Extrinsic proteins of photosystem II An intermediate member of the PsbQ protein family in red algal PS II Hisataka Ohta 1,2 , Takehiro Suzuki 1 , Masaji Ueno 1 , Akinori Okumura 1 , Shizue Yoshihara 1 , Jian-Ren Shen 3 and Isao Enami 1 1 Department of Biology, Faculty of Science and 2 Tissue Engineering Research Center, Tokyo University of Science, Japan; 3 Department of Biology, Faculty of Science, Okayama University and PRESTO, JST, Japan The oxygen-evolving photosystem II (PS II) complex of red algae contains four extrinsic proteins of 12 kDa, 20 kDa, 33 kDa and cyt c-550, among which the 20 kDa protein is unique in that it is not found in other organisms. We cloned the gene for the 20-kDa protein from a red alga Cyanidium caldarium. The gene consists of a leader sequence which can be divided into two parts: one for transfer across the plastid envelope and the other for transfer into thylakoid lumen, indicating that the gene is encoded by the nuclear genome. The sequence of the mature 20-kDa protein has low but significant homology with the extrinsic 17-kDa (PsbQ) protein of PS II from green algae Volvox Carteri and Chlamydomonas reinhardtii, as well as the PsbQ protein of higher plants and PsbQ-like protein from cyanobacteria. Cross-reconstitution experiments with combinations of the extrinsic proteins and PS IIs from the red alga Cy. calda- rium and green alga Ch. reinhardtii showed that the extrinsic 20-kDa protein was functional in place of the green algal 17-kDa protein on binding to the green algal PS II and restoration of oxygen evolution. From these results, we conclude that the 20-kDa protein is the ancestral form of the extrinsic 17-kDa protein in green algal and higher plant PS IIs. This provides an important clue to the evolution of the oxygen-evolving complex from pro- karyotic cyanobacteria to eukaryotic higher plants. The gene coding for the extrinsic 20-kDa protein was named psbQ¢ (prime). Keywords: photosystem II; oxygen evolution; extrinsic protein; psbQ; red alga. 1 Oxidation of water by photosystem II (PS II) is the source of molecular O 2 , electrons, and protons in higher plants, algae, and cyanobacteria. PS II is a multisubunit pigment– protein complex containing intrinsic and extrinsic compo- nents located in thylakoid membranes. More than 10 intrinsic, membrane-spanning proteins including CP47, CP43, D1, D2, a and b subunits of cytochrome b-559, and the psbI gene product form the transmembrane core of PS II. The extrinsic components are known to maintain and optimize the stability and activity of the water oxidation site, which is composed of a cluster of four manganese atoms located close to the luminal surface of the transmembrane domain and coordinated mainly by amino acids of the D1 protein [1–3]. The extrinsic domain of the oxygen-evolving complex is composed of three proteins of 33 kDa, 23 kDa and 17 kDa encoded by psbO, psbP, psbQ genes, respect- ively, in PS II of green algae and higher plants (reviewed in [4]). Among these three extrinsic components, the 33-kDa manganese stabilizing protein (PsbO) is highly conserved from prokaryotic cyanobacteria to eukaryotic higher plants, while the 23-kDa and 17-kDa proteins are absent in PS II from cyanobacteria and red algae, although a PsbQ-like protein was recently reported to be associated with PS II from Synechocystis sp. PCC 6803 [5]. Instead, cyanobacte- rial PS II contains two other extrinsic proteins, PsbU (12 kDa) and PsbV (cyt c-550), which functions to replace to some extent the role of PsbP and PsbQ found in green algae and higher plants [6]. Among photosynthetic organisms, red algae are one of the most primitive eukaryotic algae phylogenetically closely related to the prokaryotic oxygenic cyanobacteria. We have found that the oxygen-evolving PS II complex purified from aredalga,Cyanidium caldarium contained three extrin- sic proteins of cyanobacteria-type, i.e. the 33-kDa, 12-kDa proteins and cyt c-550 [7]. In addition to these three proteins, the red algal PS II contained a fourth extrin- sic protein of 20 kDa [7,8]. N-terminal amino acid sequence of more than 30 residues of the protein revealed that it has no significant homology with any known PS II polypeptides [7], suggesting that it is a new extrinsic component of PS II. Release-reconstitution experiments in red algal PS II showedthatthe20-kDaproteincanbindtoPSIItoa