Investigating the early stages of photosystem II assembly in Synechocystis sp. PCC 6803: isolation of CP47 and CP43 complexes.

Abstract

Biochemical characterization of intermediates involved in the assembly of the oxygen-evolving Photosystem II (PSII) complex is hampered by their low abundance in the membrane. Using the cyanobacterium Synechocystis sp. PCC 6803, we describe here the isolation of the CP47 and CP43 subunits, which, during biogenesis, attach to a reaction center assembly complex containing D1, D2, and cytochrome b(559), with CP47 binding first. Our experimental approach involved a combination of His tagging, the use of a D1 deletion mutant that blocks PSII assembly at an early stage, and, in the case of CP47, the additional inactivation of the FtsH2 protease involved in degrading unassembled PSII proteins. Absorption spectroscopy and pigment analyses revealed that both CP47-His and CP43-His bind chlorophyll a and β-carotene. A comparison of the low temperature absorption and fluorescence spectra in the Q(Y) region for CP47-His and CP43-His with those for CP47 and CP43 isolated by fragmentation of spinach PSII core complexes confirmed that the spectroscopic properties are similar but not identical. The measured fluorescence quantum yield was generally lower for the proteins isolated from Synechocystis sp. PCC 6803, and a 1-3-nm blue shift and a 2-nm red shift of the 77 K emission maximum could be observed for CP47-His and CP43-His, respectively. Immunoblotting and mass spectrometry revealed the co-purification of PsbH, PsbL, and PsbT with CP47-His and of PsbK and Psb30/Ycf12 with CP43-His. Overall, our data support the view that CP47 and CP43 form preassembled pigment-protein complexes in vivo before their incorporation into the PSII complex.

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Investigating the Early Stages of Photosystem II Assembly in
Synechocystis sp. PCC 6803
ISOLATIONOFCP47ANDCP43COMPLEXES*□ S
Receivedforpublication,November30,2010,andinrevisedform,February18,2011 Published,JBCPapersinPress,February21,2011,DOI10.1074/jbc.M110.207944
Marko Boehm‡1, Elisabet Romero§1, Veronika Reisinger¶, Jianfeng Yu‡, Josef Komenda?, Lutz A. Eichacker¶,
Jan P. Dekker§, and Peter J. Nixon‡2
Fromthe‡DepartmentofLifeSciences,ImperialCollegeLondon,SouthKensingtonCampus,LondonSW72AZ,UnitedKingdom,
the§DepartmentofPhysicsandAstronomy,FacultyofSciences,VUUniversityAmsterdam,DeBoelelaan1081,1081HVAmsterdam,The
Netherlands,the¶CenterofOrganelleResearch(CORE),UniversityofStavanger,N-4021Stavanger,Norway,andthe?Instituteof
Microbiology,AcademyofSciences,37981Tr ˇebon ˇ,CzechRepublic
Biochemicalcharacterizationofintermediatesinvolvedinthe
assemblyoftheoxygen-evolvingPhotosystemII(PSII)complex
ishamperedbytheirlowabundanceinthemembrane.Usingthe
cyanobacterium Synechocystis sp. PCC 6803, we describe here
theisolationoftheCP47andCP43subunits,which,duringbio-
genesis, attach to a reaction center assembly complex contain-
ing D1, D2, and cytochrome b559, with CP47 binding first. Our
experimental approach involved a combination of His tagging,
the use of a D1 deletion mutant that blocks PSII assembly at an
early stage, and, in the case of CP47, the additional inactivation
of the FtsH2 protease involved in degrading unassembled
PSII proteins. Absorption spectroscopy and pigment analyses
revealed that both CP47-His and CP43-His bind chlorophyll a
and ?-carotene. A comparison of the low temperature absorp-
tionandfluorescencespectraintheQYregionforCP47-Hisand
CP43-His with those for CP47 and CP43 isolated by fragmenta-
tionofspinachPSIIcorecomplexesconfirmedthatthespectro-
scopic properties are similar but not identical. The measured
fluorescencequantumyieldwasgenerallylowerfortheproteins
isolated from Synechocystis sp. PCC 6803, and a 1–3-nm blue
shift and a 2-nm red shift of the 77 K emission maximum could
beobservedforCP47-HisandCP43-His,respectively.Immuno-
blotting and mass spectrometry revealed the co-purification of
PsbH, PsbL, and PsbT with CP47-His and of PsbK and Psb30/
Ycf12 with CP43-His. Overall, our data support the view that
CP47andCP43formpreassembledpigment-proteincomplexes
in vivo before their incorporation into the PSII complex.
Photosystem II (PSII)3is the light-driven water:plastoqui-
none oxidoreductase of oxygenic photosynthesis, responsible
for producing most of the oxygen in the atmosphere (1). It is
located in the thylakoid membrane of chloroplasts and cyano-
bacteria and is a multisubunit lipoprotein complex composed
of both intrinsic and extrinsic proteins. Crystal structures of
dimeric PSII protein complexes isolated from the thermophilic
cyanobacteriaThermosynechococcuselongatus(2–5)andTher-
mosynechococcusvulcanus(6,7)haverevealedtheorganization
of the 20 subunits within each monomeric complex and the
positionsofthevariouscofactors.Theseinclude35chlorophyll
(Chl)amolecules,twopheophytinamolecules,12carotenoids,
twohememolecules,onenon-hemeiron,twocalciumions,two
chloride ions, three plastoquinones, 25 lipids, and the Mn4Ca
cluster, which catalyzes water oxidation (4).
