Isolation and characterization of photosystem II subcomplexes from cyanobacteria lacking photosystem I

Isolation and characterization of photosystem II subcomplexes fromcyanobacteria lacking photosystem I

Ildiko Szabo1, Fernanda Rigoni1, Maria Bianchetti2, Donatella Carbonera3, Francesca Pierantoni1,Roberta Seraglia4, Anna Segalla1 and Giorgio M. Giacometti1

1Department of Biology and 2Department of Environmental Sciences, University of Tuscia, Viterbo, Italy; 3Department of Physical

Chemistry, University of Padova, Italy; 4CNR Research Area, Corso Stati Uniti 4, Padova, Italy

A photosystem II (PSII) core complex lacking the internal

antenna CP43 protein was isolated from the photosystem II

of Synechocystis PCC6803, which lacks photosystem I

(PSI). CP47–RC and reaction centre (RCII) complexes were

also obtained in a single procedure by direct solubilization

of whole thylakoid membranes. The CP47–RC subcore

complex was characterized by SDS/PAGE, immunoblotting,

MALDI MS, visible and fluorescence spectroscopy, and

absorption detected magnetic resonance. The purity and

functionality of RCII was also assayed. These preparations

may be useful for mutational analysis of PSII RC and

CP47–RC in studying primary reactions of oxygenic

photosynthesis.

Keywords: CP47–RC subcomplex; cyanobacteria; photo-

system II.

The photosystem II (PSII) of oxygenic photosyntheticorganisms is composed of more than 25 protein subunits andbinds a large number of pigments. Primary chargeseparation occurs at the reaction centre (RCII) consistingof D1, D2, a and b subunits of cytochrome b559, PsbI andPsbW [1–3]. CP47 and CP43, the two internal antennachlorophyll a-binding proteins are closely associated withthe D1/D2 heterodimer. The main light-harvesting system isformed of chlorophyll a/b binding proteins in higher plantsand green algae, and of phycobilisomes in red algae andcyanobacteria. A ‘core complex’ consisting of D1, D2, CP47,CP43 and other subunits may be obtained by removing themain light harvesting system from PSII. Treatment of thePSII core complex with detergents can ‘peel’ away varioussubunits. Recent structural studies employing electronmicrographic techniques have revealed that the CP43protein is located closer to the surface of the complex thanCP47 [4]. Indeed, CP43 is more easily removed duringdetergent treatment, at least in higher plants, allowingisolation of the CP47–RC complex. RCII and CP47–RCfrom higher plants have been prepared according to severaldifferent procedures [1,5–8].

The isolation and characterization of these PSII coresubcomplexes from wild-type and mutant higher plantsrepresents an important step towards our understanding ofthe structure–function relationship of PSII. However, site-directed mutagenesis of PSII in plants is hampered by the

difficulty of chloroplast transformation. The unicellularcyanobacterium Synechocystis PCC6803 is generally usedas a model organism for the study of PSII. Of importance isthe fact that this species is able to grow photoheterotrophi-cally and is easily transformable, allowing the constructionof mutants defective in PSII function [9]. However, theirthylakoid membrane contains five times more PSI than PSII,hindering both biophysical analysis and the purification ofPSII. This problem has been resolved by constructing amutant in which part of the psaAB operon coding for thecore proteins of PSI is deleted [10]. PSI-less cells haveapproximately six times more PSII than wild-type cells, asestimated on a chlorophyll basis [10].

Attempts to isolate CP43-less PSII core complexes [11] aswell as RCII from cyanobacteria [12–14] have so far beenperformed by using PSI-containing wild-type organisms.Separation of PSI from PSII or from PSII subcomplexesrequires time-consuming and complicated procedures, andthe isolated subcomplexes often cannot be obtained insufficient quantity and purity.

The present work reports the isolation and characteriz-ation of CP47–RC and RCII from PSI-less cyanobacteria.The lack of PSI allowed us to obtain PSII subcomplexesdirectly from both thylakoid membranes and PSII.

