolution but did drive electron transport between D1-Tyr161 and QA suggesting that CP43 is not involved in these electron transfer processes. One of the functions of CP43 is thought to be the transfer of excitation energy from the Chl a/b binding proteins to the PSII reaction center, in the case of higher plants and green algae, and from phycobilisomes in the case of cyanobacteria and red algae (26, 29, 30). This difference between the two secondary antenna systems led Bricker (35) to identify amino acids in CP43 which were conserved within but not between these classes of organisms. From these analyses residues Ile135, Ser143, Val150 and Lys156 in higher plants, which are clustered in the stromally located loop B, were suggested to be potential docking sites for Lhcb proteins (35). Mutagenesis studies suggest that CP43 may be partially involved in providing an environment for the Mn cluster. Indeed, mutations in the lumenally exposed loops, especially the loop connecting helices D and E of the protein (loop E), can result in loss of oxygen evolution or altered S-state cycling properties (40, 126).
The 33 kDa, 23 kDa and 16 kDa Extrinsic Proteins |
In higher plants and green algae the products of the psbO (33 kDa subunit), psbP (23 kDa subunit) and psbQ (16 kDa subunits), together form the lumenally exposed oxygen evolving complex (OEC), which is closely associated with the Mn cluster of PSII (14, 76, 166). The 33 kDa subunit fulfills both an important structural and regulatory role in the optimization of the oxygen evolution reaction. The 23 kDa subunit allows PSII to evolve oxygen under both Ca2+ (75, 166) and Cl- limiting conditions (160, 161). This has led to the suggestion that the 23 kDa subunit acts as a concentrator of these ions (166). The 16 kDa polypeptide aids PSII to evolve oxygen efficiently under severely Cl- limiting (<3mM) conditions (3). The following sections will review the essential points relating to primary and secondary structure of these subunits and their involvement in cofactor organization. It should be noted that, as yet, no high resolution structures are available for these three proteins despite their hydrophilic nature. 33 kDa EXTRINSIC PROTEIN The psbO gene of higher plants and cyanobacteria code for polypeptides ranging in size from 292-331 amino acids. They are processed to yield mature subunits consisting of 241-247 residues (64). Between higher plant species, the mature PsbO protein is highly conserved (eg. 87% sequence homology between spinach and pea with most of the remaining amino acids being conservatively changed). Although the mature PsbO protein is often referred to as the 33 kDa extrinsic polypeptide, its calculated molecular mass is about 26.5 kDa (177). In spinach the N-terminus of the mature protein is Glu85 (254, 276). Protease digestion and crosslinking studies suggest that the N-terminal region binds to loop E of CP47 (60, 181). Region Leu101-Gly131 also appears to play a structural role within the intrinsic part of the PSII complex (182, 254, 265). Initial amino acid sequence homology studies suggested it to be a Mn binding site because of its sequence similarity with the bacterial Mn superoxide dismutases (182). However, later analysis indicated that this region had greater similarity to the Ca2+ binding site of mammalian Ca2+ binding proteins and that it formed an E-F hand (265). Indeed, the theory that region Leu101-Gly131 binds Ca2+ may be related to the fact that PSII is calculated to bind tightly, one molecule of Ca2+ (1). However, this Ca2+ is closely associated with the Mn cluster (see 39, 166) and EXAFS measurements, which provide information about the environment of metal atoms in proteins, have shown that the organization of the Mn cluster is largely unaffected by the removal of the 33 kDa extrinsic subunit (44). This suggests that the PsbO subunit and its proposed bound Ca2+ optimizes the function of the Mn cluster rather than being directly involved in water splitting. This conclusion is also supported by the finding that deletion of the psbO gene in Synechocystis 6803 does not prevent photoautotrophic growth of this organism (143, 196). Three regions within the mature PsbO protein, Lys185-Asp193, Arg262-Lys274 and Asp308-Glu322 are enriched in both acidic and basic amino acids (254), but their functional importance is not known. The PsbO subunit contains two cysteine residues (Cys112 and 135 in spinach) which are reported to form a disulfide bridge, which is essential to the function of the 33 kDa extrinsic polypeptide (242). Circular dichroism spectroscopy and Fourier transform infrared spectroscopy measurements suggest that this protein consists of about 38% b-sheet and 9% a-helices (92, 275). 23 kDa EXTRINSIC PROTEIN The spinach psbP gene is reported to code for a transit polypeptide (81 residues) and a mature protein consisting of 186 amino acids (110). It has 83% sequence identity with its pea counterpart, with most of the other amino acids being conservatively replaced (266). The molecular mass of the spinach PsbP subunit is calculated to be 20.209 kDa. About 25% of its residues are charged and the total polar amino acid content is 63%, explaining its hydrophilic nature (110, 266). It has been proposed that the charged residues are involved in either Ca2+ and Cl- sequesting or in binding the subunit to the lumenal surface of the PSII core. As yet there is no firm evidence for either a Ca2+ or Cl- binding site within the PsbP subunit. Based on the assumption that the PsbP subunit has a globular shape, it has been suggested to protrude about 4 nm into the thylakoid lumen (110). 16 kDa EXTRINSIC PROTEIN The spinach psbQ gene is reported to code for a transit polypeptide (83 residues) and a mature protein consisting of 149 amino acids (110). The PsbQ subunit is enriched in Arg, Lys and Pro residues. Although there is only 28% sequence homology between the PsbQ of spinach and Chlamydomonas, much of the homology is concentrated in regions containing these residues. One particular feature of structural interest is a four Pro repeat (Pro9-12 of mature protein). It is thought that this region is quite rigid and preceded by a region of b-sheet, followed by a b-turn (129). The cleavage of the residues 1-12 from the PsbQ protein, prevent this extrinsic subunit from rebinding to the lumenal surface of PsbQ-depleted PSII cores (128). Other potential regions of b-sheet include Asp77-84 and Leu85-90 (110). |
Low Molecular Weight Subunits |
Except for subunit PsbS, all other subunits associated with PSII have a mass under 10 kDa. The exact number of the small subunits associated with PSII in vivo is not known, primarily because some of these proteins stain poorly and/or are difficult to resolve even in high-resolution gels (138). The reader is referred to the detailed review in (64) for further information on the low molecular weight subunits of PSII, as only the structural aspects are discussed here. PsbH PROTEIN The chloroplast psbH gene codes for a primary translation product 73 amino acids in length, with a calculated molecular mass of 7.697 kDa in spinach (see 64). The mature PsbH subunits of higher plants are reported to consist of 72 amino acids beginning at Ala2 and to show 89% sequence homology between them (64, 65). Hydropathy plots suggest that the PsbH subunit has a single membrane spanning region close to its C-terminus (64, 272). The N-terminal region of the protein is reported to be stromally exposed and can be phosphorylated at Thr2 in higher plants (125, 147). The phosphorylation of this amino acid is reported to result in the stabilization of QA- when the plastoquinone pool is reduced (184). Furthermore, psbH deletion mutants of Synechocystis 6803 also show modified QA to QB transfer and are highly susceptible to photoinhibition (144). PsbI PROTEIN The PsbI