We are interested in understanding how PSII is assembled
fromitsindividualcomponents.Currentmodelssuggestastep-
wiseassemblyinbothcyanobacteriaandchloroplastsinvolving
distinct intermediates (8–10). However, as with other mem-
brane protein complexes, detailed analysis of PSII assembly
complexes is hindered by their low abundance in the mem-
brane,anduntilnow,earlyassemblyintermediatesofPSIIhave
not been isolated and biochemically characterized.
Here, we describe the isolation of the CP47 and CP43 sub-
units from the cyanobacterium Synechocystis sp. PCC 6803
(hereafter Synechocystis 6803). These PSII subunits each con-
tain six transmembrane ?-helices and, in the cyanobacterial
PSII holoenzyme, bind Chl a (16 molecules in CP47 and 13 in
CP43) and ?-carotene (4). They lie on either side of the het-
erodimeric D1/D2 reaction center complex involved in light-
induced transmembrane charge separation, and one of their
roles is to act as an inner light-harvesting antenna system (11).
CP43 is also involved in ligating the Mn4Ca cluster (3). CP47
and CP43 are tightly bound components of the larger PSII core
complex, and relatively harsh treatments are required to
remove CP47 and CP43 in vitro (12, 13).
It is known that the apopolypeptides of both CP47 (encoded
by the psbB gene) and CP43 (encoded by psbC) are synthesized
and inserted into the thylakoid membrane before their incor-
poration into the PSII complex of Synechocystis 6803 (8). How-
ever, it remains unclear whether they are able to bind pigment
molecules in this “unassembled” state (10). To address this
* This work was supported by Engineering and Physical Sciences Research
Council (EPSRC) Grant EP/F002070X/1 and in part by Czech Academy of
Science Institutional Research Concept AV0Z50200510 and Grant Project
IAA400200801.
□
SThe on-line version of this article (available at http://www.jbc.org) con-
tains supplemental Figs. 1–3.
1Both authors contributed equally to this work.
2To whom correspondence should be addressed: Dept. of Life Sciences,
Imperial College London, Wolfson Biochemistry Bldg., South Kensington
Campus, London SW7 2AZ, UK. Tel.: 44-207-594-5269; Fax: 44-207-594-
5267; E-mail: p.nixon@imperial.ac.uk.
3The abbreviations used are: PSII, Photosystem II; Chl, chlorophyll; LMM, low
molecular mass; TES, N-tris(hydroxymethyl)methyl-2-aminoethanesulfo-
nic acid; ?-DM, n-dodecyl ?-D-maltoside; BN, Blue native; BisTris, 2-[bis(2-
hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol.
THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 286, NO. 17, pp. 14812–14819, April 29, 2011
© 2011 by The American Society for Biochemistry and Molecular Biology, Inc.Printed in the U.S.A.
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issue, we describe here a novel approach for the isolation and
characterization of CP47 and CP43 complexes from Syn-
echocystis 6803.OurresultsshowthatbothCP47andCP43are
likely to bind a largely complete set of pigments and are able to
attach to neighboring low molecular mass (LMM) subunits
before assembling into larger PSII complexes.
EXPERIMENTAL PROCEDURES
Cyanobacterial Strains and Growth Conditions—The glu-
cose-tolerant strain of Synechocystis 6803 (14) and the psbA
triple deletion strain, TD41 (15), were used in this work. For
clarity, TD41 will be referred to here as ?D1. Strains were
grown in liquid BG-11 mineral medium and maintained on
solid BG-11 plates containing 1.5% (w/v) agar, both containing
5 mM TES-KOH (pH 8.2) at a light intensity of 40 or 5 microe-
instein m?2s?1of white fluorescent light, respectively, and at
29 °C. The medium was supplemented with 5 mM glucose and,
whereapplicable,chloramphenicol(30?gml?1),erythromycin
(10 ?g ml?1), gentamycin (2 ?g ml?1), kanamycin (50 ?g
ml?1), or spectinomycin (50 ?g ml?1).
Construction of Mutants—To generate the His-tagged CP47
strain (?D1/CP47-His/?FtsH2), the gentamycin resistance
cassette of plasmid pCP47His-tagGmR(16) was removed by
BamHI digestion. After blunting the ends, an erythromycin
resistance cassette was introduced to generate pCP47His-
tagEryR. Ultimately, this plasmid was transformed into the
?D1/?FtsH2strain,whichhadbeengeneratedbytransforming
the ?D1 mutant strain (15) with the plasmid used to produce
the Syn0228GENT strain described previously (17). A control
strain expressing His-tagged CP47 in a WT background (strain
PSII-His) was generated by transforming the WT with
pCP47His-tagEryR. To generate the His-tagged CP43 Syn-
echocystis 6803 mutant strain (?D1/CP43-His), a His6-coding
sequence was introduced at the 3?-end of the psbC gene
(sll0851)byoverlapextensionPCRusingthefollowingprimers:
CP43?1000-Fw, 5?-ATATTTTCCCCTTCTTCGTAGGGG-
TGC-3?; CP43?1000-Rev, 5?-CTGCCATTAAAGAATTGG-
CTAAAGAAGCAGGTC-3?; CP43HT-Fw, 5?-CATCATCAT-
CATCATCATTAGATTGAGACTTTTCTGATTTTGCAA-
3?; and CP43HT-Rev, 5?-CTAGTAGTAGTAGTAGTAGTA-
GTCGAGGTCAGGCATGAACAA-3?. The overlap extension
PCR product was cloned into the pGEMTeasy vector (Pro-
mega),andanerythromycinresistancecassettewasintroduced
at an XmnI site 170 bp downstream of the stop codon of the
gene. The genotypes of the mutants were verified by PCR anal-
ysis using gene-specific primers.
