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 IIcomplex and bicarbonate binding on its acceptor side Josef Komenda1,2, Lenka LupõÂnkovaÂ1,2and JirÏõÂ KopeckyÂ1,2 1 Photosynth

Absence of the psbH gene product destabilizes photosystem II

complex and bicarbonate binding on its acceptor side

Josef Komenda1,2, Lenka LupõÂnkovaÂ1,2and JirÏõÂ KopeckyÂ1,2

1 Photosynthesis Research Centre, University of South Bohemia, CÏeske BudeÏjovice, Czech Republic; 2 Laboratory of Photosynthesis, Institute of Microbiology, Academy of Sciences, TrÏebonÏ, 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±cyt b-559) from the

wild-type strain of the cyanobacterium Synechocystis PCC

6803 The protein was missing in the

D1±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 CO2 resulted in a reversible decrease of

the Qÿ

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 HCO3 concentration in the IC7 cells; and (d) illumina-tion of the IC7 cells lead to an extensive oxidaillumina-tion, frag-mentation and cross-linking of the D1 protein We did not

®nd any evidence for phosphorylation of the PsbH protein in the wild-type strain The results showed that in the PSII complex of Synechocystis attachment of CP47 to the D1±D2 heterodimer appears weakened and 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 and a number of low molecular mass

proteins It is believed that with an exception of

cyto-chrome b-559, the small proteins do not participate 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 role of small PSII subunits One approach is based on

the functional comparison of the intact and detergent

treated PSII complex missing a speci®c subunit

Resump-tion of the particular funcResump-tion after reconstituResump-tion of the

complex with this subunit is considered as evidence for its

role For example, using this approach the role of the PsbL

subunit in the QA 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 rarely 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, was initially found as the 10-kDa phosphoprotein in thyla-koids of higher plants by Bennett [9] From that time its homologues have been found in more than 15 photo-synthetic organisms including cyanobacteria The ®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 PCC 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 QA and QB 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

mlyÂn, 379 81 TrÏebonÏ, Czech Republic Fax: + 420 333 721246,

Tel.: + 420 333 721101, E-mail: komenda@alga.cz

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 15 August 2001, revised 16 November 2001, accepted 20

November 2001)

M A T E R I A L S A N D M E T H O D S

Strains and growth of organisms

The glucose tolerant strain Synechocystis PCC 6803 [14],

referred to as wild-type (WT), and its psbH deletion mutant

IC7 [4] were grown in BG-11 medium with

(photomixo-trophic growth) or without (photoauto(photomixo-trophic growth)

glucose (10 mM ®nal concentration) The plate medium

contained BG-11, 10 mM Tes/NaOH, pH 8.2, 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)2ás)1 of white light at

29 °C and diluted every day to maintain the chlorophyll

concentration at  8 lgámL)1 Cultures of Chlorella

soro-kiniana, Scenedesmus quadricauda and Chlamydomonas

reinhardtii were grown under the same conditions and their

density was maintained at D753ˆ  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% CO2 (CO2-enriched air), with air bubbled

through 40% NaOH (CO2-depleted air) or with pure

nitrogen In some experiments, the cell suspension was

supplemented with 10 mMNaHCO3 The light source was a

500-W tungsten ®lament bulb mounted in an aluminium

re¯ector In the experiments with 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 D753ˆ 0.2 They were exposed to 250 lmol

pho-tonsám)2ás)1of white light for 30 min either in the absence or

in the presence of 3 lCiámL)1 33P-H3PO4 Thylakoids

isolated from the cells were analysed by SDS/PAGE and

Western blotting using rabbit polyclonal

antiphosphothre-onine antibody (Zymed, USA) or by autoradiography

Preparation of membranes and PSII complexes

and their trypsinization

Cyanobacterial membranes were prepared by breaking the

cells with glass beads (150±200 lm in diameter) at 4 °C

followed by differential centrifugation For small scale

preparation, the cells (approx 150 lg of chlorophyll) were

washed and resuspended in 150 lL of 25 mM Tris/HCl

buffer, pH 7.5 containing 1 mM 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 lL of

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 mM Tris/HCl buffer,

pH 6.8 containing 1M sucrose (®nal chlorophyll

concen-tration 400±600 lgámL)1) and stored at )75 °C Large scale

preparation of membranes for isolation of PSII was

performed according to Tang & Diner [16] using a

beadbeater (Biospec Products, USA) for breaking the cells

Isolation of PSII complexes from the wild-type and mutant thylakoids was conducted according to the modi®ed procedure of Ritter et al [17] Brie¯y, membranes were spun down, resuspended in 25 mMMes/NaOH, pH 6.5 and solubilized with dodecylmaltoside (DM/chlorophyll ˆ 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 Cu2+ions and imidazole equilibrated with two column volumes of 25 mM Mes/ NaOH, pH 6.5 containing 200 mMNaCl 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 mMMes/NaOH, pH 6.5 con-taining 200 mM NaCl and 0.03% DM During this step, carotenoids and remaining small amounts of phycocyano-biliproteins and PSI were removed from the column Finally, the PSII core complex was eluted from the column

by 25 mM Mes/NaOH, pH 6.5 containing 250 mM 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 200 lgámL)1and trypsin concentration

