Cytochrome b559 content in isolated photosystem IIreaction center preparations Inmaculada Yruela1, Francisca Miota1, Elena Torrado1, Michael Seibert2and Rafael Picorel1 1 Estacio´n Exper
Trang 1Cytochrome b559 content in isolated photosystem II
reaction center preparations
Inmaculada Yruela1, Francisca Miota1, Elena Torrado1, Michael Seibert2and Rafael Picorel1
1
Estacio´n Experimental de Aula Dei (CSIC), Zaragoza, Spain;2National Renewable Energy Laboratory, Basic Sciences Center, Golden, CO, USA
The cytochrome b559 content was examined in five types
of isolated photosystem II D1-D2-cytochrome b559reaction
center preparations containing either five or six chlorophylls
per reaction center The reaction center complexes were
obtained following isolation procedures that differed in
chromatographic column material, washing buffer
compo-sition and detergent concentration Two different types of
cytochrome b559assays were performed The absolute heme
content in each preparation was obtained using the
oxidized-minus-reduced difference extinction coefficient of
cyto-chrome b559 at 559 nm The relative amount of D1 and
cytochrome b559a-subunit polypeptide was also calculated
for each preparation from immunoblots obtained using
antibodies raised against the two polypeptides The results
indicate that the cytochrome b559heme content in
photo-system II reaction center complexes can vary with the
isolation procedure, but the variation of the cytochrome b559
a-subunit/D1 polypeptide ratio was even greater This variation was not found in the PSII-enriched membrane fragments used as the RC-isolation starting material, as different batches of membranes obtained from spinach harvested at different seasons of the year or those from sugar beets grown in a chamber under controlled environmental conditions lack variation in their a-subunit/D1 polypeptide ratio A precise determination of the ratio using an RC1-control sample calibration curve gave a ratio of 1.25 cytochrome b559a-subunit per 1.0 D1 polypeptide in photo-system II membranes We conclude that the variations found in the reaction center preparations were due to the different procedures used to isolate and purify the different reaction center complexes
Keywords: chromatography; cytochrome b559; detergent; immunoblot; photosystem II
Cytochrome (Cyt) b559is a hemoprotein component of the
photosystem II (PSII) reaction center (RC) complex [1], and
it is an integral component of the minimal isolated RC
complex still capable of performing primary charge
separ-ation It is composed of two small polypeptides, the a
(9 kDa) and b (4.5 kDa) subunits, encoded by the psbE and
psbF genes, respectively Each polypeptide has a single
transmembrane a-helical domain [2,3] The heme iron is
bound to a single histidine residue on each subunit [4], and it
is located close to the stromal surface of the membrane
[2,3,5–7] However, a location for Cyt b559 heme on the
lumenal side of the PSII membrane has also been proposed,
suggesting that two hemes and two copies each of the two
subunits are present in the thylakoid membrane [8,9]
Despite numerous studies [8,10,11], the exact function of
Cyt b559is still unclear but the following are possibilities: (a) involvement in the electron transfer reactions on the oxidizing side of PSII [12,13]; (b) participation in the assembly of the water-splitting system [14]; and (c) protec-tion of PSII against photoinhibiprotec-tion [15–19] It is well known that Cyt b559can exist in a number of different redox forms At pH 6.0–6.5, PSII complexes, surrounded by their natural membrane environments, as in chloroplasts, thyla-koids and PSII membrane fragments, Cyt b559 exhibits midpoint redox potentials (E¢m) of +400 mV [the high potential (HP) form], +200–150 mV [the intermediate potential (IP) form], and +70–60 mV [the low potential (LP) form] [1,20–22] The HP form dominates in thyla-koids and PSII membranes with an intact water-oxidizing complex
A longstanding issue has been the number of Cyt b559per PSII complex Shuvalov and coworkers argued that PSII core complex from spinach with high O2-evolution activity contains two Cyt b559per PSII [8] However, the currently accepted value in isolated PSII RCs, based mainly on absorption spectroscopy techniques [1,23–26], is one heme per RC Recent data based on the crystal structure of the PSII core from Synecochoccus elongatus [2] and Thermo-synechococcus vulcanus [3] are in agreement with this proposal But a second cytochrome might have been lost during the preparation of the core material Thus the question of one or two Cyt b559 per PSII RC remains unresolved because the stoichiometry might depend on the isolation procedure used, the type of PSII preparation and/
or the organism examined
Correspondence to R Picorel, Estacio´n Experimental de Aula Dei
(CSIC), Ctra Montan˜ana 1005, Zaragoza E-50080, Spain.