Isolation of CP47-His and CP43-His—CP47-His and CP43-
His complexes were isolated from 10-liter cultures of the
respective Synechocystis 6803 mutant strain that had been
grown to stationary phase, concentrated using a cell harvester,
and pelleted by centrifugation. Cells were then washed and
finallyresuspendedinKPNbuffer(40mMpotassiumphosphate
(pH 8.0) and 100 mM NaCl) containing EDTA-free Complete
protease inhibitor (Roche) to a Chl a concentration of 1 mg
ml?1. After two passages at 1250 p.s.i. through a prechilled
French press cell (Amicon), unbroken cells were removed by
centrifugation. Subsequently, crude membranes were pelleted
by ultracentrifugation and resuspended in KPNG buffer (KPN
buffer supplemented with 10% (v/v) glycerol). Solubilization of
the sample was performed under gentle stirring for 30 min on
ice after the addition of one-ninth of the sample volume of 10%
(w/v)n-dodecyl?-D-maltoside(?-DM)inKPNbuffer.Unsolu-
bilized material was removed by ultracentrifugation, and the
supernatant was incubated for 1 h at 4 °C on an end-over-end
rotational wheel with nickel-iminodiacetic acid His affinity
purification resin (Generon). After incubation, unbound mate-
rialwasremoved,andtheresinwaswashedwithKPNGDbuffer
(KPNG buffer containing 0.04% (w/v) ?-DM) until the flow-
through became colorless (typically 40 column volumes). The
resin was then washed with 10 column volumes each of
KPNGD buffer containing increasing concentrations of imid-
azole (5, 10, and 20 mM). The His-tagged proteins were eluted
using 10 and 5 column volumes of 50 and 100 mM imidazole in
KPNGD buffer, respectively. These elution samples were then
pooled and concentrated using 10-kDa molecular mass cutoff
proteinconcentrators(Sartorius).Inasecondpurificationstep,
500-?l aliquots containing ?200 ?g of Chl a were applied to a
Superdex200FPLCcolumn(GEHealthcare)operatedataflow
rate of 1 ml min?1with KPN buffer containing 0.04% (w/v)
?-DMastherunningbuffer.Therunwasmonitoredat280nm
using a Jasco MD-2015 Plus diode array detector, and 2-ml
fractions were collected by a Frac-920 fraction collector (GE
Healthcare). Selected fractions were pooled, supplemented
with10%(v/v)glycerol,andconcentratedusing10-kDamolec-
ular mass cutoff protein concentrators. Typically, the yield of
CP47-His and CP43-His was ?0.5–1.0 mg of chlorophyll/10-
liter culture.
Isolation of His-tagged PSII Complexes—As a control, His-
tagged PSII was purified from strain PSII-His using the same
immobilizedmetalaffinitychromatographypurificationproto-
col as used for CP43-His and CP47-His except that a 100-kDa
molecular mass cutoff concentrator was used to concentrate
samples. A 500-?l aliquot containing ?200 ?g of Chl a was
then applied to 5 ml of TOYOPEARL 650S DEAE anion
exchange chromatography resin (Anachem) in a second purifi-
cation step. The anion exchange chromatography run was per-
formed at a flow rate of 0.5 ml min?1with KPN buffer contain-
ing 0.04% (w/v) ?-DM as the running buffer. Initially, for the
first 10 min, the running buffer also contained 5 mM MgSO4,
and over the next 50 min, the concentration of MgSO4was
raised linearly to 200 mM. Fractions containing PSII were
pooled, supplemented with glycerol to a final concentration
of 10% (v/v) and concentrated using 100-kDa molecular
mass cutoff protein concentrators.
Isolation of Spinach CP47 and CP43—Samples were pre-
pared from larger PSII core complexes using the methods
described previously (18, 19).
Protein Analysis—The Chl a content of samples was deter-
mined by extraction into methanol and absorption measure-
ments at 666 and 750 nm (20). Protein samples were analyzed
by Blue native (BN)-PAGE and SDS-PAGE as described previ-
ously(21).Unlessstatedotherwise,BN-8–12%(w/v)polyacryl-
amide and 18% (w/v) SDS-polyacrylamide gels containing 6 M
ureawereused.TheresultinggelswereeitherCoomassieBlue-
or silver-stained (22) or electroblotted onto PVDF membrane
using the iBlot system (Invitrogen) according to the manufac-
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turer’s instructions. Immunoblot analyses were performed
usingspecificprimaryantibodiesandahorseradishperoxidase-
conjugated secondary antibody (GE Healthcare). Signals were
visualized using a SuperSignal West Pico chemiluminescence
kit (Pierce). The primary antibodies used in this study were as
follows: anti-CP43 (directed against Chlamydomonas rein-
hardtii PsbC, serum P6, kindly provided by B. Diner), anti-
CP47 (directed against barley PsbB (residues 380–394)),
anti-D1 (directed against the C-terminal peptide) (23), anti-D2
(directed against the C-terminal peptide) (24), anti-His tag
(Invitrogen), and anti-PsbH (from Synechocystis 6803) (25).
