Báo cáo khoa học: Absence of the psbH gene product destabilizes photosystem II complex and bicarbonate binding on its acceptor side in Synechocystis PCC 6803 ppt

Absence of the
psbH
gene product destabilizes photosystem II
complex and bicarbonate binding on its acceptor side
in
Synechocystis
PCC 6803
Josef Komenda
1,2
, Lenka Lupõ
Â
nkova
Â
1,2
and Jir
Ï
õ
Â
Kopecky
Â
1,2
1
Photosynthesis Research Centre, University of South Bohemia, C
Ï
eske
Â
Bude
Ï
jovice, Czech Republic;
2
Laboratory of Photosynthesis,

Institute of Microbiology, Academy of Sciences, Tr
Ï
ebon
Ï
, Czech Republic
The PsbH protein, a small subunit of the photosystem II
complex (PSII), was identi®ed as a 6-kDa protein band in the
PSII core and subcore (CP47±D1±D2±c yt b-559) from the
wild-type s train of the cyanobacterium Synechocystis PCC
6803. The protein was missing in the D 1±D2±cyto-
chrome b-559 complex and also in all PSII complexes
isolated from IC7, a mutant lacking the psbH gene. The
following properties of PSII in the mutant contrasted with
those in wild-type: (a) CP47 was released during nondena-
turing electrophoresis of the PSII core isolated from IC7;
(b) depletion of CO
2
resulted in a reversible decrease of
the Q
À
A
reoxidation rate in the IC7 cells; (c) light-induced
decrease in PSII activity, measured as 2,5±dimethyl-benzo-
quinone-supported Hill reaction, was strongly dependent on
the H CO
3
±
concentration i n t he IC7 cells; and (d) illumina-
tion of the IC7 cells lead to an extensive oxidation, frag-
mentation and cross-linking of the D 1 p rotein. We did not

®nd any evidence for phosphorylation of the PsbH protein in
thewild-typestrain. The results showed that in the PSII
complex of Synechocysti s attachment of CP47 to the D1±D2
heterodimer appears weakened a nd binding of bicarbonate
on the PSII acceptor side is destabilized in the absence of the
PsbH protein.
Keywords: cyanobacteria; D1 protein; photosystem II; psbH
gene; Synechocystis PCC 6803.
The core of photosystem II (PSII) complex of higher plants,
algae and cyanobacteria consists of large central subunits
D1, D2, CP47, CP43 a nd a number of low molecular m ass
proteins. It i s b elieved t hat w ith a n e xception of cyto-
chrome b-559, the small proteins do not part icipate directly
in the transfer of electrons within PSII but they are
important for the optimization of electron transfer processes
and for the proper assembly of the complex (reviewed in [1]).
Two different strategies are used to get information about
the r ole of small PSII subunits. One approach is based on
the functional co mparison of the intact and detergent
treated PSII complex missing a speci®c subunit. Resump-
tion of the particular function after reconstitution of the
complex with this subunit is considered as evidence for its
role. F or example, using this approach the role o f the PsbL
subunit i n t he Q
A
binding has been proposed [2]. The
second, more frequent approach is based on the deletion of
the gene encoding a studied protein followed by a detailed
characterization of PSII complex in the resulting mutant.
This strategy has been very often successful in cyanobac-

teria, namely in the strain Synechocystis PCC 6803, which
can grow photoheterotrophically and is easily transform-
able. In this way, mutants of Synechocystis with deleted
psbK, psbH, psbI [3±5] and other genes were constructed.
These mutants contained assembled PSII complexes and
after their functional characterization possible functions
were ascribed to these subunits. Interestingly, in algae and
higher plants this approach is useful only rar ely as deletions
of PSII subunits usually lead to disappearance of the whole
PSII complex from thylakoids [6±8].
The PsbH protein, a product of the psbH gene, w as
initially found as the 10-kDa phosphoprotein i n thyla-
koids of higher plants by Bennett [9]. From that time its
homologues have been found in more than 15 photo-
synthetic o rganisms including cyanobacteria. T he ®rst
partial sequence of the cyanobacterial PsbH protein was
obtained in the thermophilic cyanobacterium Synecho-
coccus vulcanus [10], but the complete gene was
sequenced in the strain Synechocystis P CC 6803 by
Abdel-Mawgood & Dilley [11] and Mayes & Barber [12].
Construction of the Synechocystis psbH-less mutant and
its characterization in vivo provided the ®rst more solid
basis for the elucidation of the role of the protein in PSII
[4]. The mutant was more sensitive to photoinhibition in
comparison with the wild-type [4,13] and this sensitivity
has been mostly attributed to perturbations in the
electron ¯ow between Q
A
and Q
B

on the acceptor side
of PSII. In the present paper we have conducted a more
detailed analysis of the effects of the PsbH absence on
the structure and function of PSII both in vivo and
in vitro. Our results indicated a stabilizing role of the
protein for CP47 binding to the D1±D2 heterodimer and
showed its importance for bicarbonate binding and
preventing oxidative stress in PSII.
Correspondence to J. Komenda, Institute of Microbiology, OpatovickyÂ
mly n, 379 81 Tr
Ï
ebon
Ï
, Czech Republic. Fax: + 420 333 721246,
Tel.: + 420 333 721101, E-mail:
Abbreviations: cyt, cytochrome; DM, dodecylmaltoside; DCBQ,
2,5-dichloro-p-benzoquinone; DMBQ, 2,5-dimethyl-p-benzoquinone;
DNP-, dinitrophenyl-; HRA, Hill reaction activity; PSI and PSII,
photosystem I and photosystem II complexes; PSII RC, reaction
centre complex of photosystem II; ROS, reactive oxygen species.
(Received 1 5 August 2001 , revised 16 November 2001, accepted 20
November 2 001)
Eur. J. Biochem. 269, 610±619 (2002) Ó FEBS 2002
MATERIALS AND METHODS
Strains and growth of organisms
The glucose tolerant strain Synechocystis PCC 6803 [14],
referred to as w ild-type (WT), and i ts psbH deletion mutant
IC7 [4] were grown in BG-11 medium with (photomixo-
trophic growth) or without (photoautotrophic growth)
glucose ( 10 m