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

Preparation of thylakoid membranes and PSII fromPSI-less Synechocystis

Thylakoids were isolated as previously described [15].Briefly, cells were grown at 30 8C photoheterotrophicallyin BG11 medium supplemented with 5 mM glucose at5 mE:m22:s21 light intensity. At D730 ¼ 0.8, cells wereharvested by centrifugation (2000 g for 5 min at 4 8C) andresuspended in a breaking buffer containing 20 mM Mes,5 mM MgCl2, 5 mM CaCl2, 1 mM benzamidine, 1 mM

aminocaproic acid and 25% glycerol (pH ¼ 6.35 withNaOH). All subsequent steps were carried out at 4 8C. Cellswere broken on ice using a bead beater (Biospec) by

Correspondence to G. M. Giacometti, Department of Biology,

University of Padova, Viale G. Colombo 3, 35121 Padova, Italy.

Fax: 1 39 049 8276344, Tel.: 1 39 049 8276326,

E-mail: [email protected]

(Received 22 June 2001, accepted 6 August 2001)

Abbreviations: ADMR, absorption detected magnetic resonance; CP,

chlorophyll-protein; DM, n-dodecyl-[b-maltoside]; CP47–RC,

CP47-D1-D2-cytochrome b559 complex; PSI, photosystem I; PSII,

photosystem II; RCII, reaction centre comprising D1, D2, cytochrome-

b559 and PsbI proteins; T–S, triplet minus singlet; ZFS, zero field

splitting.

Eur. J. Biochem. 268, 5129–5134 (2001) q FEBS 2001

application of 15 30-s cycles at 5-min intervals. Unbrokencells were eliminated (5 min at 2000 g) and thylakoids werepelleted at 140 000 g for 30 min. Pellets were washed andresuspended in buffer B [50 mM Mes and 20 mM Na4P2O7

(pH ¼ 6.5)]. Isolated thylakoids were solubilized with 2%(w/v) n-dodecyl-(b-maltoside) (DM) for 30 min and loadedon a continuous 0–0.5 M sucrose gradient containing25 mM Mes, 10 mM NaCl, 5 mM CaCl2 and 0.03% DM(pH ¼ 6.5). Three bands, i.e. carotenoids, cytochrome b6/fand PSII, separated out after 18 h of centrifugation at130 000 g. The PSII band was collected and stored at280 8C.

Absorption spectra and chlorophyll a content

Absorption spectra and chlorophyll a content calculatedaccording to McKinney [16], were measured using aPerkinElmer Jasco 7800 spectrophotometer.

FPLC

A MonoQ HR5/5 column was equilibrated with buffer F(20 mM Bistris, pH ¼ 6.5, 20 mM NaCl, 10 mM MgCl2,1.5% taurine and 0.1% DM). The same buffer was used forsample loading.

SDS/PAGE and Western blotting

SDS/PAGE was performed as previously described [17],using a running gel containing 12–17% linear acrylamidegradient in the presence of urea (6 M) if not otherwisespecified. Proteins separated by SDS/PAGE were transferredelectrophoretically to poly(vinylidene difluoride) mem-branes in the carbonate buffer [18]. Blots were probed withpolyclonal antibodies, raised against Synechocystis D2 [15],wheat D1, and maize CP43 and CP47 [17]. Blots weredeveloped by the ECL chemiluminescence system (PharmaciaAmersham).

MALDI MS

Measurements were performed on a REFLEX time-of flightinstrument (Bruker-Franzen Analytik, Bremen, Germany).Ions formed by a pulsed UV nitrogen laser beam(l ¼ 337 nm) were accelerated to 25 kV. Sinapinic acidwas used as a matrix. Samples, were diluted in a 0.1%trifluoroacetic acid aqueous solution. For low molecularmasses (2–20 kDa), pulsed ion extraction was carried outapplying a voltage of 22.3 kV for 300 ns to the second gridand for high molecular masses (20–80 kDa) a voltage of21.9 kV for 400 ns was applied. External mass calibrationwas carried out using (M 1 H)1 ions of bovine insulin,horse myoglobin and bovine serum albumin.