Reverse-phase HPLC Pigment Analysis—Pigments were
extracted into 80% (v/v) acetone/water at 4 °C under dim light
conditions, and the sample was centrifuged at maximum speed
for 1 min in a microcentrifuge to pellet precipitated proteina-
ceous material before injection. The injection volume was 20
?l, and the pigments were resolved using a Gemini 5-?m
C6-phenyl 110-Å column (Phenomenex). The following pro-
gram was run at a flow rate of 1 ml min?1: 0–4 min at 100%
systemB,4–20minlineargradientfrom100%systemBto40%
system B and 60% system C, 20–24 min at 40% system B and
60% system C, 24–35 min at 100% system B, with system A
being65%(v/v)acetonitrileand35%(v/v)water,systemBbeing
90% (v/v) acetonitrile and 10% (v/v) water, and system C being
100% ethyl acetate. The run was monitored at 440 nm using a
JascoMD-2015Plusdiodearraydetector.Chlaand?-carotene
quantification was performed after calibration with pigment
standards (purchased from Sigma) of known concentrations.
Spectroscopic Analyses—CP43-His and CP47-His samples
were diluted in buffer containing 20 mM BisTris-HCl (pH 6.5),
20 mM NaCl, 0.09% (w/v) ?-DM, and 75% (v/v) glycerol. The
samples for the absorption experiments were diluted to an
absorbance of ?0.5 cm?1at the QYmaxima, whereas for the
fluorescence experiments, an absorbance if ?0.1 cm?1was
used. The low temperature measurements were performed in
an Utreks helium flow cryostat (5 K) or in an Oxford liquid
nitrogen bath cryostat (77 K). Absorption spectra at 5 K were
recordedinahome-builtsetupequippedwitha150-watttung-
sten-halogenlamp,amonochromator,andaphotodiodedetec-
tor. The spectra were recorded using lock-in detection with
1-nm spectral resolution. Absorption spectra at 77 K were
recorded in a PerkinElmer Lambda 40 UV-visible spectropho-
tometer with 1-nm spectral resolution. Fluorescence spectra at
77 K were recorded in a Jobin Yvon Fluorolog-3-11 fluorome-
ter. The excitation wavelength was 488 nm (10-nm full-width
half-maximum),andthespectralresolutionwas1nm.Thefluo-
rescence quantum yield was estimated by integrating the total
fluorescence and comparing with the known value of the fluo-
rescencequantumyieldforhigherplantCP43atroomtemper-
ature (19).
Mass Spectrometric Analysis—Low molecular mass proteins
were identified using the methods described previously (26).
For offline electrospray ionization MS, 20 ?l of purified CP47-
His and CP43-His complexes, respectively, were precipitated
overnightin80%(v/v)acetone/waterat?20 °C.Aftercentrifu-
gation at 13,000 ? g for 10 min, the supernatant was discarded,
and the pellet was air-dried. The dry pellet was dissolved in
solution containing 70% (v/v) acetone, 19% (v/v) water, 10%
(v/v) 2-propanol, and 1% (v/v) formic acid. Dissolved proteins
were directly applied to a nanospray emitter. Mass spectra
were obtained using a Waters Q-Tof Premier spectrometer
equipped with a nanoelectrospray ionization source. For scan-
ning LMM proteins, mass spectra at a mass range of 800–2500
m/z were acquired. Data were recorded at a capillary voltage of
0.8 kV and a cone voltage of 37 V. After MS data collection at a
rate of 1 s/scan, scans were averaged. After acquisition of frag-
ment ion spectra at a collision energy between 26 and 40 eV,
data were analyzed using MassLynx/BioLynx 4.1 software.
Sequence tags obtained from the fragment spectra were used
forsimilaritysearch(EMBL-EBIDataBank).Thesampleswere
analyzed in several repetitions.
RESULTS
Isolation of CP47-His and CP43-His from Synechocystis
6803—The low abundance of PSII assembly intermediates in
WT Synechocystis 6803 is a major barrier to their isolation. We
have adopted a 3-fold approach to aid purification of unas-
sembled CP47 and CP43 complexes. First, we incorporated
His6-tagsattheCterminusofeachproteintoallowpurification
by immobilized metal affinity chromatography. Previous work
has already shown that the presence of His-tags at these posi-
tions does not prevent assembly of active PSII (27, 28). Second,
we blocked assembly of the PSII holoenzyme using a parental
strain, ?D1, which is unable to make the D1 reaction center
subunit (8, 15), and finally, in the case of CP47, we also inac-
tivated the membrane-bound FtsH2 protease (Slr0228),
which leads to elevated levels of unassembled PSII subunits,
including the CP47 apopolypeptide, in the ?D1 strain (17).
Construction and validation of the ?D1/CP43-His and ?D1/
CP47-His/?FtsH2 strains are described under “Experimen-
tal Procedures.”
The CP43-His and CP47-His complexes were successfully
purified from detergent-solubilized thylakoid membranes by
immobilizedmetalaffinitychromatography(supplementalFig.