significant extent by itself, whereas the effective binding of cyt c-550 and the 12-kDa protein requires the presence of both the 33-kDa and 20-kDa proteins [8]. This is in contrast to the situation found in cyanobacterial PS II where cyt c- 550 could bind to PS II essentially independently of the binding of the 33-kDa protein, and where the homologous Correspondence to I. Enami, Department of Biology, Faculty of Science, Tokyo University of Science, 1-3 Kagurazaka, Shinjuku, Tokyo 162-8601, Japan. Fax: +81 471 24 2150, Tel.: +81 471 24 1501 ex5022, E-mail: enami@rs.noda.tus.ac.jp Abbreviation: PS II, photosystem II. Note: The sequence reported in this paper has been deposited in the DDBJ database (accession No. AB111526) (Received 8 July 2003, revised 21 August 2003, accepted 29 August 2003) Eur. J. Biochem. 270, 4156–4163 (2003) Ó FEBS 2003 doi:10.1046/j.1432-1033.2003.03810.x 20-kDa protein was not found. These results suggest a gradual change of the oxygen-evolving complex from prokaryotic cyanobacteria to eukaryotic red algae and higher plants. The unique 20-kDa protein found in red algal PS II may provide insights into such changes. In this work, we cloned the gene for the 20-kDa protein from the red alga, Cy. caldarium,andcomparedits sequence with those of other PS II extrinsic proteins. It was shown that the 20-kDa protein is homologous to the PsbQ protein found in green algal and higher plant PS IIs. The 20-kDa gene was successfully expressed in Escherichia coli, and cross-reconstitution with the recombinant 20-kDa protein showed that this protein is functional in place of the PsbQ protein in green algal PS II. These results provided important clues to the evolution of oxygen-evolving com- plex from cyanobacteria to higher plants. Materials and methods Preparations PS II membranes of spinach were prepared according to Berthold et al. [9]. The extrinsic proteins of PS II were extractedwith1 M CaCl 2 as described by Enami et al. [10]. Oxygen-evolving PS II from the red alga Cy. caldarium was prepared according to Enami et al. [7], and suspended in 40 m M Mes pH 6.5, 10 m M CaCl 2 , 25% glycerol. The four extrinsic proteins were released with 1 M CaCl 2 -wash and purified as described by Enami et al. [8], and finally dialysed extensively against 40 m M Mes pH 6.5 and concentrated. Green algal oxygen-evolving PS II and its extrinsic proteins were prepared from Chlamydomonas reinhardtii as described by Suzuki et al. in [11]. Cloning and sequence analysis of the extrinsic 20-kDa protein The N-terminal sequence of the 20-kDa protein was determined as described by Enami et al.[12],andthe sequence obtained was as follows: AGEPKMSFFGA DAPSSPFTYNEREGEPVYK. Based on this sequence, the gene coding for the 20-kDa protein from Cy. calda- rium was cloned by a two-step PCR method. First, two sets of degenerate oligonucleotide primers corresponding to N-terminal sequence of AGEPKM and GEPVYK were synthesized and used to amplify a 90-bp cDNA fragment by RT/PCR from a Cy. caldarium cDNA library. The cDNA fragment was sequenced to confirm that it indeed corresponded to the N-terminal sequence of the 20-kDa protein. Based on this information, the second PCR step was performed with the RACE procedure [13] using the Marathon cDNA Amplification Kit (Clontech) by which DNA fragments including the 5¢-and3¢-flanking regions of the 20-kDa protein were amplified using primers newly synthesized based on the N-terminal 90-bp cDNA frag- ment. This second-step PCR resulted in 450-bp and 600-bp cDNA fragments from the 5¢-and3¢-RACE, respectively. Sequencing of these cDNA fragments con- firmed that they contained the cDNA for the 20-kDa protein. These sequences were combined with the partial sequence of the N-terminal part to yield the whole sequence of the gene. The PCR fragments obtained were inserted into the plasmid pCRII (TA Cloning Kit, Invitrogen), and the DNA sequences were determined by the method of Dye Deoxy Terminator Cycle Sequencing with a DNA Sequencer (Applied Biosystems, model 310). Expression and purification of the recombinant 20-kDa protein The whole gene encoding the mature 20-kDa protein was cloned into the LIC site of plasmid pET-32Xa/LIC, resulting in a fusion protein with thioredoxin and (His) 6 - tag attached at its N-terminus [14,15]. The recombinant protein was expressed