The extrinsic 33-kDa protein of photosystem II
(PSII) was intramolecularly cross-linked by a zero-length cross-linker,
1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. The resulting
cross-linked 33-kDa protein rebound to urea/NaCl-washed PSII membranes,
which stabilized the binding of manganese as effectively as the
untreated 33-kDa protein. In contrast, the oxygen evolution was not
restored by binding of the cross-linked protein, indicating that the
binding and manganese-stabilizing capabilities of the 33-kDa protein
are retained but its reactivating ability is lost by intramolecular
cross-linking of the protein. From measurements of CD spectra at high
temperatures, the secondary structure of the intramolecularly
cross-linked 33-kDa protein was found to be stabilized against heat
treatment at temperatures 20 °C higher than that of the untreated
33-kDa protein, suggesting that structural flexibility of the 33-kDa
protein was much decreased by the intramolecular cross-linking. The
rigid structure is possibly responsible for the loss of the
reactivating ability of the 33-kDa protein, which implies that binding
of the 33-kDa protein to PSII is accompanied by a conformational change
essential for the reactivation of oxygen evolution. Peptide mapping,
N-terminal sequencing, and mass spectroscopic analysis of
protease-digested products of the intramolecularly cross-linked 33-kDa
protein revealed that cross-linkings occurred between the amino group
of Lys48 and the carboxyl group of
Glu246, and between the carboxyl group of Glu10
and the amino group of Lys14. These cross-linked amino acid
residues are thus closely associated with each other through
electrostatic interactions.
 |
INTRODUCTION |
PSII1 is a multisubunit
pigment-protein complex which catalyzes the light-driven oxidation of
water to molecular oxygen and the reduction of plastoquinone to
plastoquinol. The minimum unit for PSII capable of oxygen evolution
under physiological conditions contains seven major intrinsic proteins
of the reaction center peptides D1 and D2, two apoproteins of
cytochrome b559, psbI gene product,
and two chlorophyll-binding peptides CP47 and CP43, and three extrinsic
proteins of 33, 23, and 17 kDa which are associated with the lumenal
surface of thylakoid membranes (1-3). The extrinsic 23- and 17-kDa
proteins play a role in regulating the PSII affinity for calcium and
chloride, and can be removed by treatment with 1.0-2.0 M
NaCl (4-9). In cyanobacterial and red algal PSII, these two extrinsic
proteins are absent, but a low-potential cytochrome c550 and a 12-kDa protein have been found as the
alternative extrinsic components (10-12).
The extrinsic 33-kDa protein, on the other hand, is present in all
oxygenic photosynthetic organisms from cyanobacteria to higher plants
and plays an important role in stabilizing binding and maintaining
functional conformation of the manganese cluster which directly
catalyzes the H2O-splitting reaction (for reviews, see
Refs. 13-15). Removal of the 33-kDa protein from PSII membranes by
washing with high concentration of divalent cations (16) and urea plus
NaCl (17) significantly decreases the oxygen evolving activity but the
activity can be considerably restored by rebinding of the protein
(17-19). Removal of the protein also leads to a gradual liberation of
two out of the four manganese per PSII (20). Immobilization of the
protein with PSII intrinsic components by a water-soluble carbodiimide,
EDC, which directly links amino and carboxyl groups in contact,
prevents release of the protein upon CaCl2 or urea/NaCl
wash or heat treatment, thereby stabilizing binding of the manganese
cluster and the oxygen evolving activity against these treatments
(21-23). Based on these studies, the association of the 33-kDa protein
with PSII has been proposed to involve electrostatic interactions
between positive and negative charges on both the extrinsic protein and
some PSII intrinsic subunits as well as hydrogen bonding (21, 24-26).