1). Small amounts of residual high molecular mass contami-
nantswereremovedbyasubsequentsizeexclusionchromatog-
raphystep(Fig.1,AandB).BothCP47-HisandCP43-Hiswere
largely monodisperse as assessed by BN-PAGE (Fig. 1C).
Protein Composition—SDS-PAGE combined with immuno-
blot analyses confirmed the purity of each preparation and the
presence of His-tagged CP47 and CP43 (Fig. 1D). In the case of
CP47-His, we were also able to detect the presence of PsbH, a
neighboring low molecular mass subunit in the PSII holoen-
zyme(Fig.1D).Co-purificationofPsbHandCP47-Hiswasfur-
ther demonstrated by two-dimensional BN-PAGE (supple-
mentalFig.2).Together,thesedataconfirmearlierconclusions
that CP47 and PsbH are capable of forming a complex at an
early stage in PSII assembly (25). Interestingly, despite the
absence of D1, the D2 reaction center subunit, which interacts
with CP47 in the holoenzyme, was also detected in the CP47-
His complex, albeit at substoichiometric levels (Fig. 1D). Anal-
ysisbymassspectrometryconfirmedthepresenceofPsbHand
also identified PsbL and PsbT (Table 1). In the case of CP43-
His, low levels of a CP43 degradation product were detected by
immunoblotting(designatedbyanasteriskinFig.1D)(datanot
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shown), and the PsbK and Psb30/Ycf12 subunits were identi-
fied by mass spectrometry (Table 1).
Pigment Composition—A notable feature of both the CP47-
His and CP43-His complexes was the presence of bound pig-
ment. Reverse-phase HPLC pigment analyses confirmed the
presence of both Chl a and ?-carotene in CP47-His and CP43-
His complexes (supplemental Fig. 3), with Chl/carotenoid
ratios of ?7.7 for CP47-His and 5.0 for CP43-His. The wave-
lengths of maximum absorption in the red region (?max) mea-
sured at room temperature were 674 nm for CP47-His and 671
nmforCP43-His(Fig.2A),closetothevaluesreportedforCP47
and CP43 isolated by fragmentation of larger complexes (13).
TherelativeamplitudesoftheSoretandcarotenoidabsorption
bands in the room temperature absorption spectra for CP47-
HisandCP43-Hiswerealsoquitesimilartotheirspinachcoun-
terparts (Fig. 3), consistent with similar Chl/carotenoid ratios
in each type of complex.
Absorption at 5 K and Comparison with Spinach—To assess
the optical properties of the pigments bound to CP47-His and
CP43-His in more detail, we measured low temperature
FIGURE 1. Isolation and protein analysis of CP47-His and CP43-His isolated from mutants of Synechocystis 6803 blocked at an early stage of PSII
assembly. CP43-His (A) and CP47-His (B) were purified by Ni2?affinity chromatography (supplemental Fig. 1), followed by FPLC size exclusion chromatogra-
phy.ShownareelutionprofilesoftheFPLCsizeexclusionrunsmonitoredat280nm,withthefractionsthatwerelaterpooledmarkedbygrayareas(startand
stopindicatedbyarrows).a.u.,absorbanceunits.C,CP43-HisandCP47-Hissamplesbefore(pre-FPLC)andafter(post-FPLC)purificationbyFPLCsizeexclusion
chromatography were analyzed on a BN-8–12% (w/v) polyacrylamide linear gradient gel. Samples corresponding to 0.5 ?g of Chl a were loaded per lane. A
highmolecularweightmarker(HMW;GEHealthcare)wasusedtocalibratethegel.D,CoomassieBlue-stained18%(w/v)SDS-polyacrylamidegeloftheisolated
proteins and immunoblot analyses with the indicated antibodies. A low molecular mass marker (GE Healthcare) was used to calibrate the gel. WT thylakoid
membranes(0.5?gofChla)andfinalsamplesofCP47-HisandCP43-His(1?gofChla)andPSII-His(1?gofChla)wereloadedonthegels.Aminordegradation
product of CP43-His is indicated by the asterisk.
TABLE1
LMM proteins detected in CP43-His and CP47-His complexes by mass spectrometry
GeneAccession number
Detected signals
(charge state)
De novo sequenced
protein fragment
Total sequence
coverage
%
CP47-His complex
PsbHP14835998.56 (?M ? 6H?6?)
1001.57 (?M ? 6H?6?)
1003.85(?M ? 6H?6?)
1004.70 (?M ? 6H?6?)
1006.99 (?M ? 6H?6?)
1171.16 (?M ? 5H?5?)
1397.41 (?M ? 5H?5?)
1118.64 (?M ? 4H?4?)
1246.45 (?M ? 3H?3?)
892.53 (?M ? 4H?4?)
1189.33 (?M ? 3H?3?)
PVMGVFMALFLVFL
PVMGVFMALFLVFL
MGVFMALFLVFLL
TPVMGVFMALFLVF
VPGWGTTPVMGVFMA
TTPVMGVFMALFLVFLLII
GTTPVMGVFMALFLVFL
LLVAVLGILFSSYF
LLLVAVLGI
LVLTMALAVLF
SVAYILVLT
38
PsbL Q55354
36
PsbTP74787
52
CP43-His complex
PsbKP736761050.15 (?M ? 4H?4?)