Fax: + 34 976 716145; Tel.: + 34 976 716053;
E-mail: picorel@eead.csic.es
Abbreviations: Cyt, cytochrome; D1/D2 HD, heterodimer made by
crosslinking of D1 and D2 polypeptides; DM, n-dodecyl b- D
-malto-side; HP, high potential; IMAC, immobilized metal affinity
chroma-tography; IP, intermidiate potential; LP, low potential; Mes,
2-(N-morpholino) ethane-sulfonic acid; PS, photosystem;
RC, reaction center.
Enzymes: glucose oxidase (EC 1.1.3.4); catalase (EC 1.11.1.6).
(Received 23 January 2003, revised 21 March 2003,
accepted 26 March 2003)
Trang 2To address this issue, we have determined the D1 and
Cyt b559contents of various PSII RC preparations obtained
from market spinach and chamber-grown sugar beets The
cytochrome content was assayed by both
spectrophoto-metric and immunological methods The former measures
the heme content and the latter quantitates the amount of
protein present Note that it is not the aim of this work to
define the quality or performance of the different RC
preparations, but rather to examine the effect of several
isolation procedures on the Cyt b559 content of these
preparations The results presented in this work strongly
suggest that the Cyt b559 heme content and Cyt b559
a-subunit/D1 ratio are highly dependent on the RC
isolation procedure used; this ratio is close to one in all
PSII-enriched membranes tested
Materials and methods
Biological material
Sugar beet (Beta vulgaris L cv Monohill) was grown
hydroponically in a growth chamber on half-Hoagland
solution, under the following conditions: 325 lEinsteinsÆ
m)2Æs)1cool fluorescent light (16 h light period), 25C, and
80% humidity Spinach was purchased from the local
market at different times during the year
Preparation of PSII membranes
PSII-enriched membrane fragments were isolated according
to [27] with some modifications [25] Samples were
suspen-ded in 0.4M sucrose, 15 mM NaCl, 5 mM MgCl2 and
50 mM 2-(N-morpholino) ethane-sulfonic acid (Mes)/
NaOH (pH 6.0), frozen in liquid nitrogen and stored at
)80 C until use
Preparation of D1-D2-Cytb559complexes
Four RC preparations with about six chlorophyll (Chl) a per
two pheophytins (Pheo) and one preparation with about five
Chl per two Pheo were isolated from PSII-enriched
mem-brane fragments using modifications to the standard
proce-dure [23] This method solubilizes PSII-enriched membranes
at 1 mg ChlÆmL)1with 4% (w/v) Triton X-100 for 1 h After
centrifugation the resultant supernatant is loaded onto a
weak anion-exchange Toyopearl TSK DEAE-650(S)
col-umn pre-equilibrated with 50 mM Tris/HCl (pH 7.2) and
0.05% (w/v) Triton X-100 buffer The column was washed
extensively with the same buffer until the optical density of
the 417 nm peak (main Soret Pheo peak) was much higher
than that at 435 nm (main Soret Chl a peak) The material
was then eluted with a 50–200 mMNaCl linear gradient in
the same buffer Hereafter, we will call this preparation RC1
and consider it the standard control material Variations of
this procedure were performed as follows: RC2-Strong
anion-exchange Q-Sepharose Fast-Flow column and 0.1%
(w/v) Triton X-100 in 50 mMMes/NaOH (pH 6.5) washing
buffer [28]; RC3-Toyopearl TSK DEAE-650(S) column and
1% (w/v) Triton X-100 and 1.5% (w/v) taurine in 50 mM
Tris/HCl (pH 7.2) washing buffer [29]; and RC4-Toyopearl
TSK DEAE-650(S) column and 1% (w/v) Triton X-100 in
50 m Tris/HCl washing buffer [30] In each case, after
detergent exchange with 0.1% (w/v) n-dodecyl-b-D -malto-side (DM) to stabilize the RC [31], the RC complexes were eluted with a 60–300 mMlinear salt gradient in 50 mMMes/ NaOH (pH 6.5) and 0.1% (w/v) DM, and the fractions were collected at 1 mLÆmin)1 In some cases, the preparations obtained as indicated above were exposed to an additional chromatography step using a copper immobilized metal affinity chromatographic [Cu(II)-IMAC] column To pre-pare the Cu(II)-IMAC column, 100 mL of 0.1MCuSO4in distilled water were passed through a Chelating-Sepharose Fast-Flow column (Amersham-Pharmacia, 1· 10 cm) Excess copper was eliminated by washing with 100 mL distilled water The column was equilibrated with 50 mL of