Recently, we showed that positive charges on the 33-kDa protein are
important for the electrostatic interaction with PSII intrinsic
proteins, whereas negative charges on the protein do not contribute to
such interaction (27). Furthermore, we proposed that the positive
charges of
-amino groups on Lys4, Lys20,
Lys66 or Lys76, Lys101,
Lys105, Lys130, Lys159,
Lys186, and one or two Lys in
Lys230-Lys236 in the extrinsic 33-kDa protein
electrostatically interact with negative charges on PSII intrinsic
proteins (27).
Reconstitution experiments have indicated that the three extrinsic
proteins bind to the PSII complex in the order of the 33-, 23-, and
17-kDa proteins (28-30). Among these three extrinsic proteins, the
33-kDa protein is required for stoichiometric and functional binding of
the 23-kDa protein, whereas both the 33- and 23-kDa proteins are
required for functional binding of the 17-kDa protein. The extrinsic
proteins cannot, however, directly bind to each other when they are not
associated with the PSII complex (24). These results suggest that
binding of the extrinsic proteins to the PSII complex alters the
conformation of the extrinsic proteins themselves and/or that of the
intrinsic part of the complex so as to create the binding sites for the
other extrinsic proteins (24).
To examine whether conformational changes occur with the 33-kDa protein
accompanying its binding to PSII and the possible importance of such
conformational changes, we performed intramolecular cross-linking of
the 33-kDa protein with a water-soluble carbodiimide, EDC, by which the
conformational changes of the protein are expected to be suppressed.
The intramolecularly cross-linked 33-kDa protein was found to retain
the rebinding and manganese-stabilizing capabilities but not the
reactivating ability, suggesting that a suitable flexibility of the
33-kDa protein is needed for its full functioning in oxygen evolution.
 |
MATERIALS AND METHODS |
Preparation and Cross-linking--
Oxygen-evolving PSII
membranes were prepared from spinach chloroplasts with Triton X-100 as
in Ref. 31, with slight modifications as described in Ref. 21. The PSII
membranes were suspended in medium A containing 40 mM Mes
(pH 6.5), 0.4 M sucrose, 10 mM NaCl, and 5 mM MgCl2 and stored in liquid nitrogen until
use. The extrinsic 33-kDa protein was extracted from the PSII membranes
by 1 M CaCl2 treatment (16) and purified
according to Refs. 27 and 32. For cross-linking, the purified 33-kDa
protein was passed through a Sephadex G-25 column equilibrated with
distilled water. The concentration of the 33-kDa protein was determined
using an extinction coefficient of 16 mM
1
cm
1 at 276 nm (33).
Intramolecular and intermolecular cross-linking of the 33-kDa protein
was carried out in a solution containing 4 µM 33-kDa protein and 1 mM EDC at 25 °C for 12 h. After the
reaction was stopped by adding 100 mM sodium acetate, the
reaction mixture was passed through a Sephadex G-25 column equilibrated
with 20 mM phosphate buffer (pH 6.6). The pH of the protein
mixture was then adjusted to pH 10 with concentrated NaOH solution and
incubated at 25 °C for 2 h to recover carboxyl groups from
modification by EDC. After the pH was returned to 6.5 by adding 500 mM Mes (pH 6.5), the cross-linked 33-kDa protein was
concentrated by ultrafiltration and then passed through a Sephacryl
S-100HR column equilibrated with 100 mM Mes (pH 6.5) to
separate the intramolecularly and intermolecularly cross-linked
products.
Reconstitution, Oxygen Evolution, and Electrophoresis--
For
reconstitution, native PSII membranes were washed with 2.6 M urea, 0.2 M NaCl to remove the three
extrinsic proteins of 33, 23, and 17 kDa (17). The resulting PSII
membranes were incubated with either the native or the intramolecularly
cross-linked 33-kDa protein at a protein to Chl ratio of 0.6 (w/w), in
medium A at 0 °C for 30 min in the dark at a Chl concentration of
0.5 mg/ml. The reconstituted PSII membranes were collected by
centrifugation at 35,000 × g for 10 min, then washed
once with and resuspended in medium A. Oxygen evolution was measured at
25 °C with a Clark-type oxygen electrode in medium A, to which 5 mM CaCl2 and 0.4 mM
phenyl-p-benzoquinone were supplemented. Chl concentration
was determined by the method of Porra et al. (34).