Fluorescence emission spectra

Fluorescence emission spectra were recorded at 77 K at anexcitation wavelength of 437 nm with a PerkinElmerspectrofluorometer supplied by a liquid nitrogen unit. Cellsand CP47–RC were diluted to concentrations of 20 mg and2 mg Chl per mL, respectively. No glycerol was added to thesamples before measurements. The excitation and emissionslit widths were 10 and 2.5 nm, respectively.

Absorption detected magnetic resonance (ADMR)

The description of the home-built apparatus for opticallydetected magnetic resonance in a set-up that detects absor-bance changes and the triplet minus singlet (T–S) spectrawas described previously [19]. CP47–RC was diluted to afinal concentration of 2 mg Chl per mL. Glycerol (60%, v/v)was added to the samples immediately before measurementsto avoid matrix cracking, and oxygen was removed using aglucose/glucose oxidase system [20]. In some experiments,illumination at room temperature of samples, prereduced by20 mM dithionite, was performed to allow the full reductionof secondary acceptors and the formation of recombinationtriplet state located in the primary donor [19,21]. Sampleswere illuminated for 2 min by a 150-W projector lampfiltered by a 10-cm water tube and hot filters. All measure-ments were performed at 1.8 K, using 33 Hz modulationfrequency for amplitude-modulated microwaves.

HPLC

Fractions eluted from FPLC and displaying an absorbancespectra compatible with CP47–RC and RCII complexesfrom different experiments were pooled and their pigmentswere extracted by adding 9 vol. of pure (HPLC grade),acetone. After centrifugation, the supernatant was filteredthrough a 0.2-mm filter and 20-mL aliquots were analyzedon a Zorbax ODS RP-HPLC column. Samples were elutedisocratically by methanol/ethyl acetate (68 : 32, v/v) at aflow rate of 1 mL:min21. Absorbance was monitored at 410and 430 nm. For each sample the amount of chlorophyll aand pheophytin were determined by integration of thecorresponding peaks and by comparison with those ofstandard pigment solutions.

R E S U L T S

We first isolated CP47–RC from the PSII of PSI-lessSynechocystis. PSII, prepared as described above, wasdiluted to 90 mg Chl per mL and solubilized for 35 min onice in the dark, with a 5% (w/v) final concentration of thenonionic detergent DM. The CP47–RC complex wasseparated from the other solubilized PSII componentsthrough an analytical anion-exchange column. Elutioninvolved a linear MgSO4 gradient 0–200 mM MgSO4 inbuffer F containing 0.1% DM (see Materials and methods).The elution profile comprised a single peak for CP47–RC(eluted with 105–125 mM MgSO4) (not shown). The elutionprofile and the characteristics (listed below) of the resultingcomplex were reproducible for three different preparations.

The polypeptide profile of PSII and CP47–RC wasdetermined by full length SDS/PAGE (Fig. 1A) and byusing antibodies against the main components of PSII, i.e.D1, D2, CP47 and CP43. PSII used as a control containedall four proteins, whereas in CP47–RC only a negligiblereaction with anti-CP43 could be detected (Fig. 1B).