1055.64 (?M ? 4H?4?)
1202.76 (?M ? 3H?3?)
YQIFDPLVDVL
DPLVDVL
IVLAG
24
Psb30/Ycf12Q55438
13
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absorptionspectraat5Kandcomparedthemwiththoseofthe
widely studied CP47 and CP43 complexes isolated by detach-
ment from larger spinach PSII core complexes (Fig. 2B) (29–
32). In the Chl Soret region, the spectral shape is almost iden-
tical for both inner antenna complexes, with two bands at 416
and 436 nm identical in value to their spinach counterparts.
The carotenoid absorption bands for CP47-His are at 469 and
502 nm (467 and 502 nm for spinach CP47), and those for
CP43-His are at 467 and 499 nm (462 and 497 nm for spinach
CP43). Fig. 2B (inset) shows an enlargement of the Chl QY
absorption region. For CP47-His, the absorption spectrum
showed two bands at 668 and 676 nm and a shoulder around
683 nm. The absorption red tail extends to 700 nm, suggesting
the presence of a spectral component in the red edge of the
spectrum (also observed for spinach at 690 nm). Overall, when
comparing CP47-His from Synechocystis 6803 with spinach
CP47, the spectral shapes show some differences in positions
and relative amplitude, such as stronger and weaker absorp-
tions for the cyanobacterial inner antenna protein around 667
nmandintherededge,respectively.ForCP43-His,theabsorp-
tionspectrashowabroadmaximumat668.5nm,twoshoulders
at673and679nm,andanarrowbandat684.5nm.Theabsorp-
tion spectra of the spinach CP43 inner antenna (19) display a
very similar spectral shape, the main difference between the
twoorganismsbeingthe2.5-nmredshiftofthenarrowbandat
684.5 nm in Synechocystis 6803 with respect to the 682 nm
narrow band in spinach.
Absorption and Fluorescence at 77 K and Comparison with
Spinach—Fig. 4 shows the absorption and fluorescence spectra
at 77 K normalized to a value of 1 at the absorption and fluo-
rescence maxima for CP47 (Fig. 4A) and CP43 (Fig. 4B) from
Synechocystis 6803 and spinach to facilitate the comparison of
their respective spectral shapes. The second derivatives of the
absorption (multiplied by ?4) are displayed to identify the
spectral components present in the spectra. For CP47, the sec-
ond derivatives of the absorption spectra show differences at
wavelengths shorter than 675 nm, with maxima at 658.5 and
667 nm for Synechocystis 6803 and 660 and 670.5 nm for spin-
ach. At wavelengths longer than 675 nm, the peak positions at
676.5 and 682.5 nm are the same. However, the red edge is less
pronounced in cyanobacterial CP47-His, which suggests that,
although the 682.5 nm band is at the same position in both
organisms, this band is narrower in Synechocystis 6803. This
couldbeanindicationofamore“defined”proteinenvironment
oralowerdegreeofelectron-phononcoupling(couplingofthe
electronic transitions to protein lattice vibrations, which
inducebroadeningoftheabsorption)inCP47-His.Thefluores-
cencespectrumalsoshowsanarrowerandblue-shiftedspectral
distribution in Synechocystis 6803 with respect to spinach,
which indicates that the 682.5 nm pigment(s) are emitting
stateswithlargecontributionstotheoverallfluorescencespec-
trum and that the red chlorophyll (peaking around 690 nm in
spinach) gives a smaller contribution. However, a “red” chloro-
phyll is probably also present in Synechocystis 6803, in view of
the shoulder at around 690 nm in the 77 K emission spectrum.
For CP43, the second derivatives confirm that the narrow red
band is shifted from 682 nm in spinach to 684.5 nm in Syn-
echocystis6803andthattheotherabsorptionbandspeakatthe
samewavelengthsinbothorganisms.The2-nmredshiftisalso
observed in the fluorescence spectra (which peak at 682 nm in
spinach and 684 nm in Synechocystis 6803), indicating that the
pigment(s)givingrisetothenarrowabsorptionbandsarestates
FIGURE 2. CP43 and CP47 absorption spectra at room temperature (A)
and5K(B).ThespectrawerenormalizedtotheChlcontentintheQYregion
from645to710nmassuming13ChlmoleculesinCP43and16Chlmolecules
inCP47(3,5,40).TheinsetsshowanenlargementoftheabsorptionintheQY
region. RT, room temperature; a.u., absorbance units.
FIGURE 3. Comparison of the room temperature absorption spectra of
CP43-His and CP47-His isolated from Synechocystis 6803 and CP43 and
CP47 isolated from spinach. RT, room temperature; Abs, absorbance; a.u.,
absorbance units.
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withlargecontributionstothe77Ksteady-stateemissionspec-
tra (19). In contrast to spinach, the 77 K emission spectrum of
CP43-His from Synechocystis 6803 reveals a shoulder at about
679 nm, which is probably related to the feature peaking at
about 678 nm in the second derivative of the absorption spec-
trum. CP43 from spinach shows an absorption band peaking at
the same wavelength, but in the emission, this feature is not
observed, most likely as a result of overlap with the emission
from the 682 nm band. The efficiency of excitation energy
transfer to the reaction center was assessed by calculating the
fluorescence quantum yield at room temperature. The calcu-
lated fluorescence quantum yield values for the Synechocystis
6803innerantennaproteinsarebothlowerthanthoseforspin-
ach (?50 and 75% for CP47-His and CP43-His, respectively).