protein of higher plants, has an apparent molecular mass of 4.8 kDa and is present in isolated PSII reaction center preparations in near stoichiometric amounts (105, 127, 270). N-terminal sequencing revealed the organization of the first 20 amino acids (except residue 3) of the purified spinach PsbI subunit (105, 270). This sequence was used to locate the psbI gene, which in higher plants codes for 36 amino acids. Indeed, the predicted PsbI amino acid sequences of higher plants have 97% homology (64, 211). The N-terminal residue of the mature subunit is formyl-Met1, indicating that PsbI is not N-terminally processed (105, 218). Furthermore, the PsbI subunit of pea has a reported mass of 4.210 kDa (4195 kDa in spinach), as determined by mass spectrometry, confirming that the C-terminus of the protein is not processed either (218). Hydropathy plots have located a potential membrane spanning a-helix (residues 6-27) close to the N-terminus of the protein (105, 218). A ProGlyArg repeat sequence is located in the hydrophilic C-terminal region of the protein, which suggests it is exposed to the lumen. A PsbI-less mutant of Chlamydomonas reinhardtii grows photoautotrophically but is very sensitive to photoinhibition (127). Sharma et al (218) found that, of the PSII subunits of the isolated reaction center, only PsbI contains formyl-Met1 and suggested that it could act as a Chl ligand in a manner similar to formyl-Met1 of the a-subunit of the light harvesting complex of photosynthetic bacteria (145). However, PsbI does not appear to be essential for the primary photochemical reactions to occur, as shown by the work of Tang et al (243) and the fact that the psbI-less mutant of Chlamydomonas assembles PSII and evolves oxygen. PsbJ PROTEIN The open reading frame (ORF) following the psbEFL gene cluster in rice (98) and other higher plants, has been designated as ORF40 or psbJ (see 64). The psbJ product is predicted to consist of 40 amino acids and is highly conserved between higher plants and cyanobacteria (see 64). The last 30 amino acids are thought to be predominantly hydrophobic and probably span the thylakoid membrane (see 64). N-terminal sequence analyses of low molecular mass proteins associated with PSII complexes have failed to detect a subunit with the predicted amino acid sequence of PsbJ. This suggests that PsbJ is either not part of the PSII complex or that it readily dissociates from it (134). However, a mutant of Synechocystis containing an early termination sequence within psbJ, was only able to accumulate 50% of PSII complexes as compared with the wild type. The accumulated PSII complexes appear to function normally suggesting that PsbJ is involved in the assembly of PSII rather than in the primary photochemistry (134). PsbK PROTEIN It is reported that the psbK gene of spinach codes for a presequence of 61 amino acids which is cleaved to expose Lys25 at the N-terminus of the mature protein (64, 107, 167). The PsbK subunits of higher plants are predicted to be highly conserved with about 95% sequence homology between them. No information is available on the processing of the C-terminal end of this subunit and so it is currently assumed to consist of 37 amino acids and to have a molecular mass calculated to be 4.285 kDa (167). Hydropathy plots suggest that the spinach PsbK protein has a single membrane spanning segment towards the center of the protein. The function of the higher plant PsbK subunit is not yet understood and the characterization of the psbK-less mutants of Synechocystis (104, 279) and Chlamydomonas (240) give differing results. psbK-less mutants of Synechocystis sp. are reported to grow photoautotrophically and to show no enhanced susceptibility to photoinhibition. This led to the proposal that PsbK plays a role outside the normal functioning of PSII. In contrast, psbK-less mutants of Chlamydomonas were unable to grow photoautotrophically and were reported to accumulate only 10% of wild type PSII levels. These results suggest that in Chlamydomonas PsbK plays an important role in the assembly or stability of the PSII complex (240). Due to the species related differences in the effect of deleting psbK, it is difficult to predict the role of this 4.3 kDa subunit in higher plants. PsbL PROTEIN This protein was originally identified in oxygen-evolving core complexes isolated from spinach and wheat (108) and shown to have an apparent molecular mass of 5 kDa. N-terminal sequence data was used to identify ORF38 of the chloroplast genome as psbL (64). The mature polypeptide is 37 amino acids in length after the N-terminal Met residue is cleaved off to expose Thr2 (108) and is predicted to have a molecular weight of 4.366 kDa in tobacco and spinach (108, 268). C-terminal amino acids (Ser 17 - Phe37) are predicted to form a hydrophobic membrane spanning region. PsbL may be required for normal functioning of the QA site, since QA activity decreases dramatically when PSII core complexes are depleted of this polypeptide (118, 170). PsbM PROTEIN Ikeuchi et al (107) identified two polypeptides associated with oxygen evolving cores of Synechococcus vulcanus, which due to their apparent molecular masses were referred to a 4.7 kDa-I and 4.7 kDa-II. The N-terminal sequence of 4.7 kDa-I had significant similarity to the predicted products of chloroplast ORF34 of tobacco, rice and liverwort (107). Based on this, ORF34 was denoted psbM (64, 107). The 4.7 kDa-II has been designated PsbN (see below). The psbM genes of liverwort and tobacco are predicted to code for polypeptides having 85% sequence homology, with many of the remaining amino acids being conservatively replaced. The PsbM presequence of tobacco consists of 34 amino acids. Due to the lack of N-terminal sequence data, it is not known whether the presequence of higher plant PsbM is N-terminally processed. Hydropathy plots indicate that PsbM contains one hydrophobic membrane spanning region (Val3-Val27 in tobacco) (107). The function and location of this polypeptide within PSII is not known. PsbN PROTEIN The N-terminal sequence of the 4.7 kDa-II subunit showed it to have significant similarity to the predicted products of chloroplast ORF43 of tobacco, rice and liverwort (107). Based on this, ORF43 was denoted psbN (64, 107). The psbN genes of liverwort and tobacco are predicted to code for polypeptides having 86% sequence homology, with many of the remaining subunits being conservatively replaced. PsbN of tobacco is predicted to consist of 43 amino acids and to have a molecular mass of 4.722 kDa (107). Due to the lack of N-terminal sequence data on the mature PsbN subunit, it is not known whether the PsbN presequence undergoes N-terminal processing. Hydropathy plots suggest that the PsbN subunit contains a single membrane spanning region (Ile6-Gly28). The product of the psbN gene has not yet been detected in higher plants, but its deletion in Synechocystis 6803 does not appear to prevent the assembly of PSII or photoautotrophic growth (144). PsbR PROTEIN Ljungberg et al (135) resolved a 10 kDa polypeptide which copurified with PSII complexes of spinach. About 90% of this 10 kDa subunit dissociated from in-side-out PSII enriched membranes (i.e. with lumenal surface facing outwards) when they were washed with 1M NaCl/ 0.06% Triton X-100. The requirement for very low concentrations of detergent to release this subunit made it difficult to establish whether it was membrane embedded or associated with the surface of the thylakoid membrane via hydrophobic interactions. Lautner et al (132) used antibodies raised against the 10 kDa subunit to screen a cDNA library prepared from the cytosolic Poly A+ RNA of spinach. Positive clones were used to obtain the gene and hence the predicted pre-sequence of the 10 kDa subunit. The gene, which was found to be nuclear encoded, was denoted psbR (87). The PsbR presequence is predicted to consist of a N-terminal transit peptide of 41 amino acids in length and a 10.228 kDa mature protein in spinach (Ser42-Gln140) consisting of 99 residues. This prediction is consistent with N-terminal sequence data of the mature PsbR subunit (108, 132). The 4.1 kDa N-terminal transit peptide appears to target the nuclear encoded gene product through the chloroplast envelope into the chloroplast stroma. However, hydropathy plots show that the region Leu113-Tyr133 of the mature protein is hydrophobic suggesting that PsbR could be membrane embedded (132). Webber et al (269) addressed this point and showed that although this hydrophobic region was located towards the C-terminus of the protein, it actually had significant sequence homology with N-terminal transit peptides of soluble proteins of the lumen (eg. plastocyanin, PsbO, PsbP and PsbQ). For example, the Ala-Leu-Ala sequence (Ala137-Leu138-Ala139 in spinach) is thought to be part of a recognition sequence for a lumenal protease inducing the cleavage at Gln140 to form the mature spinach protein (Gln138 in wheat) (269). This finding indicates that PsbR is an extrinsic protein tightly associated with the lumenal surface of the PSII complex probably via hydrophobic interactions. Early experiments suggested that PsbR might provide a binding site for the extrinsic PsbP subunit (135). However, PSII complexes of transgenic potato plants expressing only 1-3% of wild type PsbR, bound the normal complement of the 23 kDa subunit (234). These PsbR-less PSII complexes did, however, show alteration in S-state cycling at high pHs (7.5-8.0). Furthermore, they exhibited reduced rates of QA to QB electron transfer. As PsbR affects both donor and acceptor side electron transfer processes it may be involved in maintaining optimal subunit organization. PsbS PROTEIN PSII enriched membranes of spinach are associated with a polypeptide having an apparent molecular mass of 22 kDa (135, 136). This protein is reported to remain associated with 1M NaCl washed PSII membranes, but released on detergent treatment, indicating its hydrophobic nature (135). N-terminal sequencing experiments failed, suggesting that the 22 kDa subunit is N-terminally blocked (271). To determine the primary sequence of the 22 kDa subunit, antibodies raised against it were used to screen a cDNA library of spinach. The gene which was found to be nuclear encoded (271), was denoted psbS (71). The PsbS presequence (29.2 kDa) is predicted to consist of 274 amino acids. Residues 1-69 are thought to form an N-terminal transit peptide which targets the mature protein into the thylakoid membrane (271). In the absence of information on N- or C-terminal processing of the mature subunit, it is assumed to consist of 205 amino acids (residues 70-274) having a molecular mass of 21.860 kDa in spinach. The hydropathy plot of the mature PsbS subunit predicts it to contain 4 transmembrane helices (271) and topological studies show them to be arranged in such a way that the C- and N-termini are stromally exposed (115). Alignment of the amino acid sequences of helices 1 and 2 with helices 3 and 4, shows that helices 1 and 3 and 2 and 4 are homologous. This result suggests that the four helices are the product of an internal gene duplication and this is corroborated by direct analysis of the psbS gene sequence (271). Sequence homology studies between PsbS, Lhcb1-6 and early light inducible proteins (ELIPs) show that in all cases helices 1-3 are highly conserved between them (271). This finding led to the suggestion that PsbS is a Chl binding protein although it does not appear to contain, His, Asp or Met residues which are thought to act as Chl ligands. However, recent studies do indeed indicate that the purified PsbS subunit binds approximately 5 molecules of Chl and several different kinds of carotenoids and that some of these pigments are excitonically linked (72). The functional role of PsbS, also referred to as CP22 in light of the above, has been discussed in some detail. Early religation studies suggested that PsbS might be important in the binding of PsbP (135). However core complexes containing PsbP, but lacking the PsbS subunit, have since been reported (159). The finding that the PsbS subunit binds pigments has led to the suggestion that it is part of the PSII antenna system. This hypothesis is supported by the report that PsbS is associated with PSII in a ratio of 2:1 (72). An alternative proposal of PsbS function is that it acts as a pigment chaperonin, which aids the incorporation of Chl molecules into the pigment binding proteins associated with PSII (70). This theory is consistent with the finding that PsbS is detected in developing chloroplasts (4 h after illumination of etioplasts) at a time point at which no other Chl binding proteins of PSII have accumulated. Furthermore, PsbS is reported to be stable in the absence of Chl and its association with this pigment, as it is synthesised, could prevent damaging oxidative reactions (70,72). PsbT PROTEIN Using SDS-PAGE, Ljungberg et al (137) identified a hydrophilic 5 kDa protein which copurified with the 33 kDa (PsbO) extrinsic protein. Subsequently this protein has been designated PsbT. The psbT gene of cotton has recently been sequenced and its N-terminal end found to have strong homology with the N-terminal sequences of the mature wheat and spinach PsbT proteins (108, 113). It is proposed to code for a 11 kDa presequence of 105 amino acids, which is composed of three segments; a 3.57 kDa N-terminal transit peptide (Met1-Asn32) coding for the transfer of the nuclear gene product into the chloroplast stroma; a (4.43 kDa) transit peptide (Ala33-Ala77) containing a thylakoid transfer domain responsible for the transfer of PsbT to the lumen; the (3.279 kDa in spinach) mature PsbT subunit component consisting of 28 amino acids (Glu78-Asn105) (113). This information supports the earlier results of Ljungberg (137) which suggested the PsbT is an extrinsic protein and confirms its lumenal location. Comparisons of the N-terminal sequences of the mature PsbT subunits of spinach and wheat show that the first 6 amino acids have a low degree of homology but that all the substitutions are conserved and the number of charged residues is the same. The PsbT subunit of cotton is reported to contain 2 cysteine residues (Cys94 and Cys103) which may therefore form a disulfide bridge (113). Particle induced X-ray emission studies indicate that the isolated PsbT subunit of spinach does not contain any metal ions (137) and its function is not known. PsbU PROTEIN A cyanobacterial PSII protein reported to be extrinsically located in the inner thylakoid surface and associated with the OEC and having an apparent molecular mass of 10 kDa has been denoted PsbU (186). However, there is no entry under PsbU in the SWISSPROT data base as yet. PsbV PROTEIN Isolated PSII core complexes of Synechococcus vulcanus are associated with an extrinsic polypeptide having an apparent molecular mass of 16 kDa (222). Although its apparent molecular mass and extrinsic location are similar to the properties of higher plant PsbQ, N-terminal sequencing showed it to have a high degree of sequence homology with cyt 550 of Microcystis and Aphanizonem (222, 223). The gene encoding the 16 kDa cyt 550 of Synechocystis sp PCC 6803 was identified using the N-terminal sequence of the mature protein and is now referred to as psbV (220). The predicted presequence of PsbV is thought to consist of 160 amino acids of which Met1-Ala25 form a N-terminal leader sequence. Due to the presence of the Arg23-Asn24-Arg25 motif, the leader sequence is cleaved to expose Val26 as the N-terminus of the mature protein (Vall26-Phe150) of Synechocystis PCC 6803. Similarities between the transit peptides of PsbO and PsbV suggest