50 mMNa2HPO4(pH 6.5), 50 mMNaCl, 0.2% (w/v) Triton X-100 and 1.2 mMDM Then the RC samples were diluted sixfold in 50 mM Mes/NaOH (pH 6.5), loaded onto the Cu(II)-IMAC column and washed with 50 mMMes/NaOH (pH 6.5), 50 mM NaCl, 0.2% (w/v) Triton X-100 and 1.2 mM DM buffer The samples were then eluted with 1–10 mM imidazole linear gradient in 50 mM Mes/NaOH (pH 6.5) and 0.1% (w/v) DM RC5, a preparation contain-ing about five Chl per two Pheo, was isolated from PSII-enriched membranes following the method described in [32] using the Cu(II)-IMAC column described above All the isolation steps were done at 4C in the dark The pigment composition of the isolated RC complexes was determined
as described in [33]
Spectroscopy The RC Cyt b559 heme content was measured spectro-photometrically To measure the dithionite-reduced minus ferricyanide-oxidized absorption spectra in the 510–
600 nm region, the RC samples were diluted to an optical density of 0.6–1.2 absorption units at the red maximum peak at around 675.5 nm with a buffer containing 50 mM
Mes/NaOH (pH 6.5) and 0.1% (w/v) DM (this buffer yields more transparent D1-D2-Cyt b559 complex suspensions than Tris/HCl buffers at higher pH) A differential extinc-tion coefficient of 21 mM )1Æcm)1at the maximun at 559 nm minus the minimum at around 570 nm [1] was used to determine the heme content of the different preparations Difference absorption spectra were recorded using 1 cm optical pathlength cuvettes at 10C with a Beckman DU
640 spectrophotometer Constant temperature was main-tained using a circulating bath (MultiTempII, Amersham-Pharmacia) Samples were oxidized with 2 mMferricyanide and then reduced by adding 1 lL of a saturated solution of sodium dithionite prepared in 10 mMTris/HCl, pH 7.5 (at this pH the dithionite is more stable than at lower pH) and maintained in an ice-pocket The addition of another lL of saturated solution did not further increase the absorption at
559 nm, demonstrating that the Cyt b559 content was completely reduced with the first dithionite addition It should be noticed that the same results were obtained without the addition of ferricyanide because the Cyt b559 from D1-D2-Cyt b559 complex preparations as obtained from the chromatography column is always in the oxidized state All of the measurements were carried out under anaerobic conditions maintained by adding 0.23 mgÆmL)1 glucose oxidase (Sigma, EC 1.1.3.4), 80 lgÆmL)1 catalase (Sigma, EC 1.11.1.6) and 10 m glu cose to the sample [31]
Trang 3SDS/PAGE and immunoblot
Electrophoresis was carried out as in [34] using a 4% (w/v)
acrylamide stacking gel and a 12–20% (w/v) acrylamide
linear gradient resolving gel containing 6Murea To avoid
the interference of lipids and detergents during the
electro-phoresis, the RC samples were diluted 10-fold in 1 : 1
ethanol/acetone (v/v), incubated for 1 h at 20C, and
centrifuged at 9000 g for 10 min at 4C [35] The pellet
containing the protein was resuspended in 50 mM Mes/
NaOH (pH 6.5) The samples were diluted 1 : 1 in 2% (w/v)
SDS, 2M urea, 40 mM dithiothreitol, and 50 mM Mes/
NaOH (pH 6.5), and then denaturated at room temperature
for 50 min Replicate gels were ru n u nder the same
conditions at the same time, and the proteins were
transferred onto nitrocellulose membranes for
immuno-detection using a Bio-Rad Mini Trans-Blot Cell The
transfer buffer was 25 mM Tris/HCl (pH 7.5), 192 mM
glycine and 20% methanol After protein transfer, one of
the blots was probed with rabbit antibodies raised against a
synthetic peptide homologous to the N-terminus of the
spinach PSII D1 protein and the other with rabbit antibodies