To better characterize the subunit composition of thepurified complex, we applied MALDI MS directly to thecomplex, without purification of the single subunits priorto analysis. The MALDI spectra of the CP47–RC in the20–100 kDa range (Fig. 2B) was compared with that of thePSII core (Fig. 2A). m/z Peaks at 38 070, 39 300, 51 400and 55 500 Da have recently been attributed to D1, D2,

5130 I. Szabo et al. (Eur. J. Biochem. 268) q FEBS 2001

CP43 and CP47, respectively, by comparing the molecularmasses obtained by MALDI with the protein massescalculated from the nucleotide sequences of the PSIIcomponents [22]. The peak corresponding to CP43 is almostcompletely absent in the spectra of CP47–RC, whereas therelative intensities of peaks attributed to D1, D2 and CP47remain basically unaltered. The difference in the amplitudesof the peaks assigned to CP47 and CP43 does not reflect astoichiometry different from 1 : 1 but is rather due to adifferent cross section for ionization, caused by the moreexternal position of CP43 respect to CP47 in the PSIIcomplex [22]. Figure 2C shows the proteins of the CP47–RC complex in the 2- to 20-kDa range. On the basis ofthe expected, calculated molecular masses, peaks at 4340,4806, 5739, 9324 and 12475 Da may be attributed to PsbI,the a subunit of cytochrome b559, psbW, the b subunit ofcytochrome b559, and the precursor of PsbW, respectively.The 8004-Da peak probably corresponds to a subunit of ATPsynthase (calculated mass: 7968 Da).

The room temperature absorbance spectra of the PSII coreand CP47–RC isolated subcomplex are shown in Fig. 3. Inboth cases, the maximum absorbance in the visible spectrumis at 675 nm. In the Soret region, the more pronouncedband at 417 nm in the subcomplex preparation withrespect to PSII reflects, as expected, a higher pheophytinto chlorophyll ratio, due to the absence of the chlorophyllmolecules bound to CP43. The A417/A436 ratio is 0.938 forthe CP47–RC, and ranged between 0.92 and 0.94 for thevarious preparations.

In order to avoid the cumbersome purification of PSIIthrough the sucrose gradient, PSI-less thylakoids (at80 mg:mL21 chlorophyll concentration), solubilized with10% final concentration of DM for 30 min, were used in anattempt to purify CP47–RC in a single step. The sameprocedure was performed as for PSII (see above), andresulted in the reproducible (n ¼ 3) elution profile shown inFig. 4A. This was characterized by relatively well-resolvedpigmented protein peaks (II–V), overlapping a broadunpigmented band (I). The fractions eluted between 25 and

Fig. 2. Polypeptide composition of PSII and CP47–RC by MALDI

MS. MALDI spectra of PSII (A) and CP47–RC (B) in 20–60 kDa

range, and CP47–RC in 2- to 20-kDa range (C).

Fig. 1. Polypeptide content of PSII and CP47–RC. (A) Coomassie staining of the SDS/PAGE (12%) of purified PSII (lane a) and CP47–RC (lane

b) complexes isolated from PSI-less Synechocystis. The < 55-kDa band above CP47 and < 35-kDa band between D2 and CP43 probably correspond

to the a/b and g subunits of the ATPase, respectively [27]. CP47 is present as a multiple band, as reported previously [6,11,27]; the reason for this is

unknown. (B) Immunoblots of PSII (lanes 1–3, 5–7, 9–11, 13–15) and CP47–RC complex (lanes 4,8,12,16). Lanes 1–4, 5–8, 9–12 and 13–16

were probed with antibodies specific for D1, D2, CP43 and CP47, respectively. PSII samples were loaded at 0.25 (lanes 1,5,9,13), 0.5 (lanes

2,6,10,14) and 1 mg Chl (lanes 3,7,11,15).

q FEBS 2001 RCII and CP47–RC from Synechocystis PCC 6803 (Eur. J. Biochem. 268) 5131

30 min (IV) and 30–35 min (V) exhibited absorptionspectra typical for RCII and CP47–RC, respectively.