SDS-polyacrylamide gel electrophoresis was carried out according to
Laemmli (35), with a slab gel of 11.5% acrylamide containing 6 M urea. Samples were solubilized with 5% lithium lauryl
sulfate and 75 mM dithiothreitol. After electrophoresis,
gels were stained with Coomassie Brilliant Blue and photographed.
Measurements of Manganese Contents and CD Spectra--
For
determination of manganese released from urea/NaCl-washed PSII
membranes reconstituted with the native or intramolecularly cross-linked 33-kDa protein, membrane suspensions after incubation for
0-48 h at 0 °C in the dark were centrifuged at 35,000 × g for 10 min and manganese remaining in the supernatant was
assayed with a Hitachi polarized Zeeman atomic absorption
spectrophotometer (Z-8000). Amounts of manganese per PSII were
estimated by assuming the antenna size of PSII as 250 Chl. The circular
dichroism (CD) measurements of the native or intramolecularly
cross-linked 33-kDa protein were performed with a JASCO J-500A
spectropolarimeter as described in Ref. 36.
Protease Digestion, HPLC Separation, Mass Spectroscopic, and
N-terminal Sequencing Analysis--
The untreated and intramolecularly
cross-linked 33-kDa proteins were denatured in 8 M urea and
25 mM Mes (pH 6.5) at 37 °C for 15 h, and then 3 M Tris-HCl (pH 8.5) was added to a final concentration of 2 M urea. The denatured 33-kDa protein was digested first
with lysyl endopeptidase at a protein to lysyl endopeptidase ratio of
50 (w/w) for 15 h at 37 °C, and then with another lysyl endopeptidase at the same concentration for 15 h at 37 °C. The digested mixture was subjected to a reversed phase column
(Bondasphere 5µ C4 300A, Waters Inc.) in an HPLC set-up (LC-9A,
Shimadzu Inc., Japan). The column was eluted with a gradient of 0-75%
acetonitrile in 0.1% trifluoroacetic acid at a flow rate of 1 ml/min,
and the elution pattern was monitored at 210 nm. Each fraction was
collected, dried, and resolubilized in 2 µl of 67% acetic acid out
of which, 1 µl was mixed with a same volume of matrix (a mixture of
1:1 volume of glycerol and 3-nitrobenzyl alcohol), and analyzed with a
fast atom bombardment mass spectrometer (JEOL JMS HX-110) at a voltage
of 10 kV with xenon as the ionization gas. The resulting mass spectra
were analyzed with a DA5000 data system and assigned to the known
protein sequence. The peptides obtained from HPLC were also analyzed
for their N-terminal sequence by Edman degradation of the peptides
followed by sequence analysis with a protein sequencer (Applied
Biosystem, model 477A and 476A).
 |
RESULTS |
Preparation of Intramolecularly Cross-linked 33-kDa
Protein--
To suppress the formation of intermolecularly
cross-linked products, a diluted solution of the 33-kDa protein (4 µM) was used for treatment of EDC. A small amount of
intermolecularly cross-linked product was, however, formed as shown in
Fig. 1. The polypeptide pattern of the
EDC-treated 33-kDa protein showed two Coomassie Brilliant Blue-stained
bands with apparent molecular masses of about 55 and 27 kDa (lane
1), which correspond to dimer and monomer of the 33-kDa protein
resulting from intermolecular and intramolecular cross-linking,
respectively. No trimer and polymer are formed under the conditions
employed here. The intermolecularly (dimer) and intramolecularly
(monomer) cross-linked products of the 33-kDa protein could be clearly
separated by a Sephacryl S-100HR column (lanes 2 and
3); upon electrophoresis, the intramolecularly cross-linked 33-kDa protein migrated faster and appeared as a broader band as
compared with the untreated protein.