with the host cell BL21 (Novagen) and purified by His-bind affinity chromatography according to the manufacturer’s instructions. The fusion protein was treated with Factor Xa to cleave off the thioredoxin and His-tag and then purified again by affinity column. Reconstitution Reconstitution experiments of CaCl 2 -washed PS II from red and green algae with various combinations of extrinsic proteins from different sources were performed according to Enami et al. [8,10] and Suzuki et al. [11]. SDS/PAGE was performed according to Ikeuchi and Inoue [16]. Oxygen evolution was measured with a Clark-type oxygen electrode at 25 °Cwith0.4m M phenyl-p-benzoquinone (red alga) or 2,6-dichloro-p-benzoquinone (green alga) as electron acceptor. Results Cloning and sequence analysis of the 20-kDa protein The DNA sequences obtained in the present study are shown in Fig. 1. Two in-frame ATG codons were found upstream of the N-terminal alanine residue of the mature polypeptide, one at position 40 and the other at position 91. The start codon for the 20-kDa protein gene was assigned at the first ATG codon of nucleotide number 40, because this site (AAAAATGTT) has a better match with the consensus sequence for plant translation initiation than the second site (CTTGATGAT) [17]. According to this assignment, the resulting gene encodes a polypeptide of 218 amino acid residueswithatotalmolecularmassof24028Da.Asthe N-terminal part of the mature 20-kDa protein corresponds to sequence starting at residue number 73, residues 1–72 serve as leader sequences. Hydropathy analysis (data not shown) revealed that there are two characteristic domains in this leader sequence. The first consists of residues 1–47 and is enriched in basic, hydrophilic, as well as hydroxylated residues; this is consistent with the characteristic features of transit peptides for transport across the chloroplast envel- ope [4] and suggests that this domain functions to direct the transfer of the 20-kDa protein across the chloroplast envelope. The second domain consists of residues 48–72 and has features characteristic of transit peptides for transfer of proteins through the bacterial periplasmic membranes and thylakoid membranes [4], because its central part is enriched in hydrophobic residues and its C terminus contains an alanine residue at position )1(thisis Ó FEBS 2003 Characterization of the fourth extrinsic protein in PS II (Eur. J. Biochem. 270) 4157 typically found in proteins transported across the periplas- mic and thylakoid membranes). Thus, we conclude that the 20-kDa protein is encoded by the nuclear DNA in the red alga. This is consistent with results of whole chloroplast genome sequencing of the red algae Porphyra purpurea [18] and Cy. caldarium RK1 [19], in which the gene coding for the 20-kDa protein was not found in the plastid genome. Cleavage of the transit peptides resulted in a mature polypeptide of 146 amino acid residues with a calculated molecular mass of 16 386 Da. Blast analysis with the GenBank database showed a significant homology of the 20-kDa protein gene with a cDNA clone, AV34507 from a marine red alga Porphyra yezoensis [20]. Unexpectedly, this analysis also gave low but significant scores (53–64) with oxygen-evolving enhancer (OEE) protein 3 (PsbQ) from green algae Volvox carteri [21] and Ch. reinhardtii [22]. These results suggested that the extrinsic 20-kDa protein in PS II from the red alga Cy. caldarium is a homologue of one of the PS II extrinsic proteins, PsbQ protein, in green algae. Recently, Kashino et al. reported that the sll1638 gene product of cyanobac- terium Synechocystis sp. PCC 6803 has a similarity to the PsbQ protein and is associated with the cyanobacterial PS II complex [5]. We thus aligned the red algal 20-kDa protein sequence with the PsbQ-like protein from two cyanobacteria, Synechocystis sp. PCC 6803 and Anabaena sp. PCC7120, the PsbQ protein from green algae and higher plants whose sequences are currently available, together with the homologous 20-kDa protein from the red alga P. yezoensis, using the global alignment algorithm CLU- STALW [23] (Fig. 2A). Based on these sequences, a phylo- genetic tree was constructed by the neighbour-joining algorithm as shown in Fig. 2B [24]. Generally, in contrast with the other PS II extrinsic proteins such as the PsbO protein which has a relatively high