Western blots shown in Fig. 4B were performed in orderto evaluate the D1, D2, CP47 and CP43 protein content ofthe fractions IV and V. Fraction IV retained a substantialamount of CP47, but not CP43, while fraction V lacked bothCP47 and CP43. Figure 5A shows the absorbance spectra ofthe two fractions of Fig. 4B. On the basis of polypetidefeatures and absorbance spectra, fractions IV and V were

identified as RCII and CP47–RC, respectively. Thechlorophyll a content, evaluated by HPLC, was 6.5 ^ 0.5and 22 ^ 1.2 (n ¼ 3) for RCII and CP47–RC, respectively.Figure 5B shows the fluorescence spectra of whole cells,PSII and RCII and CP47–RC subcomplexes at 77 K. In thecase of whole cells, the main peak is centered at 685 nm andan intense shoulder is also found at 695 nm. The CP47–RCfraction shows a fluorescence spectrum that lacks the695 nm shoulder and is blue-shifted by 3 nm compared withthe 685 nm maximum found in whole cells and PSII.

ADMR measurements were performed to test the func-tionality of the obtained CP47–RC complex (Fig. 5C).Continuous illumination of untreated CP47–RC samplesinduced a population of triplet states in high yield. Tripletstates may be detected by either fluorescence or absorptionchanges at their resonance frequencies. The assignment ofthe observed triplet states to P680 can be easily carried out

Fig. 4. Preparation of RCII and CP47–RC from thylakoids. (A)

Chromatographic profile of solubilized thylakoid membranes from PSI-

less Synechocystis. Elution from an anion-exchange column involved a

linear MgSO4 gradient at 0.5 mL:min21 flow rate. (B) Immunoblots of

thylakoid membranes from PSI-less Synechocystis (lanes 1–3), RCII

(lane 4) and CP47–RC (lane 5), probed by antibodies against D1, D2,

CP43 and CP47. The SDS/PAGE profile of the RCII and CP47–RC

complexes (not shown) is in agreement with the blot.

Fig. 5. Spectroscopic characterization of RC and CP47–RC. (A)

Absorbance spectra of fraction IV (CP47–RC) (solid line) and fraction

V (RCII) (dotted) isolated from thylakoid membranes. (B) Fluorescence

spectra at 77 K of whole cells, PSII, CP47–RC and RCII. (C) T–S

spectrum of CP47–RC recorded at 730 MHz. Inset: ADMR spectra

recorded at 682 nm. Mod. freq. 33 Hz; microwave power, 13 mW;

temperature 1.8 K.

Fig. 3. Room-temperature absorption spectra of CP47–RC (A)

and PSII (B). Spectrum (B) has been shifted upward by 0.02 units.

5132 I. Szabo et al. (Eur. J. Biochem. 268) q FEBS 2001

by comparison with previous results [19] concerning ZFSparameters (|D| ¼ 0.0287 cm21, |E| ¼ 0.00442 cm21)derived from resonance frequencies in the ADMR (inset)and T–S spectra. Pre-treatment with a reducing agentfollowed by preillumination of the sample at roomtemperature did not increase triplet yield. ADMRmeasurements also revealed the formation of triplet statesin the RCII complex contained in band IV (not shown).

D I S C U S S I O N

The CP47–RC complex from higher plants has beensuccessfully isolated by several different procedures.Ghanotakis et al. [5] isolated RC and CP47–RC complexesby treating PSII core complexes with DM in combinationwith high concentrations of lithium perchlorate; Dekkeret al. [8] used a modified version of the same procedure.Zheleva et al. [6] recently obtained both monomeric anddimeric forms of CP47–RC using DM together with heptylthioglycopyranoside for solubilization. However, in ourexperiments, none of these procedures yielded the pureCP47–RC complex when applied to cyanobacteria. Thesubcomplexes obtained from higher plants have beenextensively characterized, for example by EPR spectroscopy[7,23], fluorescence and thermoluminescence measurements[7], immunoblotting and electrospray ionization MS [6].Two-dimensional crystals of the complex have also beenobtained and studied by electron microscopy [24].

