Identification of domains on the extrinsic 23 kDa protein possibly involved in electrostatic interaction with the extrinsic 33 kDa protein in spinach photosystem II Akihiko Tohri 1,2 , Naoshi Dohmae 3 , Takehiro Suzuki 1 , Hisataka Ohta 1,4 , Yasunori Inoue 2,4 and Isao Enami 1 1 Department of Biology, Faculty of Science, Tokyo University of Science, Kagurazaka, Shinjuku-ku, Tokyo, Japan; 2 Department of Applied Biological Science, Faculty of Science and Technology, Tokyo University of Science, Yamazaki, Noda, Chiba, Japan; 3 Division of Biochemical Characterization, the Institute of Physical and Chemical Research (RIKEN), Hirosawa, Wako, Saitama, Japan; 4 Tissue Engineering Research Center, Tokyo University of Science, Yamazaki, Noda, Chiba, Japan To elucidate the domains on the extrinsic 23 kDa protein involved in electrostatic interaction with the extrinsic 33 kDa protein in spinach photosystem II, we modified amino or carboxyl groups of the 23 kDa protein to uncharged methyl ester groups with N-succinimidyl propionate or glycine methyl ester in the presence of a water-soluble carbodi- imide, respectively. The N-succinimidyl propionate-modified 23 kDa protein did not bind to the 33 kDa protein associ- ated with PSII membranes, whereas the glycine methyl ester- modified 23 kDa protein completely bound. This indicates that positive charges on the 23 kDa protein are important for electrostatic interaction with the 33 kDa protein associated with the PSII membranes. Mapping of the N-succinimidyl propionate-modified sites of the 23 kDa protein was per- formed using Staphylococcus V8 protease digestion of the modified protein followed by determination of the mass of the resultant peptide fragments with MALDI-TOF MS. The results showed that six domains (Lys11–Lys14, Lys27– Lys38, Lys40, Lys90–Lys96, Lys143–Lys152, Lys166– Lys174) were modified with N-succinimidyl propionate. In these domains, Lys11, Lys13, Lys33, Lys38, Lys143, Lys166, Lys170 and Lys174 were wholly conserved in the 23 kDa protein from 12 species of higher plants. These positively charged lysyl residueson the 23 kDa protein may be involved in electrostatic interactions with the negatively charged carboxyl groups on the 33 kDa protein, the latter has been suggested to be important for the 23 kDa binding [Bricker, T.M. & Frankel, L.K. (2003) Biochemistry 42, 2056–2061]. Keywords: extrinsic 23 kDa protein; extrinsic 33 kDa pro- tein; electrostatic interaction; chemical modification; oxygen evolution. Photosystem II (PSII) catalyzes the light-driven oxidation of water with concomitant reduction of plastoquinone to plastoquinol. This multisubunit protein-pigment complex contains a number of intrinsic proteins and 3–4 extrinsic proteins associated with the lumenal side of PS II. The three extrinsic proteins of 33, 23 and 17 kDa associate with higher plant and green algal PSII [1]. Their binding properties, however, are different between higher plant and green algal PSII. In higher plant PSII, the 33 kDa protein associates directly with PSII, but the 23 kDa protein cannot directly bind to PSII and associates with PSII only through its interaction with the 33 kDa protein, and the 17 kDa protein functionally associates with PSII only through its inter- action with both the 33 and 23 kDa proteins [2]. The 23 and 17 kDa proteins are easily released from higher plant PSII by washing with 1 M NaCl, indicating that the 23 kDa protein electrostatically binds to the 33 kDa protein [3], and the 17 kDa protein interacts electrostatically with both the 33 and 23 kDa proteins. In contrast, the green algal 23 and 17 kDa proteins can bind directly to PSII independent of the presence or absence of other extrinsic proteins [4]. On the other hand, cyanobacterial PSII contains three extrinsic proteins of 33 and 12 kDa, and cytochrome c550 [5], whereas, red algal PSII contains four