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Fig. 1.
SDS-polyacrylamide gel electrophoresis of
EDC-treated 33-kDa protein. The purified 33-kDa protein was
treated with EDC as described under "Materials and Methods"
(lane 1), and then passed through a Sephacryl S-100HR
column, by which two protein peaks were separated. The first peak
(lane 2) corresponded to a dimer and the second peak
(lane 3) to a monomer.
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Reconstitution of the Intramolecularly Cross-linked 33-kDa Protein
to PSII--
The three extrinsic proteins of 33, 23, and 17 kDa were
removed by washing PSII with 2.6 M urea plus 0.2 M NaCl (lanes 1, 2, Fig.
2) (17). The native 33-kDa protein was
able to rebind to urea/NaCl-washed PSII completely (lane 3).
Because the intramolecularly cross-linked 33-kDa protein migrated
faster than the native 33-kDa protein and thus comigrated with the
large amount of LHC II bands, detection of the 33-kDa protein
reconstituted was done as follows. PSII membranes reconstituted with
the native or intramolecularly cross-linked 33-kDa protein were again
treated with 2.6 M urea plus 0.2 M NaCl and
centrifuged at 35,000 × g for 10 min. The supernatants
were concentrated 5-fold by ultrafiltration as the intramolecularly
cross-linked 33-kDa protein showed a much weaker Coomassie Brilliant
Blue staining intensity than the native 33-kDa protein. The result
shown in lane 4R of Fig. 2 indicates that the
intramolecularly cross-linked 33-kDa protein rebound to PSII to a
significant amount. To estimate the amount of the intramolecularly cross-linked 33-kDa protein rebound, the 33-kDa protein corresponding to the amount when the protein was completely rebound was
electrophoresed together (lanes 3C and 4C). The
comparison of Coomassie Brilliant Blue staining intensities in
lanes 4C and 4R revealed that the intramolecularly cross-linked 33-kDa protein was completely rebound to
PSII. It should be noted that the band of the intramolecularly cross-linked 33-kDa protein rebound (lane 4R) showed a
similar broad band as the original cross-linked protein (lane
4C). If this is due to heterogeneous cross-linking, the results
suggested that the 33-kDa protein was able to rebind to PSII
irrespective of the heterogeneous cross-linking. These results indicate
that the binding sites of the 33-kDa protein remain intact after
intramolecular cross-linking.

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Fig. 2.
Reconstitution of the native or
intramolecularly cross-linked 33-kDa protein with urea/NaCl-washed PSII
membranes. Lane 1, control PSII membranes (0.02 mg of Chl);
lane 2, urea/NaCl-washed PSII membranes (0.02 mg of Chl);
lane 3, urea/NaCl-washed PSII membranes reconstituted with
the native 33-kDa protein (0.02 mg of Chl); lane 4,
urea/NaCl-washed PSII membranes reconstituted with the intramolecularly
cross-linked 33-kDa protein (0.02 mg of Chl); lane 3C, the
native 33-kDa protein (0.4 nmol); lane 3R, the native 33-kDa
protein extracted from urea/NaCl-washed PSII membranes (0.1 mg of Chl,
corresponding to about 0.4 nmol of PSII reaction center assuming there
is 250 Chl/PSII) which had been reconstituted with the native 33-kDa
protein; lane 4C, the intramolecularly cross-linked 33-kDa
protein (0.4 nmol); lane 4R, the intramolecularly cross-linked 33-kDa protein extracted from urea/NaCl-washed PSII membranes (0.1 mg of Chl) which had been reconstituted with the intramolecularly cross-linked 33-kDa protein.