homology from cyano- bacteria to higher plants, the PsbQ protein has a low homology even between green algae and higher plants. For example, the similarities (number of identical residues out of the total residues) of the PsbO protein between cyanobac- teria and higher plants range from 42% to 53% (Blast similarity score, > 200), whereas those of the PsbQ protein between the green algae Ch. reinhardtii or V. carteri and spinach are 23% and 25% (Blast similarity score, 51–55), respectively. In particular, the homology between the red algal 20-kDa protein and the cyanobacterial PsbQ-like protein is not high; blast analysis gave rise to a similarity score less than 28 (20% identity). This is reminiscent of the similarity between the cyanobacterial PsbQ-like protein and higher plant PsbQ protein (Blast similarity score, < 39). Consequently, the CLUSTALW multiple sequence alignment shows that only five residues are completely conserved in the C-terminal half of all sequences (Fig. 2A). Examination of individual sequences showed that the 20-kDa protein among red algae, and the PsbQ protein within the same category of organisms are rather conserved. The resulting phylogenic tree indicated that the PsbQ protein family could be classified into four groups: (a) cyanobacteria; (b) red algae; (c) green algae; and (d) higher plants. If we assume that all these proteins were arisen from a common ancestral protein, the PsbQ proteins of higher plants and green algae were diverged at a very early stage from those of prokaryotic cyanobacteria, whereas the red algal 20-kDa protein remains rather unchanged. As a result, the red algal 20-kDa protein has a relatively low similarity with PsbQ proteins from green algae and higher plants. Reconstitution using the recombinant 20-kDa protein For reconstitution experiments, the 20-kDa protein of Cy. caldarium was successfully expressed as a fusion protein with a His-tag using the pET expression system. The expressed protein was purified by His-bind affinity chro- matography, and the His-tag was proteolytically removed by Factor Xa. This recombinant 20-kDa protein was used for reconstitution experiments with the red algal PS II. To compare the binding and functional properties of the recombinant 20-kDa protein with those of the native 20-kDa protein, reconstitution experiments were first car- ried out with the native 20-kDa protein purified from the red algal PS II. As described previously [8,10], four extrinsic Fig. 1. Nucleotide sequence of the 20-kDa extrinsic protein of PS II from the red alga, Cyanidium caldarium. The deduced amino acid sequence is shown below the nucleotide sequence in the single-letter code. The putative chloroplast envelope transit domain (solid line) and thylakoid transfer domain (dashed line) are underlined. Arrowhead indicates the cleavage site generating the mature 20-kDa protein, and the asterisk indicates the stop codon. 4158 H. Ohta et al. (Eur. J. Biochem. 270) Ó FEBS 2003 proteins were completely released by treatment with 1 M CaCl 2 of the purified PS II particles from Cy. caldarium (Fig. 3, lane 2). The 12-kDa protein and cyt c-550 rebound to the CaCl 2 -washed PS II efficiently when they were recon- stituted together with the 33-kDa protein (Fig. 3, lane 3), but their rebinding was not complete. Reconstitution Fig. 2. Phylogenetic analysis of the 20-kDa protein sequence. (A) Alignment of the mature part of the 20-kDa protein sequence of Cyanidium caldarium with a homologous protein from a marine red alga Porphyra yezoensis [20], and those of the PsbQ related proteins from two cyano- bacteria Anabaena sp. PCC7120 [27] and Synechocystis sp. PCC6803 [28], two green algae Volvox Carteri (U22330) [21] and Chlamydomonas reinhardtii [22], and four species of higher plants Spinacia oleracea [29], Arabidopsis thaliana [30], Zea mays [31], Onobrychis viciifolia (GenBank Accession: AAB81994). The alignment was made with the global alignment algorithm CLUSTAL X [23]. Asterisks indicate identical residues among all the sequences compared; double dots indicate conserved replacement of the residue in some of the species, and single dots indicate a slightly less conserved replacement of the residue in some of the species. (B) Phylogenetic tree of the PsbQ protein family constructed based on the alignment shown above. See text for further discussions. Ó FEBS 2003 Characterization of the fourth extrinsic protein in PS II (Eur. J. Biochem. 