extrinsic proteins of 33, 20 and 12 kDa, and cytochrome c550 [6,7]. The extrinsic proteins play important roles for maximal rates of oxygen evolution under physiological ionic condi- tions [1]. The 33 kDa protein is needed to maintain the functional conformation of the Mn cluster [8,9]. Shutova et al. found that titration of the 33 kDa protein against pH in solution exhibited a striking hysteresis [10], and proposed that the protein is not only required for stabilizing the Mn-cluster but also important for proton transport to occur appropriately, accompanying oxygen evolution [11]. The functions of the 23 and 17 kDa proteins are closely related with the unique requirement of Ca 2+ and Cl – for oxygen evolution; the 23 kDa protein mitigates the demand for Ca 2+ while the 17 kDa protein does for Cl – [8,12–14]. Correspondence to I. Enami, Department of Biology, Faculty of Science, Tokyo University of Science, Kagurazaka 1-3, Shinjuku-ku, Tokyo 162-8601, Japan. Tel.: + 81 4 7124 1501 (ext. 5022), E-mail: enami@rs.noda.tus.ac.jp Abbreviations: CBB, Coomasie brilliant blue; Chl, chlorophyll; CHC, a-cyano-4-hydroxycinnamic acid; DHB, 2,5-dihydroxybenzoic acid; EDC, 1-ethyl-3-(3-(dimethylamino)propyl) carbodiimide; GME, glycine methyl ester; MBT, 2-mercaptobenzothiazole; NHS, N-hydroxysuccinimido; NSP, N-succinimidyl propionate; PSII, photosystem II. (Received 28 October 2003, revised 9 January 2004, accepted 16 January 2004) Eur. J. Biochem. 271, 962–971 (2004) Ó FEBS 2004 doi:10.1111/j.1432-1033.2004.03998.x The extrinsic proteins of 12 kDa and cytochrome c550 in cyanobacterial and red algal PSIIs have a similar func- tion to that of the 23 and 17 kDa proteins in higher plant PSII [7,15–17]. Recently, Zouni et al. [18] and Kamiya and Shen [19] published the crystal structural analysis of thermophilic cyanobacterial PSII. These studies have provided important insights into the organization of numerous subunits of cyanobacterial PSII. The 33 kDa protein and cytochrome c550 appear to interact with the large extrinsic loop E of CP47 and with the large extrinsic loop E of CP43, respect- ively. The 12 kDa protein appears to interact with both the 33 kDa protein and cytochrome c550 [19]. These studies provided, however, few insights into the structural organ- ization of the 23 and 17 kDa proteins in higher plant PSII. Three-dimensional crystals from higher plant PSII uniformly diffract poorly [20] and two-dimensional crystals examined by electron diffraction have been performed at low resolutions with PSII from higher plants devoid of an oxygen-evolving complex [21,22]. In cross-reconstitution experiments, the 23 and 17 kDa proteins bound to cyano- bacterial and red algal PSII only through non-specific interactions [16]. The CaCl 2 -washed spinach PS II mem- branes which had been reconstituted with either cyanobac- terial or red algal 33 kDa protein, could only partially rebind spinach 23 kDa protein but could not bind spinach 17 kDa protein [23]. These data indicate that there are structural determinants present on the spinach 33 kDa protein that are required for the efficient binding of the 23 and 17 kDa proteins and that are absent in cyanobacterial and red algal proteins [24]. The organization among the three extrinsic proteins in spinach PSII has been examined by cross-linking experiments. Cross-linking experiments performed with homobifunctional cross-linkers (6–14 A ˚ span) indicated that the 33 kDa protein is within a distance of 11 A ˚ of the 23 kDa protein and that the 23 kDa protein is within 11 A ˚ of the 17 kDa protein [25]. This indicates that these three extrinsic proteins must be in close proximity. Cross- linking experiments also showed that the 33 kDa protein is associated with or in close proximity to CP47 [25–28], D1 and D2 [29], a large subunit of cytochrome b559 [30] and PsbI [30]. The 33 kDa protein was shown to