M
®nal c oncentration). The plate medium
contained BG-11, 10 m
M
Tes/NaOH,pH8.2,1.5%agar
and 0.3% sodium thiosulphate [15] and in the case of the
IC7 mutant also kanamycin (25 lgámL
)1
) and atrazine
(5.10
)6
M
) were added. Liquid cultures (100±200 mL) in
conical ¯asks were aerated using an orbital shaker, irradi-
ated with 50±70 lmol photonsám
)2
ás
)1
of white light at
29 °C and diluted every day to maintain the chlorophyll
concentration at % 8 lgámL
)1
.CulturesofChlorella soro-
kiniana, Scenedesmus quadricauda and Chla mydomonas
reinhardtii were grown under the same conditions and their
density was maintained at D
753
%1.
Photoinhibitory treatment of the Synechocystis cultures
was performed at a chlorophyll concentration of 6 lgámL

)1
in 18-mm thick plate-parallel cuvettes placed in a temper-
ature controlled bath. Cultures were bubbled with air
containing 2% CO
2
(CO
2
-enriched air), with air bubbled
through 40% NaOH (CO
2
-depleted air) or with pure
nitrogen. In some experiments, the cell suspension was
supplemented with 10 m
M
NaHCO
3
. The light source was a
500-W tungsten ®lament bulb mounted in an aluminium
re¯ector. I n t he experim ents w ith a protein-synthesis
inhibitor lincomycin (Sigma, USA, 100 lgámL
)1
®nal
concentration) the culture was incubated for 10 min in the
dark before the start of light treatment.
Phosphorylation of membrane proteins in algal and
Synechocystis strains was induced in the cell suspensions
diluted to D
753
 0.2. They were exposed to 250 lmol pho-
tonsám

)2
ás
)1
of white light for 30 min either in the absence or
in the presence of 3 lCiámL
)133
P-H
3
PO
4
. Thylakoids
isolated from the cells were analysed by SDS/PAGE and
Western blotting using rabbit polyclonal antiphosphothre-
onine antibody (Zymed, USA) or by a utoradiography.
Preparation of membranes and PSII complexes
and their trypsinization
Cyanobacterial membranes were prepared by breaking the
cells with glass beads (150±200 lmindiameter)at4°C
followed by differential centrifugation. For small scale
preparation, the cells (approx. 150 lg of chlorophyll) were
washed and resuspended in 150 lLof25m
M
Tris/HCl
buffer, pH 7.5 containing 1 m
M
aminocaproic acid. The
beads were added to the suspension and the mixture was
vortexed twice for 1 min with 2 min interruption for cooling
on ice. Beads were then washed four times with 200 lLof
buffer. Aliquots were pooled and centrifuged at 3000 g for

1 min to remove unbroken cells. Membranes were collected
from the supernatant at 20 000 g for 10 min The ®nal
sediment was resuspended in 25 m
M
Tris/HCl buffer,
pH 6.8 containing 1
M
sucrose (®nal chlorophyll concen-
tration 400±600 lgámL
)1
) and stored at )75 °C. Large scale
preparation of m embranes for isolation of PSII was
performed a ccording to T ang & Diner [16] u sing a
beadbeater (Biospec Products, USA) f or breaking the cells.
Isolation of PSII complexes f rom the wild-type and mutant
thylakoids was c onducted a ccording to the modi®ed
procedure of Ritter et al. [17]. Brie¯y, membranes were
spun down, resuspended in 25 m
M
Mes/NaOH, pH 6.5 and
solubilized with dodecylmaltoside (DM/chlorophyll  20,
w/w) for 15 min. Unsolubilized material was removed by
centrifugation (40 000 g, 15 min). The supernatant was
applied on the column of chelating Sepharose (Amersham
Pharmacia, Sweden) with bound Cu
2+
ions and imidazole
equilibrated with t wo column volumes of 25 m
M
Mes/

NaOH, pH 6.5 containing 200 m
M
NaCl and 0.03% DM.
PSII and carotenoid fraction did not bind to the column and
went through directly into the second column of Q Sepha-
rose (Amersham Pharmacia, Sweden). This was washed
with several volumes of 25 m
M
Mes/NaOH, pH 6.5 con-
taining 200 m
M
NaCl and 0.03% DM. During this step,
carotenoids and remaining small amounts of phycocyano-
biliproteins and PSI were removed from the column.
Finally, t he PSII core complex was eluted from the column
by 25 m
M
Mes/NaOH, pH 6.5 containing 250 m
M
NaCl
and 0.03% DM. The preparation was concentrated in
Centricon 30 spin columns (Millipore, USA).
Trypsinization of membranes was performed at chloro-
phyll concentration 2 00 lgámL
)1
and trypsin co ncentration
50 lgámL
)1
(Serva, Germany). After 5 , 15 and 30 min
incubation at 25 °C, aliquots were withdrawn and proteo-

lysis was stopped by transfer to ice and addition of 2 m
M
Pefabloc SC (Merck, Germany).
Analysis of proteins
Isolated PSII complexes or membranes solubilized with DM
(DM/chlorophyll  20, w/w) were analysed by nondena-
turing electrophoresis at 4 °C in 5±10% polyacrylamide gel
according to Laemmli [18] except that the electrophoretic
buffers contained 12.5 m
M
Tris, 98 m
M
glycine and 0.1%
Deriphat 160, and the gel contained 0.1
M
Tris/HCl, pH 8.8
without detergent.
Protein composition of membranes and p igment protein
complexes obtained by Deriphat electrophoresis was
assessed by electrophoresis in a d enaturing 12±20% linear
gradient polyacrylamide gel containing 7
M
urea [18]. The
membranes were solubilized in 25 m
M
Tris/HCl, pH 6.8,
containing 2% SDS (w/v) and 2% dithiothreitol (w/v) at
laboratory temperature for 60 min Samples were loaded
with equal amount of chlorophyll as indicated in ®gure
legends. Analysis o f pigment proteins was p erformed

either by re-electrophoresis of individual pigment protein
bands or the whole lane from the native gel was excised
and placed on the top of the SDS gel (diagonal PAGE).
The gels with p igment proteins were incubated for one
hour in the same solubilization solution as thylakoids
prior to SDS/PAGE. Proteins separated in the gel were
either stained by C oomassie Blue or tran sferred onto
nitrocellulose membrane (0.1 lm, Schleicher-Schuel, Ger-
many) by semidry blotting. M embrane was incubated with
speci®c antibodies and then with alkaline phosphatase
conjugated secondary antibody (Sigma). Proteins were
visualized by colorimetric reac tion using B CPIP-NBT
system. Antibodies used in the study were raised against:
(a) residues 2±17 of the Synechocystis PCC 6803 D1
protein (D1-Nt); ( b) residues 5 8±86 o f the s pinach D1
protein (D1-Mp); (c) the last 29 residues of the pea
Ó FEBS 2002 Function of the PsbH protein in photosystem II (Eur. J. Biochem. 269) 611
D1 precursor (D1-Ct); (d) the last 14 residues of the
Synechocystis D2 (D2-Ct) and (e) the isolated a subunit of
the cytochrome b-559 from Synechocystis PCC 6803
(cyt b-559). For autoradiography, the membrane with
labelled proteins was exposed to X-ray ®lm at laboratory
temperature for 2 days.
Oxidation of proteins
Oxidation of the D1 protein was determined using t he
detection kit Oxyblot (Intergen, USA). Solubilized thyla-
koid m embrane proteins w ere derivatized using dini-
trophenylhydrazine, which reacts with carbonyls present
on oxidized proteins. After protein separation b y SDS/
PAGE and transfer onto the membrane, dinitrophenyl

