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.

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

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

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

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

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

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

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

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

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

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

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