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Table I shows oxygen-evolving activity of
urea/NaCl-washed PSII membranes reconstituted with the native or
intramolecularly cross-linked 33-kDa protein. Removal of the three
extrinsic proteins by urea/NaCl wash reduced the oxygen evolving
activity to 4% of the original activity. The activity was restored to
66% by reconstitution with the native 33-kDa protein, whereas it was
scarcely restored by reconstitution with the intramolecularly
cross-linked 33-kDa protein. This indicates that the intramolecularly
cross-linked 33-kDa protein lost its reactivating ability of oxygen
evolution even though it was completely rebound to PSII.
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Table I
Reactivation of oxygen evolution by reconstitution of the native or
intramolecularly cross-linked 33-kDa protein with urea/NaCl-washed PSII membranes
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When urea/NaCl-washed PSII membranes lacking the three extrinsic
proteins were incubated at 0 °C in the dark, two out of the four
manganese per PSII released after 48 h (Fig.
3) (20). Reconstitution of the native
33-kDa protein effectively suppressed the release of manganese (Fig.
3). Reconstitution of the intramolecularly cross-linked 33-kDa protein
also suppressed the manganese release equally effectively (Fig. 3).
These results indicate that the cross-linked 33-kDa protein still
retained its ability to stabilize binding of the manganese cluster.

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Fig. 3.
Stabilization of manganese binding by
reconstitution of the native or intramolecularly cross-linked 33-kDa
protein with urea/NaCl-washed PSII membranes. The urea/NaCl-washed
PSII before or after reconstitution with the native or intramolecularly cross-linked 33-kDa protein was incubated at 0 °C in the dark, and
its manganese content bound was determined at the designated time
points.
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Structural Flexibility of the 33-kDa Protein--
To examine the
changes on structural flexibility of the 33-kDa protein by
intramolecular cross-linking, the far-ultraviolet circular dichroism
spectra (CD spectra) of the native and the intramolecularly
cross-linked 33-kDa protein were measured at high temperatures (Fig.
4). The secondary structure of the native 33-kDa protein was appreciably affected at 60 °C and its random conformation appeared at 70 °C (Fig. 4A). On the
contrary, the CD spectral changes of the intramolecularly cross-linked
33-kDa protein required much higher temperature than those of the
native 33-kDa protein, and the random conformation of the
intramolecularly cross-linked 33-kDa protein appeared at 90 °C (Fig.
4B). These results indicate that structural flexibility of
the 33-kDa protein was significantly decreased by intramolecular
cross-linking.

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Fig. 4.
CD spectra of the native (A) and
intramolecularly cross-linked (B) 33-kDa protein at various
temperatures.
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Identification of the Intramolecular Cross-linking Sites--
Fig.
5 shows the peptide maps of the native
(A) and the intramolecularly cross-linked 33-kDa protein
(B) which had been digested with lysyl endopeptidase and
separated by reversed-phase HPLC. Peptide peaks 2, 3, 4, and
6 drastically decreased and new peptide peaks A
and B appeared by intramolecular cross-linking. Therefore, peaks A and B are expected to be the peptides containing
intramolecularly cross-linked sites. To identify the intramolecularly
cross-linked sites, N-terminal amino acid sequences and molecular
masses of these two peptides were determined (Table
II). Two amino acid sequences were
detected by Edman degradation of peptide peak A, which completely
agreed with amino acid sequences from
Tyr45-Lys49 and
Ile237-Gln247 of the 33-kDa protein, in which
only Lys48 and Glu246 were not detected.