(DNP)-proteins were detected by Western blotting using
anti-DNP Ig. The whole procedure was performed accord-
ing to manufacturer's instructions.
N-terminal protein sequencing
N-terminal sequence of proteins was analysed performing
eight cycles of a utomated Edman degradations using
Protein sequencer LF3600D (Beckman, USA) and program
2±39 according to manufacturer's instructions. A mino-acid
sequence was called from the comparisons of chromato-
grams. Protein in the gel was blotted onto poly(vinylidene
di¯uoride) membrane, prewetted with acetonitrile, and then
deblocked by treatment with 0.6
M
HCl for 20 h at 25 °C.
HCl was then evaporated and the membrane was inserted
into cartridge of the sequencer.
Measurement of oxygen evolution
Light-saturated steady-state rates of oxy gen evolution (Hill
reaction activity, HRA) in cell suspensions were measured
at 3 0 °C u sing a t emperature controlled chamber
[19] equipped w ith a Clark-type electrode (YSI, USA).
Arti®cial e lectron acceptors 2 ,5-dimethyl-p-benzoquinone
(DMBQ) or 2,6-dichloro-p-benzoquinone (DCBQ) (0.5 m
M
®nal concentration each) were added 1 min before the
measuring illumination (3500 lmol photonsám
)2
ás
)1
,30s)

was switched on.
Chlorophyll ¯uorescence measurement
TherateofQ
À
A
reoxidation was measured with the P.S.I.
double-modulated ¯uorometer FL-100 (P.S.I., Czech
republic). Short, nonactivating pulses of blue light were
used as the m easuring light and F
M
re¯ecting fully reduced
Q
A
was elicited by the strong saturating red ¯ash. Cells were
incubated for 5 min in the dark before measurements.
Pigment analyses
For the routine measurements of chlorophyll concentration,
the cells were collected by centrifugation and extracted w ith
100% methanol. The concentration of chlorophyll was
calculated from the absorbance values of the extract at 666
and 720 nm according to Wellburn and Lichtenthaler [20].
Detailed analysis of pigments was performed by HPLC
(Beckman, USA) using procedure of Gilmore and Yama-
moto [2 1].
RESULTS
Identi®cation of the PsbH protein in photosystem II
complexes of Synechocystis
PSII core complexes from wild-type and IC7 strains of
Synechocyst is were isolated by a combination of metal
af®nity and ionex chromatography. Absorption spectra of

preparations from each strain exhibited similar absorption
maxima at 673 nm, typical for P SII complex from this
cyanobacterial species [16]. The preparations were then
subjected to the nondenaturing electrophoresis in the
presence of Deriphat 160 (Fig. 1). In the case of wild-type,
we obtained two prominent green bands (Fig. 1A). The ®rst
band was ascribed to the monomeric PSII core consisting of
CP47, CP43, D2, D1, both cytochrome b-559 subunits, a
6-kDa protein and other smaller proteins (Fig. 1B, WT: A).
The s econd band represented PSII core lacking CP43 (PSII
subcore, Fig. 1B, WT: B). There were also two low
molecular mass pigment-containing bands that were
ascribed to free CP43 based on its protein composition
(Fig. 1B, WT: D) and free carotenoids (Fig. 1A, FP) based
on its absorption spectrum (data not shown). Similar
electrophoretic pattern of the pigment proteins was
obtained from the IC7 strain with the exception that: (a)
the band of the PSII subcore was much weaker than in wild-
type (b) there was an additional band identi®ed as the D1±
D2±cyt b-559 complex (PSII RC) (Fig. 1B, IC7: C), and (c)
the lower green band contained both CP47 and CP43
(Fig. 1B, IC7: D). The results suggest that during electro-
phoresis the PSII subcore from IC7 became unstable and
decomposed into CP47 and PSII RC c omplex.
Comparison of the protein composition of the PSII cores
and subcores (Fig. 1B) from both strains showed that there
was a protein with M
r
of % 6 kDa in the complexes from
wild-type that was absent in IC7. The band was subjected to

the automated Edman degradation. The obtained sequence
DILRPLNS corresponding to the internal sequence 8±15 of
the PsbH protein from Synechocystis PCC 6803 (SWISS-
PROT accession number P14835) con®rmed t he identity of
the protein.
Analysis of protein composition of PSII complexes from
wild-type revealed that PsbH protein was present in the core
as well as in the subcore complex lack ing CP43. Evaluation
of its presence in PSIIRC was allowed by the treatment of
the wild-type preparation with SDS in the ratio SDS/
chlorophyll  10. Deriphat PAGE of this preparation led
to the generation of PSIIRC that was devoid of the PsbH
protein (Fig. 2). It means that this subunit was released
from PSII subcore together with CP47, again suggesting a
close structural relationship between PsbH and C P47.
Effect of the PsbH absence on the accessibility
of the D1 protein to trypsin
The e ffect of the PsbH absence on the structure of the PSII
core complex was further probed by trypsinization of the
D1 protein in isolated membranes of wild-type and IC7
(Fig. 3). The initial trypsin-induced cut of the D1 protein
occurred at the N-terminus and was documented by a small
increase of the electrophoretic mobility and by a loss of
reactivity with the D1-Nt Ig (data not shown). As in
Synechococc us PCC 7942, this cutting occurred concomi-
612 J. Komenda et al. (Eur. J. Biochem. 269) Ó FEBS 2002
tantly with breakdown of the D2 protein at residue R234
and there was also trypsin-induced formation of the 35-kDa
adduct of t he D2 C-terminal fragment and D1 protein
without N-terminus [22]. Interestingly, formation of this