270) 4159 of these three extrinsic proteins together with the native 20-kDa protein resulted in a complete rebinding of all of the four extrinsic proteins (Fig. 3, lane 4). Similarly, reconsti- tution of the recombinant 20-kDa protein together with the other three proteins also resulted in the complete rebinding of the four extrinsic proteins (Fig. 3, lane 5). This indicates that the recombinant 20-kDa protein retained the same binding ability as that of the native 20-kDa protein. Table 1 shows the restoration of oxygen evolution of the CaCl 2 -washed PS II upon reconstitution with the extrinsic proteins. The native PS II of Cy. caldarium showed a high activity of 2754 lmol O 2 Æmg chl )1 Æh )1 2 in the absence of NaCl in the assay medium; this activity did not increase much upon supplemention by NaCl. Upon CaCl 2 -wash, no activity was observed in the absence or presence of NaCl. Reconstitution with all the four native proteins increased the activity to 50% and 51% of that in the native PS II, respectively, in the absence and presence of NaCl. Recon- stitution with the recombinant 20-kDa protein together with the other three native proteins restored the oxygen-evolving activity to a similar level as that with the native 20-kDa protein, indicating that the recombinant protein was functional in the red algal PS II and was as effective as the native protein. Cross-reconstitution of the 20-kDa protein and green algal extrinsic 17-kDa protein As the 20-kDa protein from Cy. caldarium has a sequence homology with higher plant PsbQ protein, we tried to cross- reconstitute the 20-kDa protein to well characterized spinach PS II in place of the 17-kDa protein. However, the 20-kDa protein was neither able to bind to CaCl 2 - washed spinach PS II in the presence of the spinach extrinsic 33-kDa and 23-kDa proteins nor contributed to increase of the Cl – binding affinity for oxygen evolution (data not shown). Recently, we have purified oxygen-evolving PS II complexes from a green alga, Ch. reinhardtii having His- tagged CP47, and reported that the extrinsic 17-kDa protein of Ch. reinhardtii directly bound to PS II independently of the other extrinsic proteins [11], which is apparently in contrast with the spinach 17-kDa protein which functionally associates with PS II only through its interaction with both the 33-kDa and 23-kDa proteins [25]. This binding property of the 17-kDa protein in green algal PS II is similar to that of the 20-kDa protein in the red algal PS II in that the latter also binds to PS II by itself and promotes the complete binding of the 12-kDa protein and cyt c-550totheredalgal PS II [8]. Thus, we performed cross-reconstitution experi- ments between the 20-kDa protein from the red alga and the 17-kDa protein from the green alga, with PS IIs from both redandgreenalgae. First, we examined whether the green algal 17-kDa protein is exchangeable for the 20-kDa protein in binding to the red algal PS II. The resulting PS II was analysed by SDS/PAGE (Fig. 4A). In agreement with the results obtained in Fig. 3, significant amounts of the 12-kDa protein and cyt c-550 bound to CaCl 2 -washed PS II from the red alga in the presence of the 33-kDa protein, but the 20-kDa protein was essential for complete binding of the 12-kDa protein and cyt c-550 (Fig. 4A, lanes 1 and 2). When the 20-kDa protein was replaced by the green algal extrinsic 17-kDa protein, the 17-kDa protein was able to bind to the red algal PS II to a moderate level, but this binding scarcely enhanced the binding of 12-kDa protein and cyt c-550 (Fig. 4A, lane 3). These results agree with the restoration of oxygen evolution which showed a decreased Cl – requirement upon reconstitution with the 20-kDa Fig. 3. Reconstitution of CaCl 2 -treatedPSIIoftheredalgawitheither the native 20-kDa protein or the recombinant 20-kDa protein, in com- binations with other three native extrinsic proteins of 33 kDa, 12 kDa and cyt c-550. Lane 1, control PS II; lane 2, CaCl 2 -treated PS II; lanes 3–5, CaCl 2 -treated PS II reconstituted with the three extrinsic proteins of 33 kDa, 12 kDa and cyt c-550 (lane 3), with the three extrinsic proteins plus the native 20 kDa protein (lane 4), and with the three extrinsic proteins plus the recombinant 20 kDa protein (lane 5). Table 1. Restoration of oxygen evolution of CaCl 2 -treated red algal PS II by reconstitution with native or recombinant extrinsic 20-kDa protein. Oxygen evolving activity (lmol O 2 Æmg chl )1 Æh )1 ) – ion (%) +10 m M NaCl (%) Cyanidium PS II 2754 ± 21 (100) 2756 ± 31 (100) CaCl 2 -treated PS II 0 (0) 0 (0) + 33 0 (0) 496 ± 22 (18) + 33 + cyt c-550 + 12 1157 ± 18 (42) 1350 ± 40 (49) + 33 + cyt c-550 + 12 + native 20 1378 ± 30 (50) 1402 ± 27 (51) + 33 + cyt c-550 + 12 + recombinant 20 1406 ± 16 (51) 1433 ± 38 (52) 4160 H. Ohta et al. (Eur. J. Biochem. 