that PsbV is exported into the thylakoid lumen where it may bind to the OEC of PSII in cyanobacteria (220). PsbV does not appear to be associated with higher plant PSII. Closer analysis of the mature PsbV subunit shows that it contains two Cys residues (Cys52 and Cys55) which may form a disulfide bond (220). psbV deletion mutants are able to grow photoautotrophically although they contain only about 60% of wild type PSII levels and show a 40% reduction in wild type oxygen evolution rates (220). Furthermore, psbV-/psbO- deletion mutants are unable to grow photoautotrophically, contain less than 20% of wild type PSII levels, evolve less than 10% of wild type oxygen levels and show increased sensitivity to photoinhibition (221). These results show that PsbV plays an important role in PSII function and assembly and that it alleviates effects associated with the loss of the PsbO subunit. PsbW PROTEIN PSII complexes of higher plants (spinach and wheat) are reported to contain a polypeptide having an apparent molecular mass of 6.1-6.5 kDa (108, 216). Antibodies raised against an N-terminal fragment of this protein were used to screen a spinach cDNA expression library and positive clones used to elucidate the sequence of the gene encoding the 6.1 kDa protein (138). The data obtained was used to show that the 6.1 kDa protein is encoded in the nucleus. It was denoted PsbW according to the nomenclature of Hallick (87). The predicted presequence of PsbW (Met1-Lys137) suggests an N-terminal bipartite transit peptide (Met1-Ala83) which targets the mature PsbW to the thylakoid membrane (138). N-terminal sequence analyses of spinach PsbW indicates that Lys84 forms the N-terminus of the mature protein and that spinach PsbW has a high degree of homology with that of wheat (108, 216). In the absence of C-terminal processing, the mature PsbW is predicted to consist of 54 amino acids (Lys84-Lys137) and to have a molecular mass of 5.925 kDa. From hydropathy plots and thermolysin digestion experiments it is concluded that PsbW contains a single membrane spanning region (Lys104-Y123) and that its N-terminus is exposed to the thylakoid lumen (138). Although PsbW appears to be tightly associated with PSII cores its stoichiometric association with isolated PSII reaction centers remains to be confirmed. The function of PsbW is now known. PsbX PROTEIN Oxygen evolving PSII cores of wheat, spinach and the cyanobacterium Synechococcus valcanus are associated with a polypeptide having an apparent molecular mass of 4.1 kDa (107, 108). The N-terminal sequences of these subunits were determined (107, 108) and used to locate the corresponding gene in Arabidopsis thaliana (116). This gene which is denoted psbX appears to be nuclear encoded in A. thaliana. The PsbX presequence is predicted to consist of 116 amino acids. Analysis of this sequence and N-terminal sequencing of the mature PsbX protein indicate that residues Met1-Ala74 form a bipartite transit peptide and Ala75-Tyr116 form the PsbX subunit. The transit peptide is very similar in structure to a family of nuclear proteins imported into the thylakoid lumen. The region Met1-about Met50 has the characteristics of a bipartite chloroplast envelope transit signal in that it is basic, hydrophilic and enriched in hydroxylated residues (116). Residues Met50-Ala74 have similarities to the thylakoid transfer signal and the Ala-x-Ala motif (Ala72-Glu73-Ala74) in A. thaliana. Together these findings indicate that PsbX is a lumenal protein. However, the hydropathy plot of the protein strongly suggest that Phe86-Val107 form a hydrophobic membrane spanning region (116). Further experiments appear to be required to determine whether PsbX is embedded in the thylakoid membrane, or lumenally exposed. Recent studies on the low molecular weight subunits have suggested that PsbX may be involved in the normal QA functioning in higher plants (170). |
|
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The known protein subunits of PSII - adapted from Barber et al., (1997) Physiol. Plantarum. 100:817-827
At present there are 25 genes which have been identified as encoding proteins for the PSII core and are referred to as psb (photosystem b) genes. In higher plants and algae, most of these genes are located in the chloroplast genome, but some are nuclear encoded. There are undoubtedly more to be discovered. In some cases these components are restricted to a particular class of organism. In addition there are the genes that encode the proteins of the outer antenna systems; cab genes in higher plants and green algae give rise to a series of chlorophyll a/chlorophyll b binding proteins (Lhcb1-6) (Bassi et al. 1997, Green et al. 1991, Jansson 1994) while the apc and cpc genes encode the protein of the phycobilisomes of cyanobacteria and red algae (Glazer 1994).
Table 1 summarises the properties of the psb genes and their origins.
Protein |
Subunit |
Mass (kDa) |
No. of transmembrane -helices |
PsbA (c) PsbB (c) PsbC (c) PsbD (c) PsbE (c) PsbF (c) PsbG PsbH (c) PsbI (c) PsbJ (c) PsbK (c) PsbL (c) PsbM (c) PsbN (c) PsbO (n) PsbP (n) PsbQ (n) PsbR (n) PsbS (n) PsbT (c) PsbT (n)* PsbU** PsbV** PsbW (n)* PsbX (n) PsbZ (n) |
Dl CP47 CP43 D2 -Cyt b559 -Cyt b559 open H protein I protein J protein K protein L protein M protein N protein 33 kDa protein 23 kDa protein 16 kDa protein 10 kDa protein Lhc-like protein ycf8 protein 5 kDa protein U protein Cyt c550 W protein X protein Z protein |
38.021 (S) 56.278 (S) 50.066 (S) 39.418 (8) 9.255 (S) 4.409 (S) 7.697 (S) 4.195 (S) 4.116 (P) 4.283 (S) 4.366 (S) 3.755 (P) 4.722 (T) 26.539 (S) 20.210 (S) 16.523 (S) 10.236 (S) 21.705 (S) 3.849 (S) 3.283 (S) ~10 (Sy) 15.121 (Sy) 5.928 (S) 4.225 (S) |
5 6 6 5 1 1 1 1 1 1 1 1 1 0 0 0 0 4 1 0 0 0 1 1 |
Table. 1 Proteins that constitute the core of PSII. These proteins are products of the psbA to psbX genes which occur in all types of oxygenic organisms except for those found exclusively in higher plants and algae (*) or cyanobacteria (**). In eukaryotic organisms the psb genes are located in either the chloroplast (c) or the nuclear (n) genomes. The molecular masses of the mature PsbA to PsbX proteins, except PsbU, are calculated from the protein sequences reported in the SWISSPROT database using the MacBioSpec (Sciex Corp., Thornhill, Ontario, Canada) for spinach (S), pea (P), tobacco (T) and Synechococcus sp. (Sy). The number of predicted transmembrane helices is based on hydropathy analyses of primary sequence.
Below we briefly discuss each protein in terms of its function and predicted secondary structure. No attempt has been made to discuss the molecular biological features of the genes involved or details associated with their targeting to the thylakoid membrane. The reader should consult the reviews by Pakrasi and Vermaas (1992) and Pakrasi (1995) for these aspects.
After N- and C-terminal modifications, this highly conserved reaction centre protein is predicted to have a molecular mass of about 38 kDa depending on species. From hydropathy plots and comparison with the L subunit of the reaction centre of purple bacteria it is assumed to contain five transmembrane helices (I to V) and two surface helices between III and IV (lumenal) and IV and V (stromal). In higher plants, but not in algae and cyanobacteria, the N-terminal threonine may be reversibly phosphorylated (Michel et al. 1988). The D1 protein is characterised by two important features:
(1) It binds the majority of the cofactors involved in PSII mediated electron transport; Tyr161 (YZ), P680 probably via His198, Phe probably via Tyr126, Tyr147, Ala150 and Glu130, QB via interactions with Tyr254, Phe255, Gly256 and others, Mn cluster possibly via Asp170, Glu189, Gln165, Ala344, His109, His332 and His377 and non-haem iron, probably via His215 and His272 (see Debus 1992, Michel and Deisenhofer 1988).