be also associated with CP43 by comparing the peptide mappings of the trypsin-digested products of NaCl-washed and CaCl 2 -washed PSII membranes [31]. Thus, the 33 kDa protein is associated with or in close proximity to essentially all of the major intrinsic proteins in higher plant PSII. Chemical modification is a useful method to elucidate which positive or negative charges on the extrinsic proteins are responsible for electrostatic interaction with the other extrinsic proteins and/or the intrinsic proteins [32,33]. We have reported that the N-succinimidyl propionate (NSP)- modified 33 kDa protein, of which the positively charged amino groups are modified to uncharged methyl ester groups [33], cannot rebind to spinach PSII, whereas the glycine methyl ester (GME)-modified protein, of which the negat- ively charged carboxyl groups are modified to uncharged methyl ester groups [32], can rebind and reactivate the oxygen evolution [34]. These results indicate that positive charges on the 33 kDa protein are important for its electrostatic interaction with PSII intrinsic proteins, whereas negative charges on the protein do not contribute to such interaction. The domains of the 33 kDa protein possibly involved in electrostatic interaction with PSII intrinsic proteins were also determined to be Lys4, Lys20, Lys66– Lys76, Lys101, Lys105, Lys130, Lys159, Lys186 and Lys230–Lys236 by a combination of V8 protease digestion and MALDI-TOF MS of NSP or 2,4,6-trinitrobenzene sulfonic acid-modified 33 kDa protein [34], or NHS-biotin modified one [35]. Furthermore, we showed that a similar number of carboxyl groups on the 33 kDa protein were modified with GME in both the protein in solution and bound to PSII [34]. This suggests that most of the carboxyl groups on the 33 kDa protein are not located in regions interacting with PSII intrinsic proteins and exposed to the lumenal side of PSII. Thus, we hypothesized that negative charges of carboxyl groups on the 33 kDa protein may be involved in electrostatic interaction with the 23 and 17 kDa proteins. In fact, Bricker and Frankel [24] showed recently, that spinach PS II membranes reconstituted with the 33 kDa protein, on which the negatively charged carboxyl groups were modified with GME, was defective in its ability to bind the 23 kDa protein of PSII. They hypothesized that the domains on the 33 kDa protein possibly involved in electrostatic interaction with the 23 kDa protein are Glu1, Glu32, Glu139 and/or Glu187, which are wholly conserved in higher plants but which are poorly conserved in cyano- bacteria. These facts in turn suggest that positive charges on the 23 kDa protein may be responsible for the electrostatic interaction with these negative charges on the 33 kDa protein. The binding domains of the 23 kDa protein, however, remain obscure. Recently, Ifuku and Sato [36] reported that the binding affinity of a recombinant mutant of the 23 kDa protein, of which N-terminal 19 residues were truncated, were apparently weaker than that of the native 23 kDa protein, and the mutant protein completely lacked the ability to retain Ca 2+ for oxygen evolution. This suggests that the N-terminal region of the 23 kDa protein is important for its binding with the 33 kDa protein. In the present study, the domains on the 23 kDa protein possibly involved in electrostatic interaction with the 33 kDa protein associated with PSII membranes were examined by chemical modification method. The results showed that positive charges on the 23 kDa protein are indeed important for its interaction with the 33 kDa protein, and we have determined the domains of positive charges on the 23 kDa protein that are possibly involved in the interaction. Materials and methods Preparations Oxygen-evolving PSII membranes were prepared from spinach chloroplasts with Triton X-100 as described in Berthold et al. [37], with slight modifications [28]. The isolated PSII membranes were suspended in medium A (40 m M Mes/NaOH,pH6.5;0.4 M sucrose; 10 m M NaCl and 5 m M MgCl 2 , and stored in liquid nitrogen until used. The extrinsic 33 and 23 kDa proteins were extracted from the PSII membranes by 1 M CaCl 2 treatment, incubated with 1 M CaCl 2 for 3 h in the dark to suppress the activity of copurified protease, dialyzed against 5 m M Mes/NaOH, Ó FEBS 2004 Interaction between the 23 and 33 kDa proteins (Eur. J. Biochem. 