raised against the spinach a-subunit of Cyt b559[36] The
standard peroxidase development procedure with
4-chloro-1-naphthol as the substrate was used to visualize the blots
The gels and blots were scanned with a Studio Scan II Si
(AGFA), and the intensity of the bands was quantified by
densitometry using US National Institute of Health
Software (NIH IMAGE) available at http://www.ncbi.nih.gov
Results and discussion
We examined the Cyt b559content of five different
D1-D2-Cyt b559PSII RC preparations containing either five or six
Chl per RC Dithionite-reduced minus ferricyanide-oxidized
absorption spectroscopy shows that the absolute Cyt b559
heme stoichiometry varied between 0.91 and 1.41 hemes per
two Pheo (Table 1) This method is commonly used to
determine the Cyt b559content in PSII RC preparations [1]
and shows that heme content can vary depending on the
specific RC isolation procedure
In RC1, the standard preparation for the purpose of this
paper, we measured slightly more than six Chl per two Pheo
(Table 1) The PSII RC is known to contain two Pheo [2,3]
RC2 contained a little less Chl (6.18) but more Cyt b559
heme (1.11) per two Pheo compared to RC1 RC3
contained less Chl (5.80) but more Cyt b559 heme (1.19)
than RC1 and RC2 RC4 contained 6.05 Chl and even more
Cyt b559heme (1.41) RC5 had about five Chl per two Pheo
as expected and 1.12 Cyt b heme per two Pheo The only
significant difference between the RC1 through the RC4 procedures was the concentration of Triton X-100 used during RC isolation wash steps, and the presence of 1.5% (w/v) taurine in RC3 Despite the different Cyt b559contents all the preparations showed normal room temperature absorption spectra, i.e the six Chl preparations with maxima at 675.5 nm and the five Chl preparation with a maximum at around 677 nm This indicates that the cytochrome content has nothing to do with the spectral quality of the preparations
In order to compare the actual Cyt b559protein content
of each preparation rather than the heme content as above,
we used polyclonal antibodies against D1 and the a-subunit
of Cyt b559to assess changes in the ratio of the polypeptide levels in the different RC preparations Figure 1 shows immunoblots using antibodies against the D1 polypeptide (upper box) and the a-subunit (lower box) No D1 degradation product, little D1/D2 heterodimer and no Cyt b559aggregate formation were detected in any of our preparations Table 2 shows the relative Cyt b559a-subunit/ D1 integrated densities of the two respective bands in the blot (Fig 1) RC1 and RC2 show similar ratios, but RC3 and especially RC4 and RC5 showed much higher ratios The results of Tables 1 and 2 demonstrate that increasing the concentration of Triton X-100 during RC washing procedure leads to a higher Cyt/D1 ratio both on a heme and a protein basis However, the variation of the Cyt b559 a-subunit to D1 ratio with the detergent concentration was more dramatic in the case of the immunoblot data (Table 2) compared to the spectrophotometric data (Table 1) This indicates that free a-subunit polypeptide (with no heme) from degraded cytochrome must bind preferentially to the column and is coeluted with the native RC complex Note
Table 1 Pigment and cytochrome b 559 (heme) content determined spectrophotometrically for various D1-D2-Cyt b 559 PSII RC complexes purified using different procedures, as described in Materials and methods Values represent means ± SE (n ¼ 4) Values in parentheses represent the Triton X-100 concentration used during the isolation washing procedure.
Cyt b 559
a
a This row also represents the Cyt b 559 heme/RC ratio.
Fig 1 Immunoblots of several isolated D1-D2-Cyt b 559 PSII RC preparations using anti-D1 (upper box) and anti-Cyt b 559 a-subunit (lower box) serum Lane 1, RC1; lane 2, RC2 ; lane 3, RC3 ; lane 4, RC4; lane 5, RC5.