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 mM Pefabloc SC (Merck, Germany)

Analysis of proteins Isolated PSII complexes or membranes solubilized with DM (DM/chlorophyll ˆ 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 mM Tris, 98 mMglycine and 0.1% Deriphat 160, and the gel contained 0.1MTris/HCl, pH 8.8 without detergent

Protein composition of membranes and pigment protein complexes obtained by Deriphat electrophoresis was assessed by electrophoresis in a denaturing 12±20% linear gradient polyacrylamide gel containing 7Murea [18] The membranes were solubilized in 25 mM 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 of pigment proteins was performed 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 pigment 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 Coomassie Blue or transferred onto nitrocellulose membrane (0.1 lm, Schleicher-Schuel, Ger-many) by semidry blotting Membrane was incubated with speci®c antibodies and then with alkaline phosphatase conjugated secondary antibody (Sigma) Proteins were visualized by colorimetric reaction using BCPIP-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 58±86 of the spinach D1 protein (D1-Mp); (c) the last 29 residues of the pea

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 the

detection kit Oxyblot (Intergen, USA) Solubilized

thyla-koid membrane proteins were derivatized using

dini-trophenylhydrazine, which reacts with carbonyls present

on oxidized proteins After protein separation by 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 automated Edman degradations using

Protein sequencer LF3600D (Beckman, USA) and program

2±39 according to manufacturer's instructions Amino-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 0.6MHCl 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 oxygen evolution (Hill

reaction activity, HRA) in cell suspensions were measured

at 30 °C using a temperature controlled chamber

[19] equipped with a Clark-type electrode (YSI, USA)

Arti®cial electron acceptors 2,5-dimethyl-p-benzoquinone

(DMBQ) or 2,6-dichloro-p-benzoquinone (DCBQ) (0.5 mM

®nal concentration each) were added 1 min before the

measuring illumination (3500 lmol photonsám)2ás)1, 30 s)

was switched on

Chlorophyll ¯uorescence measurement

The rate of Qÿ

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 measuring light and FMre¯ecting fully reduced

QAwas 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 with

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 [21]

R E S U L T S

Identi®cation of the PsbH protein in photosystem II complexes of Synechocystis

PSII core complexes from wild-type and IC7 strains of Synechocystis 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 PSII 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 second 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 complex

Comparison of the protein composition of the PSII cores and subcores (Fig 1B) from both strains showed that there was a protein with Mrof  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 the 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 lacking CP43 Evaluation

of its presence in PSIIRC was allowed by the treatment of the wild-type preparation with SDS in the ratio SDS/ chlorophyll ˆ 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 CP47 Effect of the PsbH absence on the accessibility

of the D1 protein to trypsin The effect 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 Synechococcus PCC 7942, this cutting occurred

concomi-tantly with breakdown of the D2 protein at residue R234

and there was also trypsin-induced formation of the 35-kDa

adduct of the 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 also appeared As judged 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 as indicated by inhibition of the D1± D2 adduct formation in the IC7 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 QA and QB [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 CO2 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 CO2-enriched air (Fig 4, right panel) In contrast, removal of CO2and its subsequent addition did not affect the Qÿ

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 CO2-depleted air (Fig 5, closed symbols) However, when the suspension was supplemented with 5 mM bicar-bonate and bubbled with CO2-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-type cells was not dependent on the CO2and/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 strain could be an increased formation of reactive oxygen species (ROS) in PSII that may inhibit the 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 of Deriphat 160, 8 lg of chlorophyll loaded per lane.

(B) Protein composition of pigment proteins obtained by native

Deri-phat/PAGE of PSII complexes isolated from wild-type and IC7: A,

PSII core ˆ PSII complex containing at least CP47, CP43, D1, D2 and

cytochrome b-559; B, PSII subcore ˆ PSII core complex lacking CP43;

C, PSIIRC ˆ D1±D2±cytochrome b-559 complex; D, CP47, CP43 ˆ

free pigment proteins CP47 and CP43; and FP ˆ free pigments.