Much less information is available concerning the CP47–RC subcomplex isolated from Synechocystis. In the onlyreport so far [11], two mutants of Synechocystis PCC6803lacking the psbC gene product CP43 were constructed.However, they only contained < 10% of the PSII reactioncentres of wild-type cells, hampering purification of the corecomplex to homogeneity. Protein profiles and the absor-bance and fluorescence spectra of CP47–RC have beendescribed previously [11]. The protein content of theCP47–RC complex obtained here was assayed by SDS/PAGE (Fig. 1A), immunoblot (Figs 1B and 4B) andMALDI MS (Fig. 2B,C). The chlorophyll a content,determined by HPLC (22 ^ 1.2), is slightly higher thanthat expected (20) on the basis of a recent structural studyperformed on the PSII of Synechococcus elongatus [25],reflecting a possible contamination. However, the overallpicture is compatible with a high-quality purification. Theabsorbance spectra of the CP47–RC from both PSII andthylakoid membranes are similar with those previouslyreported [5,6,11], having a A417/A436 ratio ranging from 0.92to 0.96. Concerning fluorescence spectra, a blue shift of4 nm has been reported for the Synechocystis CP47–RCcomplex with respect to PSII [11]; we also observed asimilar shift. The 695-nm emission band has been assignedto a chlorophyll a molecule bound to His114 of CP47 on thebasis of the observed effect in mutants in which His114 wassubstituted by glutamine or asparagine [26]. Its loss in theCP47–RC subcomplex seems to indicate an environmentaleffect due to prolonged detergent exposure. The 695-nmfluorescence emission is in fact observable in whole cells aswell as in thylakoid membranes and, although at lowerintensity, in PSII particles.

The question arises as to whether the CP47–RC complexobtained here is monomeric or dimeric. The A417/A436 ratioof 0.92–0.96 indicates that it is in the monomeric form [6].

This hypothesis is supported by the MALDI spectra. In factthe PsbL and PsbK subunits, which have been observed onlyin the dimeric but not in the monomeric CP47–RCcomplexes from spinach [6], are lacking in our preparation.It is worth noting that most of the small subunits of PSII andof the subcomplexes of cyanobacteria, in agreement toprevious reports [12,27], cannot be resolved by SDS/PAGE(see also Fig. 1A of the present work), but their presence canbe easily detected on the MALDI spectrum.

As regards the functionality of the isolated complex,ADMR results clearly indicate that quinone QA is lostduring the isolation procedure. This is not surprising, as alldetergent solubilized complexes are known to lose thebound quinone. The recombination triplet state becomeseasily populated under illumination at low temperaturewithout sample pretreatment. In contrast, in whole cells aswell as in intact membranes, little recombination triplet statecan be detected unless prereduction followed by samplepreillumination is carried out. This effect is well-knownand has been previously reported [19,21]. The T–Sspectrum of P680 shows main bleaching at 683 nm, andresembles the spectrum previously reported by Carboneraet al. [19] for TP680 in BBY particles. The features found atshorter wavelengths (670–680 nm) are retained in the T–Sspectrum of CP47–RC and only slightly blue shifted, but arecompletely lost in the isolated D1-D2 cytb559 complex[19]. This suggests that, although quinone QA is lost, inCP47–RC the environment of the primary donor isreasonably well conserved.

An important advantage of the procedure described in thiswork is that both RC and CP47–RC complexes of highpurity are obtainable directly by solubilization of wholethylakoid membranes, without need to isolate PSII. TheRCII obtained here displays an absorbance spectrum similarto those recorded by Oren-Shamir et al. [13] and Giorgi et al.[14]. The A417/A436 ratio is slightly lower than that of theselatter RCII complexes. Immunoblot assays revealed that theRCII obtained here completely lacks both CP43 and CP47(see Fig. 4B); in addition, its chlorophyll content was6.5 ^ 0.5, indicating no major contamination. This value isin good agreement with those obtained by Oren-Shamir andGiorgi and colleagues [13,14] (5–7 and 7–8 chlorophyllsfor two pheophytin a molecules, respectively).