270) Ó FEBS 2003 protein but this effect was not obvious upon reconstitution with the green algal 17-kDa protein, in the presence of the 33-kDa, 12-kDa proteins and cyt c-550 (Table 2). Taken together, these results suggest that the green algal 17-kDa protein is not able to bind and function in the red algal PS II in place of the 20-kDa protein. Second, cross-reconstitution of the 20-kDa protein with the green algal PS II was carried out. Fig. 4B shows reconstitution of the 20-kDa protein with the green algal PS II depleted of all its three extrinsic proteins by CaCl 2 - wash. Interestingly, the 20-kDa protein significantly bound to the CaCl 2 -washed green algal PS II in the presence of the 33-kDa and 23-kDa proteins (Fig. 4B, lane 5). This binding lowered the Cl – requirement of oxygen evolution remark- ably (Table 2), suggesting that the red algal 20-kDa protein is at least partially functional in replacing the extrinsic 17-kDa protein in the green algal PS II. Discussion We cloned the gene for the 20-kDa protein from the red alga, Cy. caldarium and demonstrated that the gene carries a transit peptide with two characteristic domains, one for transfer across the chloroplast envelope and the other for transfer into the lumen of the thylakoid membrane. This indicates that the gene is located in the nuclear genome of the red alga, consistent with the fact that homologous sequence of the gene was not found in the plastid genome of two species of red algae, P. purpurea [18] and Cy. caldarium RK1 [19], whose complete plastid sequences have been determined. The present study thus represents the first report on the detailed analysis of the 20-kDa protein gene found in the red algal PS II. The 20-kDa protein is unique in that it is not found in PS II of the prokaryotic cyanobacteria, other eukaryotic algae and higher plants. To our surprise, the derived amino acid sequence of the mature 20-kDa protein showed some similarities with the PsbQ protein from green algae and higher plants and also the PsbQ-like protein from cyano- bacteria which has been reported to be associated with purified cyanobacterial PS II [5]. Phylogenetic analysis clearly showed that the 20-kDa protein is a member of the PsbQ protein family; which, according to their sequence similarities, can now be divided into four groups, namely, cyanobacteria, red algae, green algae, and higher plants. The sequence similarities of the PsbQ protein within the same group are reasonably high. However, the sequence similarities of the PsbQ protein among different groups are relatively low. One may therefore ask whether the red algal 20 kDa protein is functionally related with the PsbQ protein of green algal or higher plant PS II. In order to clarify this question, we performed cross-reconstitution experiments Fig. 4. Cross-reconstitution of red algal or green algal PS II with the green algal 17-kDa protein or the red algal 20-kDa protein. (A) Red algal PS II from Cy. caldarium was washed with 1 M CaCl 2 and then reconstituted with the green algal extrinsic 17-kDa protein. In the figure, R33, Rc550, R12 and R20 represent the extrinsic 33-kDa protein, cyt c-550, 12-kDa and 20-kDa proteins of the red alga Cy. caldarium, respectively, whereas G33, G23, G17 represent the extrinsic 33-kDa, 23-kDa and 17-kDa proteins of the green alga Ch. reinhardtii, respectively. Lane 1, CaCl 2 -washed PS II reconstituted with R33, Rc550 and R12; lane 2, R33, Rc550 and R12 plus R20; lane 3, R33, Rc550 and R12 plus G17. Each of the extrinsic proteins was labelled with specific signs as indicated in the left and right sides of the figure. For details of the reconstitution experiment, see text. (B) Green algal PS II from Ch. reinhardtii waswashedwith1 M CaCl 2 and then reconstituted with the red algal 20-kDa extrinsic