271) 963 pH 6.5 and further against 20 m M phosphate buffer, pH 6.5 and then purified by column chromatography with a DEAE-Sepharose CL-6B column (Pharmacia Biotech Inc., NJ, USA) [16,38]. The concentrations of the 33 and 23 kDa proteins were determined using an extinction coefficient of 16 m M )1 Æcm )1 at 276 nm [39] and 26 m M )1 Æcm )1 at 277 nm [38], respectively. Chemical modification For modification of amino groups of lysyl residues and the free amino terminus of the 23 kDa protein, the purified protein (30 l M ) was incubated in a reaction mixture containing 20 m M phosphate buffer, pH 6.5 and 0.5–6.0 m M NSP at 25 °C for 90 min. The reaction mixtures were dialyzed against 10 m M Mes/NaOH, pH 6.5 to remove unreacted NSP. Chemical modification of carboxyl groups on the purified 23 kDa protein was performed in 100 m M GME, pH 6.2 containing 30 l M of the 23 kDa protein and 2 m M 1-ethyl-3-(3-(dimethylamino)propyl) car- bodiimide (EDC) at 25 °C for 12 h. The reaction mixture was dialyzed against 1 M NaCl and 20 m M phosphate buffer, pH 6.5 to remove unreacted and electrostatically attached reagents, and then against 10 m M Mes/NaOH, pH 6.5. NSP was purchased from Wako Pure Chemicals (Tokyo, Japan), and GME and EDC were purchased from Nacalai Tesque Chemicals (Tokyo, Japan). Reconstitution and electrophoresis For reconstitution, PS II membranes were washed with 2.6 M Urea, 0.2 M NaCl in the dark to remove the three extrinsic proteins of 33, 23 and 17 kDa [8]. The resultant PSII membranes were incubated with the 33 kDa protein and with either the unmodified or modified 23 kDa protein at a protein-Chl ratio of 0.6 (w/w), in medium A at 0 °Cfor 30 min in the dark at a Chl concentration of 0.5 mg mL )1 . The reconstituted PSII membranes were collected by centrifugation at 35 000 g for 10 min and then washed once with and resuspended in medium A. The reconstituted PSII membranes were again treated with 2.6 M urea, 0.2 M NaCl in the dark for 30 min and the centrifuged super- natants were applied on SDS/PAGE to estimate the amounts of the 33 kDa and 23 kDa proteins rebound by the reconstitution. SDS/PAGE was performed with a gradient gel of 16– 22% acrylamide containing 7.5 urea [40]. Samples were solubilized with 5% lithium lauryl sulfate and 75 m M dithiothreitol. The amounts of rebound 23 kDa protein were determined from the integrated optical densities of the 23 kDa bands using the program NIH IMAGE (National Institutes of Health, USA) after the SDS/PAGE was scanned using a CanoScan N656U (Canon, Tokyo). Isoelectric focusing was performed using a 5.5% poly- acrylamide containing homogenous gel covering a pH range of 3.5–10.0 or 4.0–6.0 using 5% (v/v) ampholine (Amer- sham Pharmacia Biotech AB, Sweden). Proteins were stained with 0.048% CBB in 30% methanol and 10% acetic acid. Oxygen evolution was measured with a Clark-type oxygen electrode in 40 m M Mes/NaOH, pH 6.5 and 0.4 M sucrose (medium B) at 25 °C in the absence and presence of 10 m M NaCl or 5 m M CaCl 2 , with 0.4 m M phenyl- p-benzoquinone as the electron acceptor. Chl concentration was determined by the method of Porra et al. [41]. Protease digestion The 23 kDa protein (3 nmol) modified with 0.5 or 4 m M NSP was dried and solubilized in 10 lLof1 M Tris/HCl, pH 8.5, 8 M guanidine/HCl, 1 m M EDTA and 1% dithio- threitol, and incubated at 37 °C for 2 h to denature the 23 kDa