Peptide peak A had a measured mass of 1927.10 Da, which is consistent
with the predicted mass of Tyr45-Lys49 plus
Ile237-Gln247 minus one molecule of
H2O (1927.18 Da). These results clearly indicate that
peptide peak A is the intramolecularly cross-linked product between the
amino group of Lys48 in Tyr45-Lys49
and the carboxyl group of Glu246 in
Ile237-Gln247. The N-terminal sequence of
peptide peak B agreed with Arg5-Lys20 of the
33-kDa protein, in which Glu10 and Lys14 were
not detected. A measured mass of peptide peak B (1968.00 Da) was
consistent with the predicted mass of
Arg5-Lys20 minus one molecule of
H2O (1968.20 Da). These indicate that peptide peak B is the
intramolecularly cross-linked product between the carboxyl group of
Glu10 and the amino group of Lys14 in
Arg5-Lys20. These results indicate that amino
acid residues between Lys48 and Glu246 and
between Glu10 and Lys14 are closely associated
with each other through electrostatic interaction.

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Fig. 5.
HPLC elution pattern of the lysyl
endopeptidase digests of the native (A) or intramolecularly
cross-linked (B) 33-kDa protein after digestion with lysyl
endopeptidase.
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Table II
N-terminal amino acid sequences and molecular masses of the peptide
peaks A and B shown in Fig. 5 and their assignments
Amino acids which were not detected under sequencing analysis are shown
in parentheses.
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Peptide peaks 2, 3, 4, and 6, which were significantly decreased by
intramolecular cross-linking, were found to be
Gly160-Lys186,
Ile237-Gln247,
Thr15-Lys44, and
Leu77-Lys101, respectively, by mass
spectrometeric analysis (data not shown). Peptide peaks 3 and 4 are
involved in the intramolecularly cross-linked peptide peaks A and B. The cross-linked products containing peptide peaks 2 and 6 were not,
however, found on the peptide map of lysyl endopeptidase digests of the
intramolecularly cross-linked 33-kDa protein (Fig. 5B). It
is likely that these cross-linked products were retained in and not
eluted from the reversed-phased HPLC column. Thus, in addition to the
intramolecular cross-linking between Lys48 and
Glu246 and between Glu10 and Lys14,
intramolecular cross-linkings containing the peptide of
Gly160-Lys186 and
Leu77-Lys101 seem to be formed.
 |
DISCUSSION |
Reactivation Mechanism of Oxygen Evolution by Binding of the 33-kDa
Protein--
The present results demonstrated that the rebinding and
manganese-stabilizing capabilities of the 33-kDa protein were retained but its reactivating ability was lost by intramolecular cross-linking of the protein with EDC. This implies that different mechanisms exist
for the stabilization of manganese binding and reactivation of oxygen
evolution; the latter but not the former was impaired by the
intramolecular cross-linking. Several possibilities may be considered
as responsible for loss of the reactivating ability, e.g. an
inhibition of homodimerization of the 33-kDa protein which might be
essential for its functioning, a loss of the function of the 33-kDa
protein in maintaining binding of Ca2+ and/or
Cl
(37), and a suppression of conformational changes of
the 33-kDa protein possibly accompanying its binding to PSII. Our
present results support the hypothesis that a conformational change
occurred accompanying binding of the 33-kDa protein, and this
conformational change was suppressed by the intramolecular
cross-linking, since the secondary structure of the protein was
significantly stabilized against heat treatment by intramolecular
cross-linking (Fig. 4). This suggests that the structural flexibility
of the 33-kDa protein was remarkably reduced. In addition, while
urea/NaCl-washed PSII membranes reconstituted with the native 33-kDa
protein completely rebound the 23-kDa protein, the PSII reconstituted
with the intramolecularly cross-linked 33-kDa protein scarcely rebound
the 23-kDa protein (data not shown). Since the 23-kDa protein cannot
directly associate with the 33-kDa protein in solution (24), these
results suggest that binding of the 33-kDa protein to PSII alters the
conformation of the protein itself which is essential for binding of
the 23-kDa protein. The occurrence of a structural change of the
protein is consistent with results of pH-dependent
structural changes (38), effects of genetic or chemical modification of
its disulfide-forming cysteines (39, 40), or the effects of
conformational constrains resulting from other amino acid substitutions
(41).