DISCUSSION
Although the structure of the cyanobacterial PSII holoen-
zyme is now known to atomic resolution (6), there is still much
tolearnabouthowthecomplexisassembledandthenrepaired
following irreversible damage (reviewed in Ref. 10). Here, we
have addressed a very basic question: at what stage can chloro-
phyll and carotenoid molecules be inserted into the inner
antenna proteins CP47 and CP43 of PSII? To address this, we
have generated His-tagged strains of Synechocystis 6803 to
allow rapid isolation of unassembled CP47-His and CP43-His
protein complexes from the membranes of a ?D1 deletion
strain. In the case of CP47, we also exploited the observation
that the levels of unassembled CP47 increase from 10 to 20% of
WTlevelsto100%ofWTlevelswhentheFtsH2thylakoidpro-
tease is inactivated (17). Thylakoid FtsH proteases appear to
play an important role in removing unassembled protein sub-
units (17). Consequently, the use of FtsH mutants might be a
usefulstrategytoincreaselevelsofotherunstablecomplexesor
assemblycomplexesinthethylakoidmembraneinbothcyano-
bacteria and chloroplasts.
Our results indicate that the isolated His-tagged CP47 and
CP43 complexes contain a very similar set of chlorophyll and
carotenoid pigments compared with spinach CP47 and CP43
isolated by fragmentation of larger core complexes. Evidence
comes from the close similarity in spectral shape and relative
amplitudes in the absorption spectra in the visible range from
400 to 750 nm between the Synechocystis 6803 complexes and
spinachPSIIcorecomplexes(Fig.3)aswellassimilaritiesinlow
temperature absorption and fluorescence spectra (Figs. 2 and
4). Thus, we can conclude that the insertion of pigment into
CP47 and CP43 in Synechocystis 6803 is able to occur before
their incorporation into a larger PSII complex, at least in the
PSII assembly mutants studied here. Previous work has shown
that the availability of Chl a is important for the synthesis and
accumulation of full-length CP47 apopolypeptide (33, 34) and
that absence of carotenoid leads to impaired synthesis of CP47
and especially of CP43 (35). Also, accumulation of CP47 and
CP43 is severely impaired in site-directed mutants lacking an
appropriate amino acid ligand to chlorophyll (36, 37). These
datathereforesuggestthatbindingofpigmentstartstooccurat
an early stage in the assembly of CP47 and CP43 in the WT,
perhapsevencotranslationallyduringinsertionoftheapopoly-
peptides into the membrane to help stabilize the complex. A
close synchronization between pigment binding and the syn-
thesis and folding of CP47 and CP43 would also help to mini-
mizepotentialtoxiceffectsoffreechlorophyllinthemembrane
(38).Whetheralloronlyafractionofthechlorophyllmolecules
bindtoCP47andCP43atthisearlystageofPSIIassemblyinthe
WT is unclear. How chlorophyll and carotenoid are presented
to the CP47 and CP43 apopolypeptides is also currently
unknown, although there is some suggestion that enzymes
involved in chlorophyll biosynthesis might be docked onto the
thylakoid membrane (39).
The precise Chl/carotenoid ratio to be expected for CP47-
HisandCP43-Hisisstillnotclear.Inthemostrecentstructure,
35 Chl a molecules are present per PSII monomer, with 16
molecules bound to CP47 and 13 to CP43 (4). However, the
assignment of carotenoids within the PSII structural models
has proved to be more difficult. With improved resolution, the
number has increased from 7 (3) to 11 (5) and most recently to
12molecules(4).analysisbyMu ¨hetal.(40)concludedthatfive
carotenoids are associated with CP47 and four with CP43.
However, many of these carotenoids are located at the inter-
FIGURE 4. Comparison of the low temperature absorption and fluores-
cence spectra in the QYband region between the CP47 and CP43 inner
antennaproteinsofSynechocystis6803andspinach.Absorptionandfluo-
rescence spectra for CP47-His (A) and CP43-His (B) from Synechocystis 6803
andforCP47andCP43fromspinachwererecordedat77Kandnormalizedto
1 at the absorption and fluorescence maxima. Second derivatives of absorp-
tion spectra (2nd Der Abs) are also shown.
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faces between subunits, such as between CP43 and PsbK/PsbZ
(41), and at the interface of the two monomers between CP47
and D1-PsbT (40). The Chl/carotenoid ratios determined here
(7.7 for CP47-His and 5.0 for CP43-His) are much greater than
those predicted by Mu ¨h et al. (40) (3.2 for CP47 and 3.25 for
CP43) and might reflect absence of carotenoid in the smaller
assembly complexes described here and/or loss of carotenoid
during purification.