(2) It turns over more rapidly than any other protein in the thylakoid membrane (Mattoo et al. 1984). This remarkable feature is linked to the fact that PSII is susceptible to photoinduced damage (Barber and Anderson 1992). This damage can lead to photoinhibition and reduction in photosynthetic efficiency. The degradation, synthesis and reinsertion of the D1 protein into the complex represent a very important aspect of the dynamics of PSII and have been extensively studied (Andersson and Aro 1997, Aro et al. 1993). This unique property will almost certainly place special conditions on the structural organisation of PSII.
In its mature form this highly conserved PSII core protein consists of about 5% amino acids and has a molecular mass of approximately 56 kDa, dependent on species. It is often known as CP47 and predicted to have six transmembrane helices (I to V) with the N- and C-termini exposed at the stromal surface (Bricker 1990). The lumenal loop joining putative transmembrane helices V and VI is large, containing about 200 amino acids. The protein binds about 15 chlorophyll a and 3 b-carotenes. It contains 14 conserved histidines of which 12 are located within the predicted membrane spanning regions and are prime candidates for chlorophyll ligands (Bricker 1990, Shen et al. 1993). The pigments form a core light harvesting system for the reaction centre but the large lumenal loop may function directly or indirectly in the water oxidation reaction. Deletion of the psbB gene and a wide range of site-directed mutational studies have emphasised the importance of this protein in PSII assembly and function. The evidence to date indicates that the PsbB protein is an absolute requirement for photoautotrophic growth (Vermaas et al. 1986, 1988).
After post-translation processing, the PsbC or CP43 protein, depending on species, contains about 470 amino acids and has a molecular mass of approximately 50 kDa. It is in many ways homologous with PsbB (CP47) in that it is likely to have 6 transmembrane helices, containing a considerable number of conserved histidine residues, binds about the same level of chlorophyll and carotenoids and has a large lumenal loop (composed of about 150 amino acids) between helices V and VI (Bricker 1990). It differs from CP47 in two main respects.
(1) Its N-terminal, threonine, can be irreversibly phosphorylated in the case of higher plants (Michel et al. 1988) (not so for algae or cyanobacteria).
(2) It is more weakly associated with the PSII reaction centre and can be removed from the isolated core to yield a CP47-RC complex (Dekker et al. 1990, Ghanotakis et al. 1989). This feature may also apply in vivo when the D1 protein is degraded and replaced during the photoinhibitory repair cycle (see below). Despite these differences, CP43, like its counterpart CP47, acts as an antenna to the PSII core and its presence also seems to be necessary to maintain water splitting activity. Again, deletion of the psbC gene and its modification can have a serious impact on both PSII assembly and the water oxidation function. However, its impact is less severe than that encountered with the PsbB protein. For example, deletion of the psbC gene in Synechocystis 6803 did not completely stop the partial assembly of PSII although it did inhibit oxygen evolution and photoautotrophic growth (Carpenter et al. 1998, Rogner et al. 1991).
The PsbD (D2) protein is homologous to the D1 protein. Although it has a slightly higher molecular mass of about 39.5 kDa it almost certainly consists of five transmembrane helices and has surface helices analogous to those predicted for the D1 protein (Michel and Deisenhofer 1988). In higher plants the N-terminal threonine can undergo reversible phosphorylation (Michel et al. 1988). Compared with the D1 protein it is involved to a lesser extent in binding active cofactors although it does contain inactive cofactors. The second ligand for P680 is likely to be D2-His198 while the binding of QA is believed to involve at least Thr218, Phe253 and Trp254 based on analogies with QA binding in the M subunit of purple bacteria (Michel and Deisenhofer 1988). D2-His215 and His269 are proposed to form ligands for the non-haem iron while D2-Glu69 has been implicated as a Mn ligand (Vermaas et al. 1993). Normally the D2 protein is not rapidly turning over, but under exceptional conditions of photoinhibition it does (Schuster et al. 1988).
The PsbE and PsbF proteins are the a- and b- subunits of cytochrome b559 (Cyt b559), respectively. The two proteins are closely associated with the D1 and D2 proteins and probably form a heterodimer so as to bind a haem via the single histidine residue contained in their sequences (Babcock et al. 1985). After processing, the PsbE and PsbF proteins contain about 82 and 38 amino acids in most higher plants and have molecular masses in the region of 9.3 and 4.4 kDa, respectively (Sharma et al. 1997). Hydropathy plots suggest that each forms one single transmembrane helix and there is probably one heterodimer per reaction centre (Alizadeh et al. 1995). There have been many speculations about the function of Cyt b559, but the most favoured at present is that it plays a protective role by acting as an electron acceptor or electron donor under conditions when electron flow through PSII is not optimised. Under these conditions potentially harmful reactions can occur either by singlet oxygen production involving the P680 triplet (formed by recombination of P68O+Phe- when QA is doubly reduced) or by secondary oxidations due to increased lifetime of P680+ (occurring when electron donation from water is insufficient) (Barber 1995, Barber and Andersson 1992).
Recently, a light induced cross-linkage between the N-terminus of the a-subunit of Cyt b559 and the D1 protein (to form an adduct with an apparent molecular mass of 41 kDa) has been discovered. The results indicate that Cyt b559 is located close to the D1 protein since crosslinkng occurred between the N-terminus of the a-subunit and the hydrophobic loop near to the QB binding site (Barbato et al. 1995).
PsbG
Reported initially to be a PSII protein but shown later by Nixon et al. (1989) to be the product of a ndh gene and therefore a component of a NADPH/quinone oxido-reductase.
The mature PsbH protein contains 72 amino acids and occurs in all oxygenic organisms. This 7.7 kDa protein is predicted to have a single transmembrane helix. In higher plants it undergoes reversible N-terminal phosphorylation (Farchaus and Dilley 1986) but the reason for this and the function of the protein as a whole is unknown. It contains no redox reactive centres and has been suggested to play a role in regulating QA to QB electron transfer (Packham 1988). Deletion of the psbH gene in Synechocystis did not prevent PSII assembly and photoautotrophic growth although the deletion mutant was more sensitive to photoinhibition (Mayes et al. 1993). Interestingly, this sensitivity was mainly due to inhibition of the repair process rather than to an increase in photochemical damage of PSII (Komenda and Barber 1995).
This 4.2 kDa protein contains about 35 amino acids and is highly conserved between species. It is predicted to contain a single transmembrane helix and like Cyt b559 is located very close to the D1 and D2 heterodimer (Ikeuchi and Inoue 198S, Webber et al. 1989b). Its function is unknown. The psbI gene can be deleted in Chlamydomonas without impairing PSII assembly and photoautotrophic growth (Kuenster et al. 1995). Sharma et al. (1997) found that the mature PsbI protein retains formyl-Met1 and suggested that perhaps it could act as a chlorophyll ligand in a manner similar to the N-terminus of the a-subunit of LH2 of purple synthetic bacteria (McDermott et al. 1995).