protein. Lane 1, control PS II; lane 2, PS II washed with 1 M CaCl 2 ; lanes 3–5, CaCl 2 - washed PS II reconstituted with G33 and G23 (lane3), G33 and G23 plus G17 (lane 4), G33 and G23 plus R20 (lane 5). Table 2. Restoration of oxygen evolution of CaCl 2 -treated red algal or green algal PS II by cross-reconstitution with red algal (R) or green algal (G) extrinsic proteins. Oxygen evolving activity (lmol O 2 Æmg chl )1 Æh )1 ) – ion (%) +10 m M NaCl (%) Red algal PS II (R-PS II) 2663 ± 33 (100) 2670 ± 35 (100) CaCl 2 -treated R-PS II 0 (0) 0 (0) + R33 + Rc550 + R12 1065 ± 33 (40) 1282 ± 30 (48) + R33 + Rc550 + R12 + R20 1252 ± 32 (47) 1335 ± 32 (50) + R33 + Rc550 + R12 + G17 1118 ± 30 (42) 1308 ± 35 (48) Green algal PS II (G-PS II) 1100 ± 55 (100) 1178 ± 58 (100) CaCl 2 -treated G-PS II 0 (0) 0 (0) + G33 + G23 330 ± 16 (30) 554 ± 24 (47) + G33 + G23 + G17 506 ± 20 (46) 589 ± 28 (50) + G33 + G23 + R20 484 ± 25 (44) 577 ± 22 (49) Ó FEBS 2003 Characterization of the fourth extrinsic protein in PS II (Eur. J. Biochem. 270) 4161 with combinations of the 20 kDa and green algal or higher plant PS II. Although the 20 kDa protein was not able to bind to and function in the higher plant PS II, it was able to bind to the green algal PS II and functions to diminish the Cl – requirement of oxygen evolution in place of the green algal PsbQ protein. This confirms the conclusion from sequence analysis that the red algal 20 kDa protein is a member of the PsbQ family; the inability of this protein to bind and function in higher plant PS II can be attributed to a relatively distant relationship between red algae and higher plants. Based on these results, we designate the gene for the extrinsic 20 kDa protein psb Q¢ (prime). The red algal PS II contains, in addition to the 20-kDa protein, 33-kDa, 12-kDa proteins and cyt c-550 as its extrinsic proteins in the oxygen-evolving complex [7,8]. The latter two extrinsic proteins are similar to those found in PS II from the prokaryotic cyanobacteria [6] but not from eukaryotic algae and higher plants [4], suggesting that the red algal PS II is closely related to that of cyanobacteria rather than that of eukaryotic algae or higher plants. Although the PsbQ-like protein was also found to associate with cyanobacterial PS II [5], there is so far no evidence indicating that this protein is functional in the cyanobac- terial PS II. PS II purified from thermophilic cyanobacteria has been found to contain no significant amount of the PsbQ-like protein [2,3,6,26]; yet the 12-kDa protein and cyt c-550 are able to bind completely and function fully in PS II from the thermophilic cyanobacteria as long as the 33-kDa protein is present. On the other hand, the full binding and functioning of the 12-kDa protein and cyt c-550 requires the presence of the 20-kDa protein in the red algal PS II [8]. Thus, the red algal PS II has evolved from the cyanobacterial PS II by incorporating the 20-kDa (PsbQ) protein as one of its functional members. The present results suggest that upon the loss of the 12-kDa protein and cyt c-550 in the green algal and higher plant PS II, the 20-kDa protein evolved further to the 17-kDa (PsbQ) protein in functioning to keep the Cl – affinity for oxygen evolution. Naturally, it will be interesting and important to determine where the psbQ¢ gene in red algae has been converted to the PsbQ protein completely in the green algal and higher plant PS IIs during evolution. Along with this, the process of conversion of cyanobacte- rial-type PS II with the 12-kDa protein (PsbU) and cyt c-550 (PsbV) as extrinsic proteins to the green algal and higher plant-type PS II containing PsbP and PsbQ as extrinsic proteins may be clarified. References 1. Debus, R.J. 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This binding property of the 17-kDa protein in green algal PS II is similar to that of the 20-kDa protein in the red algal PS II in that the latter also binds to PS II by. requires the presence of the 20-kDa protein in the red algal PS II [8]. Thus, the red algal PS II has evolved from the cyanobacterial PS II by incorporating the 20-kDa (PsbQ) protein as one of its. Characterization of the fourth extrinsic protein in PS II (Eur. J. Biochem. 270) 4159 of these three extrinsic proteins together with the native 20-kDa protein resulted in a complete rebinding of all of the four