D1±D2 adduct was inhibited in the IC7 mutant. After these
initial events the D1 protein was subsequently cut at residue
K238 and later also at R257 generating the C -terminal 12
and 10-kDa Ct1 and Ct2 fragments. A 20-kDa Nt1
fragment reacting with D1-Mp and very weakly with
D1-Nt represented the D1 subfragment between residues
R8 and K238. However, in the IC7 mutant, a 16-kDa Nt2
fragment reacting with D1-Mp Ig a lso appeared. As j udged
from the amino-acid sequence of the protein, the Nt2
fragment originated from the cut at R225. In summary,
trypsinization of thylakoids showed that in the absence of
the PsbH protein the accessibility of the D1 protein to
trypsin was changed and also mutual position between D1
and D2 was modi®ed a s i ndicated b y i nhibition of the D1±
D2 adduct formation in the I C7 strain.
The PsbH protein affects the bicarbonate
binding on the acceptor side of PSII
A characteristic feature of PSII in the IC7 strain is a slow
electron transfer between Q
A
and Q
B
[4]. This was
demonstrated in the Fig. 4 (compare solid lines in the left
and right panels). Additional retardation of the electron
transfer could be induced by removal of CO
2
from the
medium during dark incubation of the mutant cells. This
retardation was fully reversed after subsequent addition of

bicarbonate and /or bubbling with the CO
2
-enriched air
(Fig. 4, right panel). In contrast, removal of CO
2
and its
subsequent addition did not affect the Q
À
A
reoxidation rate
in the wild-type strain (Fig. 4, left panel). It indicated that
the binding of bicarbonate to the PSII acceptor side was
weakened in the IC7 mutant as a consequence of the missing
PsbH protein. This conclusion was supported by the
following experiment. We have shown previously that after
exposure to high irradiance, the Hill reaction activity of the
IC7 cells measured using 2,5-dimethyl-benzoquinone as an
arti®cial electron acceptor (DMBQ-HRA) was very quickly
inhibited. In contrast, the decline of activity measured
using 2,6-dichloro-benzoquinone (DCBQ-HRA), was much
slower [13]. We found that this difference was further
enhanced when the illuminated IC7 cells were bubbled with
the CO
2
-depleted air (Fig. 5, closed symbols). However,
when the suspension was supplemented with 5 m
M
bicar-
bonate and bubbled with CO
2

-enriched air, the decline of
DMBQ- and DCBQ-HRA was parallel (Fig. 5, open
symbols). The rate of DMBQ- and DCBQ-HRA decline
in the wild-typ e cells was not dependent on the C O
2
and/or
bicarbonate concentration (not shown, see [ 13]).
The D1 protein is extensively photooxidized
in the mutant
It was shown previously that the turnover of the D1 protein
in the illuminated IC7 cells is retarded and also the recovery
from photoinhibition is slow as compared with the wild-
type cells [13]. Possible explanation for this feature of the
IC7 s train could be a n increased formation o f r eactive
oxygen species (ROS) in PSII that m ay inhibit t he D1
replacement process [23]. The ®rst supporting evidence for
this came from the analysis of pigment content in the
autotrophically grown wild-type and IC7. We assumed that
increased formation of ROS could lead to increase of
cellular carotenoid content, as these pigments are able to
eliminate to some extent the ROS effect. HPLC analysis
revealed almost four times higher ratio of myxoxantho-
phyll/chlorophyll in the IC7 cells as compared to the wild-
type cells (Table 1). The increase in content of other
carotenoids was not as signi®cant. Increased generation of
Fig. 1. Identi®cation of the PsbH protein by analysis of PSII complexes
isolated from wild-type and IC7 mutant. (A) Pigment protein pro®le of
PSII complexes isolated from wild-type and IC7 after the native PAGE
in the presence o f Deriphat 160, 8 lg o f chlorophyll loade d per l ane.
(B) Protein composition of pigment proteins obtained by native Deri-

phat/PAGE of PSII complexes isolated from wild-type and IC7: A,
PSII core  PSII complex containing at least CP47, CP43, D1, D2 and
cytochrome b-559; B, PSII subcore  PSII core comp lex lac king CP43;
C, PSIIRC  D1±D2±cytochrome b-559 complex; D, CP47, C P43 
free pigment p roteins CP47 and CP43; and F P  free pigments.
Ó FEBS 2002 Function of the PsbH protein in photosystem II (Eur. J. Biochem. 269) 613
ROS in the PSII complex of IC7 was further supported b y
the r esults of the D1 analysis in cells exposed to high
irradiance. The Western blot showed, in addition to the
typical 32-kDa D 1 b and, formation of a 40-kDa band that
also reacted with the antibody raised against the a subunit
of cytochrome b-559 (Fig. 6). Although this band was
present even in control cells, high irradiance induced
formation of an a dditional, slightly smaller D1±cyto-
chrome b-559-reactive band. We propose that this band
was identical to that found by Barbato et al. [24] in
illuminated plant thylakoids which seems to be induced by
the action of ROS [25]. Effect of high irradiance was further
accompanied by decreased i ntensity of the original 32-k Da
band and in the case of IC7 mobility of the remaining
protein was decr eased in an oxygen-dependent manner.
Such a shift often re¯ects protein oxidation [26] and this was
con®rmed by O xyblot, a co mmercially available kit devel-
oped to detect oxidized proteins. Indeed, after light treat-
ment of IC7 cells the D1 protein with lower mobility
exhibited signi®cant oxidation that was partially inhibited in
the cells bubbled with nitrogen during illumination. In
addition, a 23-kDa N-terminal D1 fragment was detected
in the IC7 cells and its mobility was also shifted by high
irradiance in the presence of oxygen. As showed by Miyao