Depending on its species of origin, this protein has about 39 amino acids and a calculated molecular mass of 4.2 kDa forming one single membrane helix. Deletion of the psbJ gene in Synechocystis diminished, but did not prevent the assembly of PSII or the growth of this organism under photoautotrophic conditions (Lind et al. 1993).
The highly conserved 4.3 kDa PsbK protein contains about 37 amino acids and is found in all types of oxygenic organisms. It is predicted to have one transmembrane a-helix, but its function is unknown. Deletion of the psbK gene in Synechocystis had little or no effect (Ikeuchi et al. 1991, Zhang et al. 1993) while a corresponding deletion in Chlamydomonas resulted in poor assembly of PSII and loss of ability to grow photoautotrophically (Takahashi et al. 1994).
The PsbL protein is highly conserved (~65%) in both higher plants and algae (Ikeuchi et al. 1989a). The mature protein contains 37 amino acids with a molecular mass of 4.4 kDa and is predicted to have one transmembrane helix. PsbL seems to be required for normal functioning at the QA site, since QA activity decreases dramatically when isolated PSII core complexes are depleted of this polypeptide (Kitamura et al. 1994, Nagatsuka et al. 1991). Recently we have shown (D. Zheleva, J. Sharma and J. Barber, unpublished results) that a loss of PsbL and QA occurs when an isolated dimeric form of a CP47-RC complex undergoes monomerisation.
The psbM and N genes encode mature proteins predicted to contain 34 and 43 amino acids, respectively. Their molecular masses are 3.7 and 4.7 kDa. Although found in all types of oxygenic organisms their functions are unknown. Both are predicted to contain one transmembrane helix (Ikeuchi et al. 1989a). Deletion of psbN in Synechocystis 6803 did not prevent PSII assembly or photoautotrophic growth (Mayes et al. 1993).
Between higher plants and cyanobacteria this protein is highly conserved, containing after processing 241 to 247 residues. Although the mature PsbO protein is often referred to as the 33 kDa protein, its calculated molecular mass is about 26.5 kDa (Nixon et al. 1992). It is an extrinsic protein with a high b-sheet content (Xu et al. 1992; W.-Z. He 1991. Thesis, Univ. of London, London) and plays an important role in maintaining and optimal environment for water oxidation to occur. Various studies indicate that it does so by stabilising the Mn cluster, but there is no evidence that it binds Mn directly. Indeed, deletion of the psbO gene in Synechocystis 6803 does not inhibit oxygen evolution or photoautotrophic growth (Burnap and Sherman 1991, Mayes et al. 1991, Philbrick et al. 1991). Under these conditions the function of the 33 kDa protein may be carried out by the PsbV protein (Shen et al. 1995b). Crosslinking studies indicate that it is closely located to the lumenal loop of CP47 (Odom and Bricker 1992) and to the PsbE and PsbI proteins (Enami et al. 1992).
After processing the PsbP protein consists of about 186 amino acids with a calculated molecular mass of about 20 kDa. Although found in higher plants and algae, this protein is not conserved in cyanobacteria. Its function seems to be to optimise the Ca2+ and Cl- levels needed for the water oxidising reaction (Debus 1992) and is located in the vicinity of the 33 kDa protein.
The PsbQ mature protein contains about 149 amino acids and, like PsbP, is located close to the 33 kDa protein and the Mn cluster. It too, seems to be involved in optimising the ionic environment necessary for oxygen evolution (Debus 1992). PsbQ, however, is not found in cyanobacteria.
The role of PsbR is unknown. It has a molecular mass of 10.2 kDa and consists of about 99 amino acids (Lautner et al. 1988, Ljungberg et al. 1986a). It seems to be an extrinsic protein which is bound relatively tightly to the lumenal surface in the vicinity of the water splitting site, whether it has transmembrane helix is a matter for debate (Webber et al. 1989a). It has not been observed in cyanobacteria.
The PsbS protein consists of about 205 amino acids and has a molecular mass of 22 kDa (Funk et al. 1994, Ljungberg et al. 1986a). It is predicted to have 4 transmembrane helices (Kim et al. 1992, Wedel et al. 1992). Helices I and K and H and IV are homologous, indicating that the protein is derived from internal gene duplication. Sequence homology studies suggest that PsbS is related to Lhcb1-6 proteins (cab gene products) and is likely to be a chlorophyll binding protein (Funk et al. 1995, Wedel et al. 1992), A functional role, therefore, for PsbS could to act as a pigment chaperone which aids the incorporation of chlorophyll molecules into the pigment binding proteins. It does not, however, exist in cyanobacteria.
The ycf8 gene, which is located in the chloroplast genome on the same operon as psbB of higher plants, Chlamydomonas (Monod et al. 1994) and Cyanophora paradoxa (V.L. Stirewalt, C.B. Michalowski, W. Luffelhardt), has now been called psbT (Hong et al., 1995). This gene encodes a protein having a molecular mass of about 3.8 kDa which was suggested to be a component of PSII (Hong et al. 1995, Monod et al. 1994). Recently this psbT (c) product was identified as a low molecular mass component of the isolated CP47-RC subcomplex from spinach (D. Zheleva, J. Sharma and J. Barber, unpublished results). Deletion of the psbT (c) gene results in increased sensitivity to photoinhibitory stress (Monod et al. 1994).
A nuclear encoded hydrophilic 5 kDa protein (Ikeuchi et al. 1989a) which copurifies with PsbO (33 kDa) (Ljungberg et al. 1986b) has also been called PsbT (Kapazoglou et al. 1995). Although its function is unknown it seems to be an extrinsic protein located on the lumenal surface of PSII, The mature psbT (n) protein consists of about 28 amino acids in higher plants and the presence of two cysteine residues suggests that it contains a disulphide bridge (Ikeuchi et al. 1989a, Kapazoglou et al. 1995).
PsbU
This is a cyanobacterial protein reported to be extrinsically located on the lumenal surface of PSII close to the 33 kDa protein. It has an apparent molecular mass of 10 kDa (Pakrasi 1995).
PsbV
PsbV is also known as cytochrome c550 and is found only in cyanobacteria. It has a molecular mass of 15.1 kDa (Shen et al. 1992), is an extrinsic protein on the lumenal surface of PSII and plays a role in water oxidation. Its deletion, however, does not prevent photoautotrophic growth (Shen et al. 1995a) although it is required if the psbO gene is also deleted (Shen et al. 1995b).
PsbW is found in higher plants but not in cyanobacteria. It has an apparent molecular mass of 6.1 kDa (Ikeuchi et al. 1989a, Schroder et al. 1988) containing 54 amino acids (Lorkovic et al. 1995). It is predicted to have one membrane spanning region with the N-terminus exposed to the lumen. Its function is unknown but it seems to be located close to the reaction centre (Hagman et al. 1995, Irrgang et al. 1995).
PsbX has a molecular mass of 4.2 kDa (Ikeuchi et al. 1989a,b) and is found in all classes of oxygenic organisms. It may have one membrane spanning domain (Kim et al. 1996) and, like PsbL, plays some role in QA functioning (Nagatsuka et al. 1991).