From the present and previous results, however, we cannot determine
whether the conformational change of the 33-kDa protein itself or a
further structural rearrangement of intrinsic PSII proteins
allosterically induced by binding of the 33-kDa protein is responsible
for the reactivation of oxygen evolution. The possible structural
changes of intrinsic PSII proteins upon binding of the 33-kDa protein
have been previously reported; for example, we recently showed that
removal of the 33-kDa protein makes the C-terminal region of CP43
accessible to trypsin, thus suggesting that removal of the protein at
the lumenal side induces a conformational change of the CP43 protein at
the stromal side (42). An effect on properties of the acceptor side of
PSII upon either biochemical removal of the 33-kDa protein (43) or
genetic deletion of the psbO gene encoding the 33-kDa
protein (44, 45) has also been reported based on thermoluminescence and
fluorescence measurements. Based on these results, we propose the
following mechanism for reactivation of oxygen evolution by binding of
the 33-kDa protein: binding of the 33-kDa protein to PSII alters the
conformation of the 33-kDa protein itself which allosterically results
in structural changes of intrinsic PSII proteins ligating the manganese
atoms, leading to formation of a functional conformation of the
manganese cluster and then the reactivation of oxygen evolution.
Structure of the 33-kDa Protein--
Although the primary
structure of the 33-kDa protein has been determined in various species
of plants (46), there is only very limited information concerning the
tertiary structure of the protein. Two Cys residues (Cys28
and Cys51) of the protein form a disulfide bond important
for maintaining the functional structure of the protein (40, 45, 47).
The secondary structural analysis of the 33-kDa protein in solution by
far-UV CD spectroscopy revealed that the protein contains a large
proportion of
-sheet and a relatively small amount of
-helical structure (48). Recently, we reported that the positive charges of
-amino groups on Lys4, Lys20,
Lys66 or Lys76, Lys101,
Lys105, Lys130, Lys159,
Lys186 and one or two Lys in
Lys230-Lys236 in the 33-kDa protein
electrostatically interact with negative charges on PSII intrinsic
proteins (27). This implies that these Lys residues are located on the
surface of the protein which interacts with PSII intrinsic proteins
when it binds to PSII. The present study revealed that the positive
charges of the amino group of Lys48 and Lys14
electrostatically associate with the negative charges of the carboxyl
group of Glu246 and Glu10, respectively, when
the protein is free in solution. This is very interesting, since we
have reported that the carboxyl group of Glu246 or C
terminus electrostatically interacts with the amino group of
Lys190 of the 33-kDa protein when the protein is associated
with PSII (49). This indicates that the carboxyl group in the
C-terminal region of the 33-kDa protein electrostatically interacts
with different amino groups, depending on whether the protein is free in solution or binds to PSII, which again suggests that the 33-kDa protein alters its conformation upon binding. Fig.
6 summarizes the localization of amino
acid residues interacting with each other in the spinach 33-kDa protein
which have been determined in the present and previous studies (27, 40,
47). Amino acids associated with each other through electrostatic
interaction or disulfide bond are connected with a line when
the protein is free in solution and with a dotted line when
the protein binds to PSII. Domains involving Lys residues which are
located in the regions interacting with PS II intrinsic proteins are
boxed. These results provide an important information for
the tertiary structure of the 33-kDa protein.

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Fig. 6.
Localization of amino acids closely
associated with each other (connected with a line) and Lys
residues located in the regions that interact with PSII intrinsic
proteins (boxed) in the primary structure of the 33-kDa
protein from spinach. Residues interacting with each other through
either electrostatic interaction or disulfide bond are connected with a
solid line when the protein is free in solution and with a
dotted line when the protein is associated with PSII.
Domains involving Lys residues interacting with PSII intrinsic proteins
are boxed.
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We thank Dr. Jian-Ren Shen of the Institute of
Physical and Chemical Research (RIKEN) for critical reading of the
manuscript.