[25], also fragmentation of the D1 protein may be induced
by ROS. Taken together, the above results provide strong
experimental support for enhanced generation of ROS in
the PSII complex lacking the PsbH protein.
The PsbH of
Synechocystis
is not phosphorylated
in vivo
The PsbH protein has originally been identi®ed in higher
plants due to its phosphorylation in light [9]. This
phosphorylation also exists in green algae and two
N-terminal threonine residues seem to be phosphorylated
in these organisms [27,28]. However, th e question concern-
ing phosphorylation of the cyanobacterial PsbH remains
still open. There is a single report documenting in vitro
phosphorylation of PsbH in Synechocystis by Race &
Gounaris [29]. However, in this report identi®cation of the
phosphorylated ban d as the PsbH protein was ambiguous
as in thylakoids there is a dozen of polypeptides below
Fig. 2. The PsbH protein is absent in the PSII
RC complex o f wild-type. PSII complex of
wild-type isolated by chromatography has
been analysed after an addition of SDS in the
ratio (w/w) SDS/chlorophyll  1or10bythe
native PAGE in the presence of Deriphat 160
in the ®rst dimension (8 lg of chlorophyll
loaded per lane) and SDS/PAGE in t he sec-
ond dimension according t o Materials and
methods (diagonal PAGE).
Fig. 3. Time course of the D1 trypsinolysis in membranes isolated from

wild-type and IC7 cells. Isolated membranes were incubated with
trypsin and samples were taken at indicated time intervals for elec-
trophoresis of proteins and immunoblotting as described in Materials
and methods (5 lg of chlorophyll loaded per lane). Nitrocellulose
membrane with separated thylakoid pr oteins was probed with a mix-
ture of anti-(D1-Mp) Ig and anti-(D1-Ct) Ig according to Materials
and methods. Nt1  fragment of D1 between R8 a nd K238;
Nt2  fragment of D1 between R8 and R225; Ct1  fragmentofD1
between K238 and A344; Ct2  fragment of D1 between R257 and
A344; a nd D1D2 adduct  adduct between D 1 (R8-A344) and frag-
ment of D2 (R234-L352).
614 J. Komenda et al. (Eur. J. Biochem. 269) Ó FEBS 2002
10 kDa. In addition, the cyanobacterial PsbH lacks the
N-terminal sequence with threonine 2 and 4 residues
phosphorylated in plants and algae [30,31]. To clarify this
point, we attempted to id entify phosphorylation of the
PsbH in vivo using antiphosphothreonine Ig that proved to
react well with PSII phosphoproteins in plant chloroplasts
[32]. For comparison, we also analysed PsbH phosphory-
lation in green algae Scenedesmus quadricauda, Chlorella
sorokiniana and Chlamydomonas reinhardtii. A single 6.5-
kDa band in Scenedesmus and two closely migrating 8 and
9-kDa bands in Chlorella and Chlamydomonas could be
detected in the PSII cores (Fig. 7A). In contrast, absolutely
no reaction of cyanobacterial proteins with the antibody in
membranes and PSII core complexes of both wild-type and
IC7 suggested that the threonine phosphorylation does not
occur in thylakoids of Synechocystis. In order to extend this
conclusion for phosphorylation of other residues, the cells of
Synechocystis were labelled with

33
P-H
3
PO
4
. We found
weak phosphorylation of two bands with M
r
values of 3 and
4 kDa clearly distinct from the PsbH protein (Fig. 7B).
In addition, th ese two bands were p resent in both wild-type
and the IC7 mutant. In summary, we did not obtain any
experimental evidence for the phosphorylation of the PsbH
protein in Synechocyst is cells.
DISCUSSION
Packham [33] proposed that the PsbH protein of photo-
system II is a functional homologue of the H subunit from
the reaction centre of photosynthetic bacteria. This is in line
with the effect of the protein on the herbicide binding in
PSII [34] and a lso in line with the data in this paper.
The r ecent 3.8 A
Ê
model o f P SII [35] tentatively s ituated the
membrane helix of PsbH on the side o f CP47 a nd D2 in the
proximity of the Q
A
±Q
B
region. This position provides a
good justi®cation for the stabilizing effect of PsbH o n the

binding of CP47 to the heterodimer D1±D2 as well as on the
bicarbonate binding to the acceptor side of PSII. Never-
theless, there is also recent report showing the PsbH protein
in Chlamydomonas on the periphery of the PSII dimeric core
[36]. However, detection of the protein was based on the
attachment of gold particles to His-tagged N-terminus that
can be positioned at the different region of the core than the
membrane helix.
In Synechocystis, the PsbH protein was found not only in
the PSII core but also in the subcore (CP47±D1±D2±cyt)
Fig. 4. Eect of CO
2
depletion on t he reoxi-
dation of t he reduced PSII primary electron
acceptor Q
A
in wild-type and IC7 cells. Cul-
tures of wild-type (left p anel) and IC7 (right
panel) gr own in the presence of glucose
were bubbled in the d ark at 30 °Cwith
CO
2
-enriched air for 3 0 min (solid lines),
then with CO
2
-depleted air for another 30 min
(dotted line) and ®nally again w ith CO
2
-
enriched air f or 30 min (dashed line). The

kinetics of the Q
A
reoxidation was me asured
by P.S.I. ¯uorometer as described in Materials
and methods.
Fig. 5. Eect of CO
2
on the DMBQ-HRA and DCBQ-HRA during
illumination of IC7 cells. Culturegrowninthepresenceofglucosewas
bubbled with CO
2
-depleted (closed symbols) or CO
2
-enriched air
(open symbols) a t 30 °C for 10 min in the dark and then during illu-
mination with 1000 lmo l photonsám
)2
ás
)1
, aliquots of cells were taken
at the times indicated for measurement of DMBQ-HRA (circles) and
DCBQ-HRA (squares). Culture bubbled w ith C O
2
-enriched a ir co n-
tained 10 m
M
bicarbonate in addition. Means of a t least three mea-
surements are shown, s.e. did not exceed 8%. T he initial values of
DMBQ-HRA and DCBQ-HRA were 180  30 and 270  40 lmol
O

2
mg (chlorophyll)
)1
h
)1
, respectively.
Table 1. Carotenoid composition in cells of wild-type and IC7 strains grown in the absence of glucose. Numbers represent the percentage of the total
carotenoids, numbers in parenthesis represent the percentage of the particular carotenoids taking content per chlorophyll unit in wild-type cells as
100%.
Myxoxanthophyll Zeaxanthin Echinenon b-caroten
Wild-type cells 22 (100) 35 (100) 19 (100) 23 (100)
IC7 cells 47 (347) 23 (107) 13.5 (115) 16 (115)
Ó FEBS 2002 Function of the PsbH protein in photosystem II (Eur. J. Biochem. 269) 615
complex. In contrast, the PsbH protein was not detected in
the subcore isolated from spinach [37]. The reason for this
discrepancy is unclear, but it could be related to the
difference either between the species or between the methods
of the subcore preparation.
The instability of t he IC7 s ubcore during electrophoresis
of the isolated PSII core can be relevant to the situation
in vivo during the PSII core assembly. Weak binding of
CP47 to the D1±D2 heterodimer may destabilize these
subunits to the extent that they are degraded before the
whole c omplex can be assembled. This proteolysis seems to
be less ef®cient in cyanobacteria than that in algae and
therefore the assembly of PSII complexes occurs in the
psbH-deletion mutant of Synechocystis, but not in the
similar mutant of Chlamydomonas [6,7]. On the other hand,
the PsbH protein may also represent an important factor
regulating process of PSII repair. Its removal from PSII