PsbY has been suggested to be a manganese cluster stabilising protein, present in two possible forms (PsbY-1 or 2) having a molecular masses of 4.673 kDa or 4.893 kDa respectively after processing (Gau et al. 1998; Mant and Robinson, 1998).
ARRANGEMENT OF PSII IN THE THYLAKOID MEMBRANE |
By thin sectioning of fixed chloroplasts, electron microscopy has revealed the overall architecture of the thylakoid membrane of higher plants. The thylakoid membrane consists of two main compartments, the grana and the stroma lamellae. The stroma membranes form unstacked (non-appressed) regions, whereas the grana membranes are mostly present as stacked (appressed) membranes. There is a marked heterogeneity in lateral distribution of the major complexes of this membrane, PSII, PSI and ATP synthase. It is generally accepted that PSII, PSI and ATP synthase are mainly laterally segregated, with PSI and ATP synthase excluded from the appressed grana membranes and PSII abundantly present in the stacked parts of the thylakoid membrane (15, 22). The total picture, however, is more complex, because there is also heterogeneity amongst both PSII and PSI in subunit composition and lateral distribution and the cytochrome b6-f complex is found in both appressed and non-appressed regions (5). The detailed ultrastructure of the chloroplast and the thylakoid membrane has been studied mainly using the techniques of freeze-etching and freeze-fracture electron microscopy, which avoid chemical fixation, dehydration and stain artifacts. Together, these two approaches have been used to determine the location of PSII and its antenna components within the thylakoid membrane and to gain low (40-50Å) resolution information about their organization and size. In freeze-etching studies, thylakoids are flash frozen prior to the evaporation of surface water under vacuum (typically at -100oC). This process exposes the membrane surface and so allows the visualization of extrinsic components in their near to native state. The organization of the membrane embedded parts of PSII and the antenna proteins have been studied using the freeze-fracture technique, which involves the cleavage of the lipid bilayer along its internal hydrophobic plane, prior to image analysis. The terminology (ESs, PSs, ESu, PSu) developed by Staehelin (231, 232) has generally been adopted to describe the endoplasmic (E) and protoplasmic (P) surfaces (S) of stacked (s) and unstacked (u) freeze-etched thylakoid membranes. The corresponding freeze-fracture (F) planes are referred to as EFs, PFs, EFu and PFu. The ultrastructure of thylakoid membranes of barley (31, 32, 94, 152), spinach (157, 190, 230, 231, 233), maize (156), lettuce (95), soybean (114) Portulaca (229), Alocasia (12) and pea (16, 199) have all been analysed by freeze-etch and freeze-fracture electron microscopy and show marked similarities (see 152, 232). The protein complexes detected in these analyses were named according to the surfaces with which they were associated (e.g. ESs or PFu particles). They differed in size and shape and the constituent components of many were identified by the analysis of mutant membranes. For example, the analysis of PSI deficient mutant thylakoid membranes showed that this photosystem formed part of the large PFu particles (227). Localization of complexes in the stacked and unstacked thylakoid membranes was further facilitated by antibody labelling (183). |
Localization of PSII and LHCII |
Comparative freeze-etch and freeze-fracture studies of wild type and PSII deficient mutants of tobacco showed that the thylakoid membranes of the latter were depleted of ESs and EFs particles (153). From these results, it was concluded that the ESs and EFs particles corresponded to the extrinsic and internal parts of PSII, respectively. Support for this conclusion came from parallel studies of PSII deficient barley mutants (xantha-b12, viridis-c12, viridis e-64 and viridis zd69) which showed that their granal membranes were also greatly depleted of EFs particles (e.g. 225, 232). The antenna proteins were located using Lhcb protein deficient mutants (e.g. barley mutants xantha-l35 and viridis-k23 and chlorina f2) and by comparing thylakoid membranes from light and dark grown plants, the latter being depleted in these antenna proteins (17, 155, 226, 228). These studies showed that membranes depleted of Lhcb proteins lacked the PFs particles found in wild type membranes. Freeze-etch images of Lhcb protein depleted membranes also showed the ESs particles to be smaller. Together these results suggested that the PFs particles contained the antenna proteins and that they were closely associated with PSII (EFs and ESs) particles. The close association of PSII and the Lhcb proteins was characterized in more detail by the analysis of 2-D crystalline arrays of ESs complexes. Images of such ordered arrays showing a section of their freeze-etch surface (ESs surface) and a part of the protoplasmic fracture face (PFs surface), were presented in (152, 226). They showed that the Lhcb proteins (PFs particles in the PFs fracture face) fitted in register into the grooves between the PSII complexes (ESs particles in the freeze-etched ESs surface). |
SUBUNIT ORGANIZATION OF PSII
This section reviews the subunit organization of PSII and its antenna system using information that has been obtained by electron crystallography, single particle image averaging as well as crosslinking and other biochemical techniques. The combined data is summarized in the form of two currently favored subunit organization models as the available information is still insufficient to confirm which of these is correct. Top and side view projection maps of the largest PSII-LHCII supercomplex structurally characterised to date, are used as the framework for the two possible models of subunit organization. These contoured projection maps are very similar to those presented in Boekema et al (33) but improved in that they are the sums of larger data sets (1,925 vs. 500 top views; 2213 vs 80 side views) and have a higher resolution (approximately 20Å). They also differ in that they contain the densities of two 23 kDa subunits in addition to those of the other core (CP47, CP43, D1, D2, the 33 kDa subunit, cytb559 and the antenna (Lhcb1, Lhcb2, Lhcb4 and Lhcb5) (33, 176) proteins. Both models are identical in terms of their depiction of the extrinsic and Lhcb protein components. They differ only in the attributed locations of the PSII core components, CP47, D1, D2 and cyt b559.
Localization of D1-D2-Cyt b559-CP47 Complex and CP43
Regions within each of the projection maps are shaded in dark, mid and light gray. The dark gray regions represent aligned monomeric D1-D2-cytb559-CP47 complexes (and associated low molecular weight subunits) of the type reported by Dekker et al (52) and Nakazato et al (172). Together, the two dark and two mid gray regions depict the shape of the PSII core dimer consisting of the integral membrane protein components D1, D2, cyt b559, CP47 and CP43 and associated low molecular weight subunits. By elimination, it follows that the two mid gray regions each contain CP43 (205).
Localization of the 33 kDa Extrinsic Subunits
Top and side view projection maps of PSII complexes (+/-33 kDa extrinsic subunit) were used to produce subunit difference maps (33, 50). Each monomeric portion of the dimer is associated with a region of density attributed to the 33 kDa subunit and these densities overlap to form a single central protrusion in the side view. The model implies that the 33 kDa subunit: PSII core monomer stoichiometry is 1:1. There are reports which indicate the presence of two copies of the 33 kDa subunit per reaction centre (133, 274, 277). However, the dimensions of the 33 kDa subunit protrusions shown in Fig. 5 are consistent with single copies of the protein monomer (110).