Comparison of the low temperature absorption and fluores-
cence spectra of the inner antenna proteins isolated from Syn-
echocystis6803andspinachshowsthatthespectraldistribution
of absorption bands is similar but not identical in both organ-
isms. Regarding the functionality of the antenna complexes
with respect to their efficiency of excitation energy transfer to
the reaction center, the fluorescence quantum yields for CP47-
His and CP43-His were calculated and found to be generally
lower than those for their spinach counterparts. The differ-
ences may be of several origins. One explanation might derive
from the fact that the Synechocystis 6803 antenna complexes
are assembly complexes isolated at a stage preceding their
assembly into the core complex (not isolated from the core
complex,asinthecaseofspinach).Thisimpliesthattheenergy
absorbed by the antenna protein pigments is not “meant” to be
used by the reaction center for photochemistry and that there
might therefore be an intrinsic energy dissipation pathway,
possibly mediated by LMM PSII subunits. Alternatively, the
assembly complexes could be in a protein conformation that is
not favorable for efficient excitation energy transfer (because it
isnotneeded),whichmightchangetoanoptimizedconforma-
tion once the complexes are integrated into the core complex.
Another possibility is that the differences may arise from vari-
ations in the macrostructure of the inner antenna systems
between the two organisms and sequence differences between
them. In spinach, the core antennas play a role in transferring
excitation energy absorbed by other peripheral antennas: the
minor antennas CP24, CP26, and CP29 and the main light-
harvestingcomplexII.InSynechocystis6803,aroleasan“inter-
mediary”inexcitationenergytransfercouldbebypassedbythe
presence of the dynamic phycobilisomes, which can funnel
excitationenergydirectlytothereactioncenter.Therefore,the
requirementofhighlyefficientexcitationenergytransferwould
be less of a determinant in Synechocystis 6803 than in spinach.
We note that these possibilities are not mutually exclusive and
that we cannot conclude from the available data which possi-
bility or possibilities are the right one(s). To shed light on these
issues,itwillbenecessarytoperformfurthercomparativespec-
troscopic studies between assembly complexes and complexes
isolated from the core complex.
An additional novel aspect of our work is the detection of
LMM subunits in the isolated CP43-His and CP47-His com-
plexes. Although our data show that these subunits are capable
ofbindingtounassembledCP47andCP43,additionalevidence
isrequiredtoconfirmthatsuchcomplexesalsoformintheWT
strainandarenotmerely“dead-end”complexesthatonlyaccu-
mulate in assembly-defective mutants. For instance, we have
shown here that PsbH is capable of forming a complex with
CP47-His(Fig.1andsupplementalFig.2).LowlevelsofaCP47-
PsbH complex have also been detected in WT thylakoids (25),
and accumulation of full-length unassembled CP47 apopoly-
peptideisimpairedinaPsbH-nullmutant(25).Together,these
data suggest a stabilizing effect of PsbH on CP47 accumulation
during the early steps of PSII assembly in the WT (25). The
small chlorophyll-a/b-binding-like protein ScpD can also bind
to CP47 in the vicinity of PsbH (42), but ScpD is not expressed
under the illumination conditions used here (data not shown),
which would explain its absence in the CP47-His complex.
Overall,thecurrentlyavailabledatasuggestthatthelowlevelof
unassembled CP47 found in the WT thylakoid membrane
existspredominantlyasaCP47-PsbHcomplexandcaninclude
ScpDunderhighlightconditions.Importantly,wewereableto
detect the co-purification of PsbT and PsbL with CP47-His.
These LMM subunits are located next to CP47 in the PSII
holoenzymeandlieattheinterfaceofthetwomonomersinthe
dimeric complex. Whether PsbT and PsbL are present at stoi-
chiometric levels in the CP47-His complex and attach to CP47
early in assembly in the WT is presently unclear.
CP43 has been detected in two distinct complexes (CP43a
andCP43b)insolubilizedthylakoidmembraneextractsofSyn-
echocystis 6803, so it is likely that CP43 also binds LMM sub-
units at an early stage of PSII assembly in the WT (8). Based on
the PSII crystal structures, three LMM subunits might form a
stablecomplexwithCP43:PsbK,PsbZ,andPsb30/Ycf12(3–5).
Usingmassspectrometry,wehavebeenabletoidentifyPsbKin
the CP43-His complex. A tight association between PsbK and
CP43 has previously been documented in fragmentation stud-
iesofPSIIcorecomplexes(39),andtheearlybindingofPsbKto
CP43 during assembly has been inferred from analysis of Chla-
mydomonasPSIImutants(43).Inaddition,wehaveidentifieda
LMM polypeptide in the His-CP43 complex containing a short
sequence match with Psb30/Ycf12 (Table 1). Hence, we assign
Psb30/Ycf12asanadditionalcomponentoftheCP43-Hiscom-
plex. Whether PsbZ is also a component of the CP43-His com-
plex remains to be clarified.
Inconclusion,wehavedescribedthefirstisolationandchar-
acterization of unassembled CP47 and CP43 subunits from the
thylakoid membrane. Our experimental results indicate that
CP47 and CP43 are capable of forming preassembled pigment-
protein complexes containing neighboring LMM subunits
found in the holoenzyme. On the basis of our data here and
earlierresults,weproposeamodularassemblyofPSIIinwhich
D1-PsbI(44),D2-cytochromeb559(8),CP47-PsbH-PsbL-PsbT,
and CP43-PsbK-Psb30 subcomplexes, the latter possibly
includingPsbZ,arecombinedtogethertoformfirstaPSIIreac-
tion center-like complex, then the RC47 complex (a PSII core
complex lacking CP43), and finally a monomeric core complex
(reviewedinRef.10).Subsequentformationofoxygen-evolving
PSII would require light-driven assembly of the Mn4Ca cluster
and attachment of the extrinsic proteins.
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