could result in a complete disassembly of PSII during the D1
replacement w hile in its p resence the D1 replacement could
proceed in the subcore complex as suggested by Zhang et al.
[38].
We have identi®ed formation of the D1±cyto-
chrome b-559 adduct and the D1 fragments together with
the apparent oxidation of the D1 protein in the cells of IC7.
This shows that the impaired function of PSII in IC7 leads
to increased probability of the formation of ROS. These
species oxidize the D1 protein which can be subsequently
cross-linked with the a subunit of cytochrome b-559, or
even fragmented. However, ROS may also attack other
PSII subunits as well as protein synthesis apparatus and
then the r ecovery from photoinh ibition i s slow a s observed
in IC7 [13]. Oxidative damage was also implicated in the
slow restoration of PSII activity after photoinhibition of
Synechocyst is [39] and Synechococcus elongatus cells [23].
We were not able to accelerate r ecovery from photoinhibi-
tion in IC7 by bubbling the cell suspension with nitrogen
during high irradiance treatment. Nevertheless, this does not
negate our hypothesis as even under these conditions,
oxidation of the D1 protein still occurred although to a
lesser extent (Fig. 6).
Importance o f t he PsbH protein for the proper f unction-
ing of the PSII complex in h igher plants and algae is c losely
related to phosphorylation of its threonine residues on the
N-terminus. However, in Synechocystis we did not ®nd any
evidence for the phosphorylation of this protein. Looking at
the N-terminal s equences of PsbH in organisms containing
phycobilisomes attached to the stromal side of the mem-

brane (e.g. Synechocystis, Synechococcus, Porphyra and
Cyanidium), it is apparent that they contain the PsbH
protein with shorter N -terminal part w ithout the phosph o-
rylable threonines. As the common feature of these organ-
isms is the absence of grana, it is possible that the
Fig. 6. Oxidation , cross-linking and fragmen-
tation of the D1 protein during illumination of
the IC7 cells. Cells of wild-type and IC7 grown
in the presence of g lucose were illuminated
with 1000 lmol photonsám
)2
ás
)1
for 90 min,
and after breaking the cells m embrane
proteins were analysed by SDS/PAGE and
Western blotting. (A) Degradation and o x i-
dation of D1: D1  content of the 32-kDa
D1 band [anti-(D1-Mp) I g], 0.5 lg of c hloro-
phyll loaded per lane; Oxy D1  oxidation of
the 32-kDa D1 band ( an ti-DNP Ig), 5 lgof
chlorophyll loaded per lane. (B) Fragmenta-
tion and cross-linking of D1, 5 lg of c hloro-
phyll loaded per lane; D1  32 kDaD1band;
D1fr  N-terminal 23-kDa D1 f ragment;.
D1ad, cyt ad  41 kDa D1±a cyto-
chrome b-559 double band; cyt  a-subunit
of cytochrome b-559.
616 J. Komenda et al. (Eur. J. Biochem. 269) Ó FEBS 2002
phosphorylation of PsbH is important for the function of

PSII in the Se appressed regions of the membrane. In line
with this, G iardi et al. [40] showed that af ter PsbH
dephosphorylation by alkaline phosphatase an extremely
fast inactivation of the PSII activity occurred in isolated
spinach membranes while the phosphatase had n o effect on
the activity of the cyanobacterial membranes.
Xiong et al. [41] postulated a hypothesis suggesting that
arginine residues o f the D1 protein (especially Arg257)
stabilize b inding of bicarbonate on the PSII acceptor side.
However, similar role could be ful®lled by arginines of the
PsbH protein as suggested by Sundby et al. [42]. They found
that phosphorylation of the PsbH protein is indirectly
proportional to t he binding of bicarbonate on the acceptor
side of PSII. Based on this correlation they proposed that
the basic residues on the stromal side of the PsbH protein
are i nvolved in the bicarbonate binding. From this point of
view it is interesting that our results indicated destabilization
of the bicarbonate binding in PSII as a consequence of the
missing PsbH protein. However, it is not clear if the protein
binds bicarbonate directly or whether it has long-distance
effect on the conformation of D1 and/or D2 that is
important for the binding of this anion. It is worth to note
that fast light-induced inactivation of DMBQ-HRA, which
most probably re¯ects release of bicarbonate, has been also
found in the PEST-deletion mutant of Synechocystis by
Nixon et al. [43]. It may i ndicate that the PEST sequence of
the D1 protein is in close c ontact with PsbH and also
contributes to the formation of the bicarbonate binding site.
In line with this hypothesis our trypsinization experiment
showed that in the absence of the PsbH protein the P EST

region of the D1 p rotein was more exposed to stroma. The
fact that the release of bicarbonate completely inhib ited the
DMBQ-HRA but only slowed down the DCBQ-HRA
suggests that DCBQ may accept electrons before the
bicarbonate binding site. Therefore, it is tempting to
speculate that the difference between the active
(Q
B
-reducing) and inactive (Q
B
-nonreducing) PSII centres,
having distinct af®nity to DMBQ and DCBQ [44], is given
by the occupancy of the bicarbonate binding site and/or the
state of the PsbH protein (e.g. phosphorylation) that affects
the bicarbonate binding.
ACKNOWLEDGEMENTS
This work was supported by the projects no. LN00A141 of
The Ministry of Education, You th and Sports of the C zech Republic,
203/00/1257 of the Grant Agency of the Czech Republic and also
by Institutional Research Concept no . AV0Z5020903. W e thank
Dr K. Bezous
Ï
ka for the sequencing of the Psb H protein, Ms. Jana
Hofhanzlova
Â
forableassistanceandProf.J.Ma
Â
lek for critical reading
of the manuscript. We are grateful to Prof. J. Barber for a kind gift
of the IC7 mutant as well as Prof. E M. Aro, Dr Peter Nixon,