Localization of the 23 kDa Extrinsic Subunits
When isolated in the presence of glycine betaine PSII-LHCII supercomplexes additionally bind the 23 kDa subunit (Boekema, Nield, Hankamer, Barber Eur J Biochem., 1998). Difference mapping experiments, suggested that the two lumenal protrusions in the side view projection map, located on either side of the 33 kDa components, each contain a 23 kDa subunit. Their positions in the top views also identified by difference mapping. These proposed positions are in agreement with analysis of crystalline PSII arrays containing the 23 kDa subunit (142). Freeze-etching studies of the lumenal surface of the grana regions of thylakoid membranes (ESs surfaces), also show four lumenal protrusions (217) which could correspond to the four densities (see section entitled Heterogeneity of PSII in vivo). If the PSII complexes studied, before and after the removal of extrinsic proteins by Ford et al (66), are interpreted as dimers the positions attributed to the extrinsic subunits would be consistent with both models.
Localization of the Lhcb Proteins
Western blot analyses of PSII-LHCII supercomplexes showed them to be enriched in Lhcb1, Lhcb2, Lhcb4 (CP29) and Lhcb5 (CP26), but depleted of Lhcb3 and Lhcb6 (CP24) (176). Crosslinking studies have shown that CP47 and CP43 are both in close contact with CP29 (Lhcb4) and that CP43 is also in close contact with CP26 (Lhcb5) (46, 202). By superimposing high resolution electron density map of the Lhcb1/Lhcb2 heterotrimer (267) upon the top view projection maps shown in Fig. 5a and 5b, the LHCII complex can be seen to fit snugly into the two tips of the PSII-LHCII supercomplex. Furthermore as Lhcb4 and 5 are similar in mass and have strong sequence homology with the LHCII proteins, two monomeric electron density maps based on the data of (267) were positioned in the PSII-LHCII supercomplex (in both Models 1 and 2) between the PSII core and the LHCII heterotrimer. There appears to be insufficient space to fit any additional monomeric Lhcb proteins. This conclusion is in agreement with pigment analyses of the isolated PSII-LHCII supercomplex which showed it to be associated with 156 Chl a and 46 Chl b molecules (see Fig. 2; refs. 88, 89, 176). In Boekema et al (33) it was suggested that Lhcb6 (CP24) might also be present in these isolated PSII-LHCII supercomplexes. This was a precautionary conclusion based on the detection of low levels of the protein present in early and less pure preparations of the complex. This conclusion has been amended in the light of the western blot data presented in (89, 176). The positions of Lhcb4 and 5 between the LHCII trimer and the PSII core is in agreement not only with the available crosslinking data (46, 202), but with the hypothesis that Lhcb4, 5 and 6 are involved in the regulation of the rate of excitation energy transfer from the outer Lhcb (Lhcb1, 2 and 3) proteins to the reaction centre via CP47 and CP43 (see 111). However the depletion of Lhcb3 and 6 in these complexes with respect to granal membranes, must be explained. One explanation is that the isolated PSII-LHCII supercomplex characterized, originates from the population of ESs particle equivalent to those observed as arrays in freeze-etching studies of grana regions and that Lhcb3 and Lhcb6 are associated predominantly with the non-arrayed ESs particles in the grana (see section on Heterogeneity of PSII in vivo). This hypothesis is consistent with the isolation of a Lhcb6-Lhcb4-LHCII supramolecular complex (26). It is also consistent with the finding that PSIIa complexes in the grana consist of more than one sub-population differing in antenna size (6, 7). Furthermore, the large distances between the non-arrayed ESs particles means that they may have larger antenna systems than their arrayed counterparts and could additionally bind Lhcb3 and Lhcb6 (see section on Heterogeneity of PSII in vivo).
The low-resolution subunit organization models presented in Fig. 5, though incomplete, give two models which it is hoped will aid the more detailed determination of subunit organization and PSII structure in the future.
PSII structure
The Psb proteins make up the core of PSII which is excitonically linked to an outer antenna system consisting of the Lhcb proteins in the case of higher plants and green algae (chlorophytes) or the phycobiliproteins in the case of cyanobacteria and red algae (cyanophytes and rhodophytes). Other types of secondary antenna systems occur in chromophytes composed of a variety of carotenoid-chlorophyll complexes (Gantt, 1996).
There is every reason to believe, however, that the basic structure of the PSII core is conserved amongst all types of oxygenic organisms except for some minor differences in subunit content. Low resolution information (20 Å or poorer) is available for the PSII core structure and has recently been reviewed (Hankamer et al. 1997a). Much of this information has emerged from electron microscopy. Indeed, single particle analyses of PSII cores isolated from higher plants (spinach) and from cyanobacteria (Synechococcus) have revealed almost identical structures (Boekema et al. 1995). In both cases the complexes had comparable sizes (approximately 170 x 100 Å), and had similar protein compositions. Also of significance, is that these cores were dimeric, having molecular masses of about 450 kDa. The dimers seem to represent the most stable form of PSII although monomerisation of the isolated dimer does not inhibit the water splitting function (Hankamer et al. 1997b). The isolated and solubilised PSII core dimer of spinach has been reconstituted with thylakoid lipids and induced to form ordered 2D crystals by dialysing out the detergent. These crystals have been used to obtain both 2D and 3D maps after negative staining (Morris et al. 1997). Since the extrinsic proteins had been lost during crystallization the lumenal exposed protein mass is likely to be mainly composed of the extrinsic loops of CP47 and CP43.
In the absence of high resolution data, molecular by analogies between the D1 and D2 proteins and the L and M subunits of the purple bacterial reaction centre (Ruffle et al. 1992, Svensson et al. 1990). More recently the emerging high resolution structure of PSI offers an additional opportunity to predict high resolution features for PSII structure. The work of Kraub et al. (1996) has shown that the 22 transmembrane helices of PsaA and PsaB are arranged with 10 forming a central core with structural analogy with the L and M subunits of purple bacteria and the remaining 12 transmembrane segments fan out with 6 helices on either side of the central core. This structural arrangement contains therefore also suggests homology with D1 and D2 protein organisation and thus with PSII. The additional 6 helices on each side of the central core could be, in e case of PSII, CP47 and CP43 (Fromme et al. 1996). As pointed out by Fromme et al. (1996), there are some sequence homologies between CP47, CP43 (transmembrane segments I and IV) and transmembrane segments a and d of PsaA and B. Moreover, there is crosslinking data which indicates that CP47 is more closely located to the D2 than the D1 protein (Moskalenko et al. 1992). Taking this into account, comparison of the PSI and PSII proteins is consistent with 8 Å data obtained from 2D crystals of the CP47-RC subcore using electron crystallography (Rhee et al. 1997).
The postulated similarities between PSI and PSII emphasise the fact that photosynthetic reaction centres are derived from a common origin whether it be by gene splitting or gene fusion. In the case of PSII, the evolution of the water splitting system has placed additional requirements on the structure: the necessity for binding the Mn cluster and creating the environment for water oxidation to occur and the requirement to degrade and replace the D1 protein. In the former case a gene addition has been made to create CP43 and CP47 with large lumenal located hydrophobic loops. In the context of turnover of the D1 protein, the location of CP43 next to D1 and the ease by which it can be removed from isolated cores compared to CP47 should be noted. Indeed, Barbato et al. (1992) have presented evidence that the turnover of the D1 protein requires not only a conversion of the core dimmer to a monomer but also the removal of CP43. We now await high-resolution structural information in order to appreciate further the dynamics of PSII function and the role of the many subunits which it contains.
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