Báo cáo khoa học: Extrinsic proteins of photosystem II An intermediate member of the PsbQ protein family in red algal PS II ppt

Extrinsic proteins of photosystem IIAn intermediate member of the PsbQ protein family in red algal PS II Hisataka Ohta1,2, Takehiro Suzuki1, Masaji Ueno1, Akinori Okumura1, Shizue Yoshih

Extrinsic proteins of photosystem II

An intermediate member of the PsbQ protein family in red algal PS II

Hisataka Ohta1,2, Takehiro Suzuki1, Masaji Ueno1, Akinori Okumura1, Shizue Yoshihara1, Jian-Ren Shen3 and Isao Enami1

1

Department of Biology, Faculty of Science and2Tissue 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 eupro-karyotic 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 O2, 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

a red alga, 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 showed that the 20-kD a protein can bind to PS II to a 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)

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, and compared its

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

extracted with 1MCaCl2as 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 mMMes pH 6.5, 10 mMCaCl2, 25% glycerol The four

extrinsic proteins were released with 1MCaCl2-wash and

purified as described by Enami et al [8], and finally dialysed

extensively against 40 mM 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], and the

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¢- and 3¢-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¢- and 3¢-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 CaCl2-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 25C with 0.4 mM 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 residues with a total molecular mass of 24 028 D a As the 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 (this is

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 caldariumis 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, theCLUSTALWmultiple 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 caldariumwas 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

fromthe 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.

proteins were completely released by treatment with 1M

CaCl2 of the purified PS II particles from Cy caldarium

(Fig 3, lane 2) The 12-kDa protein and cyt c-550 rebound

to the CaCl2-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.

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

CaCl2-washed PS II upon reconstitution with the extrinsic

proteins The native PS II of Cy caldarium showed a high

activity of 2754 lmol O2Æmg chl)1Æh)1

NaCl in the assay medium; this activity did not increase

much upon supplemention by NaCl Upon CaCl2-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 CaCl2 -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-550 to the red algal

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 red and green algae

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 CaCl2-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 -treated PS II of the red alga with either

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)1Æh)1)

–

ion (%) +10 m M NaCl (%) Cyanidium PS II 2754 ± 21 (100) 2756 ± 31 (100)

+ 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)

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 CaCl2 -wash Interestingly, the 20-kDa protein significantly bound

to the CaCl2-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 was washed with 1 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)1Æh)1)

–

ion (%) +10 m M NaCl (%) Red algal PS II (R-PS II) 2663 ± 33 (100) 2670 ± 35 (100)

+ 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)

+ G33 + G23 + G17 506 ± 20 (46) 589 ± 28 (50) + G33 + G23 + R20 484 ± 25 (44) 577 ± 22 (49)

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

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