Dr A. Mattoo and Dr R. B arbato who donated speci®c antisera.
REFERENCES
1. Hankamer, B., Barber, J . & Boekema, E.J. (1997 ) Structure and
membrane organization of p hotosystem-II in green plants. Annu.
Rev. Plant Physiol. Plant Mol. Biol. 48, 641±671.
2. Kitamura, K., Ozawa, S., Shiina, T. & Toyoshima, Y. (1994)
L protein, e ncoded by psbL, restores normal functioning of t he
primary quino ne acce ptor, Q(A), in isolated D1/D2/CP47/Cytb-
559/I photosystem II reaction center c ore complex. FEBS Lett.
354, 113±116.
Fig. 7. Analysis of phosphorylation of the PsbH protein by Western blot using antiphosphothreonine antibodies (left panel) and by radioactive labelling
with
33
P-H
3
PO
4
(right panel). (A) Western blot. Membranes isolated from illuminated cells of green algae Chlorella (Chlo), Scenedesmus (Scen) and
Chlamydomonas (Chla), and from Synechocystis strains w ild-type and IC7 were solubilized with DM and subjected to PAGE in the presence o f
Deriphat 160 (8 lg of chlorophyll loaded per lane.). PSII core complexes were excised from the gel and further analysed by SDS/PAGE. Separated
proteins were transferre d o nto n it rocellul ose mem brane w hich was subsequently used f or immu nodetec tion u sing an tiphospho threonine rabbit
polyclonal antibodies (Zymed). (B) Autoradiogram. Membranes isolated from wild-type and IC7 cells were labelled with
33
P-H
3
PO
4
and analysed
by SDS/PAGE (5 lg of chlorophyll loaded per lane). Separated proteins were transferred onto nitrocellulose membrane, which was subsequently
used for autoradiography.

Ó FEBS 2002 Function of the PsbH protein in photosystem II (Eur. J. Biochem. 269) 617
3. Ikeuchi, M., Eggers, B., Shen, G.Z., Webber, A., Yu J.J.,
Hirano, A., Inoue, Y. & Vermaas, W. (1991) Cloning of the
psbK gene from Synechocystis sp. PCC-6803 and characterization
of photosystem-II in mutants lacking PSII-K. J. Biol. Chem. 266,
11111±11115.
4. Mayes, S.R., Dubbs, J.M., Vass, I., Hideg, E., Nagy, L. &
Barber, J. (1993) Further characterization of the psbH locus of
Synechocystis sp. PCC-6803 ± inactivation o f psbH impairs Q(A)
to Q(B) electron-transport in photosystem-2. Biochemistry 32,
1454±1465.
5. Ikeuchi, M., Shukla, V.K., Pakrasi, H.B. & Inoue, Y. (1995)
Directed inactivation of the psbI gene do es not a ect photosystem -
II in the cyanobacterium Synechocystis sp PCC 6803. Mol. General
Genet. 249, 6 22±628.
6. Summer, E.J., Schmid, V.H.R., Bruns, B.U. & Schmidt, G.W.
(1997) Requirement f or the H phosphoprotein in photosystem II
of Chlamydomona s reinhardtii. Plant Ph ysiol. 113, 1 359±1368.
7. O'Connor, H.E., Rue, S.V., Cain, A.J., Deak, Z., Vass, I.,
Nugent, J.H.A. & Purton, S. (1998) The 9-kDa phosphoprotein of
photosystem II. Generation and characterisation of Chlamydo-
monas mutant lacking PSII-H and a site-directed mutant lacking
the phosphorylation site. Biochim. Biophys. Acta 1364, 6 3±72.
8. Takahashi, Y., Matsumoto, H., Goldschmidt-Clermont, M. &
Rochaix, J.D. (1994) Dire cted disruption of the Chlamydomonas
chloroplast psbK gene destabilizes the photosystem II reaction-
center co mplex. Plant. Mol. B iol. 24, 7 79±788.
9. Bennet, J. (1977) Phosphorylation of c hloroplast membrane
polypeptides. Nature 269, 344±346.
10. Koike, H., Mamada, K., Ikeuchi, M. & Inoue, Y. (1989) Low

molecular mass proteins in cyanobacterial photosystem II: iden-
ti®cation of psbH and psbK gene products by N-terminal
sequencing. FEBS Lett. 244, 3 91±396.
11. Abdel-Mawgood, A.L. & Dilley, R.A. (1990) Cloning and
nucleotide sequence o f the psbH gene from cy anobacterium
Synechocystis 6803 . Plant Mol. Biol. 14, 445±446.
12. Mayes, S.R. & Barber, J. (1990) Nucleotide sequence of the psbH
gene of the cyanobacterium Synechocystis 6803. Nucleic Acid Res.
18, 194.
13. Komenda, J. & B arber, J. (1995) C omparison of psbO and psbH
deletion mutants of Synechocystis PCC 68 03 indicate that degra-
dation of D1 protein is regulated by the Q
B
site and is dependent
on protein s ynthesis. Biochemist ry 34, 9 625±9631.
14. Williams, J.G.K. (1988) Construction of speci®c mutations in
photosystem II photosynthetic reaction center by genetic engi-
neering. Methods E nzymol. 167, 7 66±778.
15. Pakrasi, H.B., Williams, J .G.K. & Arntzen, C.J. (1 988) Targeted
mutagenesis of the psb E and psbF genes blocks photosynthetic
electron transport: evidence for a fu nctional role of cytochrome
b559 in photosystem II. EMB O J. 7, 325±332.
16. Tang, X S . & D iner, B.A. ( 1994) Biochemical a nd spectroscopic
characterization of a new oxygen-evolving ph otosystem II core
complex from the cyanobacterium Synechocystis PCC 6803. Bio-
chemistry 33, 4594±4603.
17. Ritter, S., Komenda, J., S
Ï
etlõ
Â

kova
Â
,E.,S
Ï
etlõ
Â
k, I. & Welte, W. (1992)
Immobilized metal a nity chromatography for the separation o f
photosystems I and II from the thermophilic cyanobacterium
Synechococcus elongatus. J. Chr omatogr. 625, 21±31.
18. Laemmli, U.K. (1970) Cleavage of structural proteins during
the assembly of the head of bacteriophage T4. Nature 227,
680±685.
19. Bartos
Ï
,J.,Berkova
Â
,E.&S
Ï
etlõ
Â
k, I. (1975) A versatile chamber for
gas exchange measurements in suspensions of algae and chloro-
plasts. Photosynthetica 9, 395±406.
20. Wellburn, A.R. (1984) The spectral determination of chlorophyll-
a and chlorophyll-b, as well as carotenoids, using various solvents
with spectrophotometers of dierent resolution. J. Plant. Physiol.
144, 307±313.
21. Gilmore, M.A. & Yamamoto, H.Y. (1991) Resolution of lutein
and zeaxanthin using non-endcapped, lightly carbon-loaded C