Trang 4that the difference spectroscopy employed only detects the
heme in native Cyt b559, but the antibody blots detect all
a-subunit polypeptide present Taurine used to prepare
RC3 is a chaotropic agent that is sometimes used to strip off
loosely bound contaminants to give cleaner protein
pre-parations, and thus RC3 exhibits lower Cyt b559 protein
content than RC4 (prepared the same way, but without
taurine) In the case of the IMAC procedure (RC5), the
heme content was a little higher than in RC1, but the
a-subunit/D1 ratio was even higher than that in RC4 This
result occurs despite the lower Triton X-100 concentration
used (0.2% [w/v] in RC5 compared to 1% [w/v] in RC4)
The simplest explanation for the much higher level of
a-subunit polypeptide content compared to heme content
in RC5 results from a consideration of the chemistry of the
Cu(II)-IMAC column The Cu(II), linked to the column
matrix, binds to any available histidine residue on the
surface of a protein As Cyt b559is on the surface of the
D1-D2-Cyt b559complex [2,3,6], the two histidines that bind
the heme are prone to be attack by Cu(II) As a consequence
the heme is displaced and washed out of the column, but the
free polypeptide subunits remain bound to the
Cu(II)-IMAC column by the histidine residues until coeluted with
the native RC complex after the application of the imidazole
elution step In order to examine this hypothesis, RC2 and
RC3 were passed through a Cu(II)-IMAC column, and the
results are shown in Fig 2 Both preparations exhibit a
higher a-subunit/D1 ratio after passing through the
Cu(II)-IMAC column, i.e the RC2 and RC3 showed a
densito-metric ratio of 1.02 and 1.32, respectively, after the column
compared to 0.62 and 1.09 before the column
The results reported here indicate that the Cyt b559
content in the PSII RC complex can be altered by the
purification procedure In order to confirm that these variations did not result from variations in Cyt b559content
in the starting PSII membranes, we analysed several batches
of market spinach harvested during different times of the year and sugar beets grown under controlled environmental conditions in a growth chamber Figure 3 shows the blots using antibodies against D1 (upper box) and Cyt b559 a-subunit (lower box) All of the PSII membrane batches had similar Cyt b559/D1 ratios, independent of the harvest time of the year, growth conditions or plant species (the spectrophotometric method also confirmed the presence of similar concentrations of Cyt b559in all PSII membranes, data not shown) The absolute Cyt b559/D1 ratio in the membranes was calculated using a calibration curve gener-ated with different amounts of RC1 control material Figure 4A shows the blot from the gel containing different amounts of RC1 control sample that was used to generate the standard correlation curve represented in Fig 4B The gel also contains a duplicate of amounts of PSII membranes (Fig 4A) corresponding to the Cyt b559 and D1 content that fits within the correlation curve The densitometric values obtained from the blot for the a-subunit and D1 from the membranes were introduced in the generated calibration curve to calculate the absolute ratio of these polypeptide content present in membranes considering that ratio 1 : 1 in RC1 control sample This absolute ratio resulted in 1.25 Cyt b559a-subunit per D1 polypeptide Assuming that RC1 control sample, obtained using the lowest Triton X-100 concentration (0.05%, w/v), contains one Cyt per D1, we can conclude that: (a) PSII-enriched membranes from higher plants contain a little more than one but certainly less than two Cyt b559a-subunits per D1 polypeptide; (b) both the Cyt b559 heme and a-subunit contents of D1-D2-Cyt b559complex depend on the puri-fication procedure used to obtain the preparations; (c) a
Table 2 Relative cytochrome b 559 a-subunit/D1 integrated density ratios of the Western blot bands in Fig 1 All the ratios were normalized to the value of RC1 (the standard control preparation) in Table 1 The absolute densitometric band ratio of RC1 was 0.81 Values represent means ± SE (n ¼ 4).
a
Contained Taurine.
Fig 2 Immunoblots of RC2 and RC3 preparations before and after
passing through a Cu(II)-IMAC column Lane 1, RC2; lane 2, RC2
after IMAC column; lane 3, RC3; lane 4, RC3 after
Cu(II)-IMAC column.
Fig 3 Immunoblots of PSII membrane preparations from market spinach obtained in autumn (lane 1), winter (lane 2), spring (lane 3), and from sugar beets (lane 4) grown in a growth chamber under controlled environmental conditions Upper box: immunodetection with antibody against D1 protein; lower box: immunodetection with antibody against Cyt b a-subunit.
Trang 5high Triton X-100 concentration during the
chromato-graphic washing steps clearly increases both the heme and
the a-subunit content per RC; and (d) RC preparations
using Cu(II)-IMAC exhibit a very high a-subunit
poly-peptide compared to their heme content
Acknowledgements
The authors thank M V Ramiro for her helpful technical assistance.
We are indebted to Drs A K Matto and R Barbato for their kind gifts
of antibodies against the D1 and Cyt b 559 polypeptides, respectively.
E T was recipient of a predoctoral fellowship from the CONSI + D
(Diputacio´n General de Arago´n) This work was supported by the
Ministry of Science and Technology of Spain (Grant PB98-1632 and
BMC2002-00031) (RP) and by the Division of Energy Biosciences,
Office of Science, U.S Department of Energy (under Contract
#DE-AC36–99G010337) (MS).
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