ROS in the PSII complex of IC7 was further supported by

the results of the D1 analysis in cells exposed to high

irradiance The Western blot showed, in addition to the

typical 32-kDa D1 band, 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 additional, 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 intensity of the original 32-kDa band and in the case of IC7 mobility of the remaining protein was decreased in an oxygen-dependent manner Such a shift often re¯ects protein oxidation [26] and this was con®rmed by Oxyblot, a commercially 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 ofSynechocystis 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, the 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 band 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 of 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 ˆ 1 or 10 by the native PAGE in the presence of Deriphat 160

in the ®rst dimension (8 lg of chlorophyll loaded per lane) and SDS/PAGE in the sec-ond dimension according to 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 proteins was probed with a

mix-ture of anti-(D1-Mp) Ig and anti-(D1-Ct) Ig according to Materials

and methods Nt1 ˆ fragment of D1 between R8 and K238;

Nt2 ˆ fragment of D1 between R8 and R225; Ct1 ˆ fragment of D1

between K238 and A344; Ct2 ˆ fragment of D1 between R257 and

A344; and D1D2 adduct ˆ adduct between D1 (R8-A344) and

frag-ment of D2 (R234-L352).

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 identify 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 33P-H3PO4 We found weak phosphorylation of two bands with Mrvalues of 3 and

4 kDa clearly distinct from the PsbH protein (Fig 7B)

In addition, these two bands were present 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 Synechocystis cells

D I S C U S S I O N

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 also in line with the data in this paper The recent 3.8 AÊ model of PSII [35] tentatively situated the membrane helix of PsbH on the side of CP47 and D2 in the proximity of the QA±QBregion This position provides a good justi®cation for the stabilizing effect of PsbH on 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 the

reoxi-dation of the reduced PSII primary electron

acceptor Q A in wild-type and IC7 cells

Cul-tures of wild-type (left panel) and IC7 (right

panel) grown in the presence of glucose

were bubbled in the dark at 30 °C with

CO 2 -enriched air for 30 min (solid lines),

then with CO 2 -depleted air for another 30 min

(dotted line) and ®nally again with CO 2

-enriched air for 30 min (dashed line) The

kinetics of the Q A reoxidation was measured

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 Culture grown in the presence of glucose was

bubbled with CO 2 -depleted (closed symbols) or CO 2 -enriched air

(open symbols) at 30 °C for 10 min in the dark and then during

illu-mination with 1000 lmol 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 with CO 2 -enriched air

con-tained 10 m M bicarbonate in addition Means of at least three

mea-surements are shown, s.e did not exceed 8% The 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%.

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 the IC7 subcore 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 complex 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 while in its presence 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 recovery from photoinhibition is slow as observed

in IC7 [13] Oxidative damage was also implicated in the slow restoration of PSII activity after photoinhibition of Synechocystis [39] and Synechococcus elongatus cells [23]

We were not able to accelerate recovery 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 of the PsbH protein for the proper function-ing of the PSII complex in higher plants and algae is closely 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 sequences 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 without the phospho-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 glucose were illuminated with 1000 lmol photonsám )2 ás )1 for 90 min, and after breaking the cells membrane proteins were analysed by SDS/PAGE and Western blotting (A) Degradation and oxi-dation of D1: D1 ˆ content of the 32-kDa D1 band [anti-(D1-Mp) Ig], 0.5 lg of chloro-phyll loaded per lane; Oxy D1 ˆ oxidation of the 32-kDa D1 band (anti-DNP Ig), 5 lg of chlorophyll loaded per lane (B) Fragmenta-tion and cross-linking of D1, 5 lg of chloro-phyll loaded per lane; D1 ˆ 32 kDa D1 band; D1fr ˆ N-terminal 23-kDa D1 fragment; D1ad, cyt ad ˆ 41 kDa D1±a cyto-chrome b-559 double band; cyt ˆ a-subunit

of cytochrome b-559.

phosphorylation of PsbH is important for the function of

PSII in the Se appressed regions of the membrane In line

with this, Giardi et al [40] showed that after PsbH

dephosphorylation by alkaline phosphatase an extremely

fast inactivation of the PSII activity occurred in isolated

spinach membranes while the phosphatase had no effect on

the activity of the cyanobacterial membranes

Xiong et al [41] postulated a hypothesis suggesting that

arginine residues of the D1 protein (especially Arg257)

stabilize binding 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 the 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 involved 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 indicate that the PEST sequence of

the D1 protein is in close contact 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 PEST

region of the D1 protein was more exposed to stroma The

fact that the release of bicarbonate completely inhibited 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 (QB-reducing) and inactive (QB-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

A C K N O W L E D G E M E N T S

This work was supported by the projects no LN00A141 of The Ministry of Education, Youth and Sports of the Czech Republic, 203/00/1257 of the Grant Agency of the Czech Republic and also

by Institutional Research Concept no AV0Z5020903 We thank

Dr K BezousÏka for the sequencing of the PsbH protein, Ms Jana Hofhanzlova for able assistance and Prof J MaÂlek 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 Barbato who donated speci®c antisera.

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