18
high-performance liquid chromatograph ic column. J. Chromatogr.
543, 137±145.
22. Komenda, J. (2000) Role of two forms of the D1 protein in the
recovery from photoinhibition of pho tosystem II in the cyano-
bacterium Synechococcus PCC 7942. Biochim. Biophys. Acta 1457,
243±252.
23. Komenda, J. & Masojõ
Â
dek, J. (1995) Oxygen retards recovery
from phetoinhibition due to a secondary damage to the PSII
complex. In Photosynthesis: From Light to Biosphere (Mathis, P. ed)
Vol. IV, p p. 203±206. Kluwer Academic Press, The N etherlands.
24. Barbato,R.,Friso,G.,Rigoni,F.,Frizzo,A.&Giacometti,G.M.
(1992) Characterization of a 41 kDa photoinhibition adduct in
isolated photosystem II reaction centres. FEBS Lett. 309, 165±169.
25. Miyao,M.,Ikeuchi,M.,Yamamoto,N.&Ono,T.(1995)Speci®c
degradation of the D1 protein of photosystem II by treatment with
hydrogen peroxide in darkness: implications for the mechanism of
degradation of the D1 protein under illumination. Biochemistry
34, 10019±10026.
26. He, W.Z., Newell, W .R., Haris, P.I., Chapman, D. & Barber, J.
(1991) Protein secondary structure o f the isolated photosystem-II
reaction center and conf ormational ch anges studied by Fou rier-
transform infrared-spectroscopy. Biochemistry 30, 4 552±4559.
27. Dedner, N., Meyer, H.E ., Ashton , Ch & Wildner, G.F. (1988)
N-terminal sequence analysis of the 8 kDa protein in Chlamydo-
monas reinhardii. Localization of the phosphothreonine . FEBS
Lett. 236, 77±82.
28. Hamel, P., Olive, J., Pierre, Y., Wollman, F A. & De Vitry, C.

(2000) A new subunit of cytochrome b6f complex undergoes
reversible phosphorylation up on state transition. J. Biol. Chem.
275, 17072±17079.
29. Race, H.L. & Gounaris, K. (1993) Identi®cation of the psbH gene-
product as a 6-kDa phosphop rotein in the cyanobac terium
Synechocystis 680 3. FEBS Lett. 323, 3 5±39.
30. Michel, H.P., Hunt, D.F., Shabanowitz, J. & Bennett, J. (1988)
Tandem mass spectrometry reveals that three photosystem II
proteins of spinach chloroplasts contain N-acetyl-O-pho spho-
threonine a t their NH
2
termini. J. Biol. Chem. 263, 1123±1130.
31. Vener, A.V., Harms, A ., Sussman, M.R. & Vi erstra, R.D. (2001)
Mass spectrometric r esolution of reversible protein phosphoryla-
tion in photosynthetic membranes of Arabidopsis thaliana. J. Biol.
Chem. 276, 6959±6966.
32. Rintama
È
ki, E., Salonen, M., Suoranta, U.M., Carlberg, I.,
Andersson, B. & Aro, E.M. (1997) Phosphorylation of light-har-
vesting complex II and photosystem II core proteins shows
dierent irradiance-d ependent regulation in vivo ± application of
phosphothreonin e antib odies to analysis of thylakoid phospho-
proteins. J. Biol. Chem. 272, 30476±30482.
33. Packham, N.K. (1988) Is the 9 kDa thylakoid membrane phos-
phoprotein functionally and structurally analogous to the H
subunit of bacterial reaction centres? FEBS Lett. 231, 2 84±290.
34. Chiaramonte, S., Giacometti, G.M. & Bergantino, E. (1999)
Construction and characterization of a functional mutant of
Synechocystis 6803 harbouring an eukaryotic PSII-H subun it. Eur.

J. Biochem. 260, 833±843.
35. Zouni, A., Witt, H.T., Kern, J., Fromme, P., Krauss, N., Saenger,
W. & Orth, P. (2001) Crystal structure of photosystem II from
Synechococcus elongatus at 3.8 A
Ê
resolution. Nature 409, 739±743.
36. Buchel, C., Morris, E., Orlova, E. & Barber, J. (2001) Localisation
of the PsbH subunit i n photosystem II: a new approach using
labelling of His-tags with a Ni
2+
-NTA gold cluster and single
particle analysis. J. Mol. Biol. 312, 371±379.
37. Zheleva, D., Sharma, J., Panico, M., Morris, H.R. & Barber, J.
(1998) Isolation and characterization of monomeric and d imeric
CP47- reaction center photosystem II complexes. J. Biol. Chem.
273, 16122±16127.
618 J. Komenda et al. (Eur. J. Biochem. 269) Ó FEBS 2002
38. Zhang, L.X., Paakkarinen, V., Vanwijk, K.J. & Aro, E.M. (1999)
Co-translational assembly of the D1 protein into photosystem II.
J. Biol. C hem. 274, 16062±16067.
39. Constant, S., Perewoska, I., Alfonso, M. & Kirilovsky, D. (1997)
Expression of the psbA gene during photoinhibition and recovery
in Synechocystis PCC 6714: inhibition and damage of transcrip-
tional and translational machinery prevent the restoration of
photosystem II activity. Plant M ol. Biol. 34, 1 ±13.
40. Giardi, M.T., Komenda, J. & Mas ojidek, J. (1994) Involvement
of protein phosphorylation in the sensitivity of photosystem II to
strong illumination. Physiol. Plantarum 92, 181±187.
41. Xiong, J ., Subramaniam, S. & Govindjee (1996 ) Modeling of t he
D1/D2 proteins and cofactors of the photosystem II reaction

center: implications for herbicide and bicarbonate binding. Protein
Sci. 5, 2054±2073.
42. Sundby, C., Ulla, K., Henrysson, L. & Henrysson, T. (1989)
Eects of bicarbonate on thylakoid protein phosphorylation.
Biochim. Biop hys. Acta 975 , 277±282.
43. Nixon, P.J., Komenda, J., Barber, J., Deak, S., Vass, I. & D iner,
B.A. (1995) Deletion of the PEST-like region of p hotosystem II
modi®es the QB-binding pocket but does not p revent rapid
turnover of D1. J. Biol. C hem. 270, 14919±14927.
44. Graan, T. & Ort, D.R. (1986) Detection of oxygen evolving
photosystem II centers in active in plastoquinone redu ction. Bio-
chim. Biophys. A cta 852, 320±330.
Ó FEBS 2002 Function of the PsbH protein in photosystem II (Eur. J. Biochem. 269) 619