In summary, we report the successful isolation andcharacterization of CP47–RC and RCII from PSI-lesscyanobacterial thylakoid membranes and PSII. The use ofthis strain offers numerous advantages, including highefficiency of isolation in terms of yield (40–60% in termsof loaded chlorophyll), no PSI contamination, and thepossibility of isolating the complexes directly fromthylakoid membranes. The resulting subcomplexes may beuseful for future structure–function studies.

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

The authors are grateful to Prof W. Vermaas for making the PSI-less

cyanobacteria strain available. They thank Prof R. Barbato and

Dr E. Bergo for discussions and assistance. This work was financed

by Inco-Copernicus EU grant (PL971176), Italian MURST, under

program PRIN99, and CNR Target Project on Biotechnology. A ‘Young

Researcher Grant’ of the University of Padova to I. S. is also

acknowledged.

q FEBS 2001 RCII and CP47–RC from Synechocystis PCC 6803 (Eur. J. Biochem. 268) 5133

R E F E R E N C E S

1. Nanba, O. & Satoh, K. (1987) Isolation of a photosystem II reaction

center consisting of D1 and D2 polypeptides and cytochrome

b-559. Proc. Natl Acad. Sci. USA 84, 109–112.

2. Irrgang, K.D., Shi, L.X., Funk, C. & Schroeder, W.P. (1995) A

nuclear-encoded subunit of the photosystem II reaction center.

J. Biol. Chem. 270, 17588–17593.

3. Sharma, J., Panico, M., Barber, J. & Morris, H.R. (1997)

Characterization of the low molecular weight photosystem II

reaction center subunits and their light-induced modifications by

mass spectrometry. J. Biol. Chem. 272, 3935–3943.

4. Barber, J. (1998) Photosystem two. Biochim. Biophys. Acta 1365,

269–277.

5. Ghanotakis, D.F., dePaula, J.C., Demetriou, D.M., Bowlby, N.R.,

Petersen, J., Babcock, G.T. & Yocum, C.F. (1989) Isolation and

characterization of the 47 kDa protein and the D1–D2- cytochrome

b-559 complex. Biochim. Biophys. Acta 74, 44–53.

6. Zheleva, D., Sharma, J., Panico, M., Morris, H.R. & Barber, J.

(1998) Isolation and characterization of monomeric and dimeric

CP47-reaction center photosystem II complexes. J. Biol. Chem.

273, 16122–16127.

7. Bianchetti, M., Zheleva, D., Deak, Z., Zharmuhamedov, S.,

Klimov, V., Nugent, J., Vass, I. & Barber, J. (1998) Comparison of

the functional properties of the monomeric and dimeric forms of

the isolated CP47-reaction center complex. J. Biol. Chem. 273,

16128–16133.

8. Dekker, J.P., Bowlby, N.R. & Yocum, C.F. (1989) Chlorophyll and

cytochrome b-559 content of the photochemical reaction center of

photosystem II. FEBS Lett. 254, 150–154.

9. Vermaas, W. (1993) Molecular-biological approaches to analyze

photosystem II structure and function. Annu. Rev. Plant Physiol.

Plant Mol. Biol. 44, 457–481.

10. Shen, G., Boussiba, S. & Vermaas, W. (1993) Synechocystis sp.

PCC6803 strains lacking photosystem I and phycobilisome

function. Plant Cell 5, 1853–1863.

11. Rogner, M., Chisholm, D.A. & Diner, B.A. (1991) Site-directed

mutagenesis of psbC gene of photosystem II: isolation and

functional characterization of CP43-less photosystem II core

complexes. Biochemistry 30, 5387–5395.

12. Gounaris, K., Chapman, D.J. & Barber, J. (1989) Isolation and

characterization of a D1/D2/cytochrome b-559 complex from

Synechocystis sp.6803. Biochim. Biophys. Acta 973, 296–301.

13. Oren-Shamir, M., Sai, P.S.M., Edelman, M. & Scherz, A. (1995)

Isolation and spectroscopic characterization of a plantlike

photosystem II reaction center from the cyanobacterium Synecho-

cystis sp.6803. Biochemistry 34, 5523–5526.

14. Giorgi, B.L., Nixon, P.J., Merry, S.A.P., Joseph, D.M., Durrant,

J.R., De Las Rivas, J., Barber, J., Porter, G. & Klug, D.R. (1996)

Comparison of the primary charge separation in the photosystem II

reaction center complex isolated from wild-type and D1-130

mutants of the cyanobacterium. Synechocystis PCC 6803. J. Biol.

Chem. 271, 2093–2101.

15. Barbato, R., Polverino de Laureto, P., Rigoni, F., deMartini, E. &

Giacometti, G.M. (1995) Pigment–protein complexes from the

photosynthetic membrane of the cyanobacterium Synechocystis sp.

PC6803. Eur. J. Biochem. 234, 459–465.

16. McKinney, G. (1941) Absorption of light by chlorophyll solutions.

J. Biol. Chem. 140, 315–322.

17. Barbato, R., Shipton, C.A., Giacometti, G.M. & Barber, J. (1991)

New evidence suggests that the initial cleavage of the D1-protein

may not occur near the PEST sequence. FEBS Lett. 290, 162–166.

18. Dunn, S.D. (1986) Effects of the modification of transfer buffer

composition and the renaturation of protein in gel on the

recognition of proteins on Western blots by monoclonal antibodies.

Anal. Biochem. 157, 144–153.

19. Carbonera, D., Giacometti, G. & Agostini, G. (1994) A well

resolved ODMR triplet minus singlet spectrum of P680 from PSII

particles. FEBS Lett. 343, 200–204.

20. Crystall, B., Booth, P.Y., Klug, D.R., Barber, J. & Porter, G. (1989)

Resolution of a long lived fluorescence component from D1/D2/

cytochrome b-559 reaction center. FEBS Lett. 249, 75–78.

21. Vass, I. & Styring, S. (1993) Characterization of chlorophyll triplet

promoting states in photosystem II sequentially induced during

photoinhibition. Biochemistry 32, 3334–3341.

22. Szabo, I., Seraglia, R., Rigoni, F., Traldi, P. & Giacometti, G.M.

(2001) Determination of photosystem II subunits by matrix-assisted

laser desorption/ionization mass spectrometry. J. Biol. Chem. 276,

13784–13790.

23. Petersen, J., Dekker, J.P., Bowlby, N.R., Ghanotakis, D., Yocum,

C.F. & Babcock, G.T. (1990) EPR characterization of the CP47–

D1–D2–cytochrome b-559 complex of photosystem II. Biochems-

try 29, 3226–3231.

24. Dekker, J.P., Betts, S.D., Yocum, C.F. & Boekema, E.J. (1990)

Characterization by electron microscopy of isolated particles and

two-dimensional crystals of the CP47–D1–D2-cytochrome b-559

complex of photosystem II. Biochemistry 29, 3220–3225.

25. 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 resolution. Nature 409,

739–743.

26. Shen, G. & Vermaas, W.F. (1993) Mutation of chlorophyll ligands

in the chlorophyll binding CP47 protein as studied in a

Synechocystis sp. PCC6803 photosystem I-less background.

Biochemistry 33, 7379–7388.

27. Rogner, M., Nixon, P.J. & Diner, B.A. (1990) Purification and

characterization of photosystem I and photosystem II core

complexes from wild-type and phycocyanin-deficient strains of

the cyanobacterium Synechocystis PCC 6803. J. Biol. Chem. 265,

6189–6196.

5134 I. Szabo et al. (Eur. J. Biochem. 268) q FEBS 2001