protein. Then, 5 lL of 5% iodoacetamide was added and incubated at 37 °C for 30 min to block SH groups. The reaction mixtures were added to a final concentration of 10% of cold trichloroacetic acid and centrifuged, and the resulting precipitates were washed twice with acetone. The final precipitates were dried and resolubilized in 20 lLof0.1 M ammonium bicarbonate. After 1 lgofStaphylococcus V8 protease (ICN Biomedicals, OH, USA) was added, the 23 kDa protein was digested at 37 °C, overnight and then desalted by Ziptipl-C18 (Millipore, MA, USA). Mass spectroscopic analysis The protease-digested protein was applied directly to a MALDI-TOF MS (Reflex; Bruker Daltonics, MA, USA), with a matrix of a-cyano-4-hydroxycinnamic acid (CHC), 2-mercaptobenzothiazole (MBT) or 2,5-dihydroxybenzoic acid (DHB). The mass of each measured peptide fragment was assigned to the known 23 kDa protein sequence. Results As described above, Bricker and Frankel [24] showed that negatively charged carboxyl groups on the extrinsic 33 kDa protein are important for electrostatic interaction with the extrinsic 23 kDa protein. This suggests that positive charges on the 23 kDa protein may electrostatically interact with the negative charges on the 33 kDa protein. To confirm this, we modified positively charged amino groups on the 23 kDa protein to uncharged methyl ester groups with NSP. Figure 1A shows the isoelectric focusing of the NSP- modified 23 kDa protein. The pI value shifted toward acidic pH with increasing NSP concentration. For exam- ple, the pI value downshifted from 6.8 (unmodified protein, lane 1) to 4.8–5.5 (0.5 m M NSP-modified protein, lane 2) and 4.3–4.8 (4 m M NSP-modified protein, lane 5). These changes were estimated to result from modification of 1–5 amino groups in 0.5 m M NSP-modified protein and 5–10 amino groups in 4 m M NSP-modified protein to uncharged groups, as calculated using a computer pI/Mr tool [42]. It should be noted here that the band of the modified protein appeared much broader than the unmodified protein upon isoelectric focusing, implying that the resulting protein products may be composed of proteins with different numbers of amino residues modi- fied. This is similar to the results obtained by modification of the 33 kDa protein with NHS-biotin [35], NSP and 2,4,6-trinitrobenzen sulfonic acid [34], or GME [24]. In order to determine whether elimination of surface positive charges affected binding of the 23 kDa protein, the ability of the NSP-modified protein to rebind with the 964 A. Tohri et al. (Eur. J. Biochem. 271) Ó FEBS 2004 33 kDa protein associated with PSII membranes was examined. Urea/NaCl-washed PSII membranes in which the three extrinsic proteins of 33, 23 and 17 kDa had been removed, were reconstituted with the unmodified and NSP- modified 23 kDa protein together with the 33 kDa protein. The reconstituted PSII membranes were again treated with 2.6 M urea plus 0.2 M NaCl, and the supernatants after centrifugation were analyzed by SDS/PAGE to determine the amounts of the 33 and 23 kDa proteins rebound. As shown in Fig. 2, the native 33 and 23 kDa proteins completely rebound to urea/NaCl-washed PSII membranes (lane 4), whereas the binding abilities of NSP-modified 23 kDa protein decreased with increasing NSP concentra- tion (lanes 5–9) and this ability was completely lost with NSP treatments above 4 m M (lanes 8 and 9). This suggests that positive charges on the 23 kDa protein are important for electrostatic interaction with the 33 kDa protein. Table 1 shows the reactivation of oxygen evolution by reconstitution of the 23 kDa protein modified with various concentrations of NSP. When the 33 kDa protein was reconstituted with urea/NaCl-washed PSII membranes in which no oxygen evolution was detected even in the presence of CaCl 2 , the oxygen evolution was reactivated to 0, 96 and 252 lmol O 2 Æmg chl )1 Æh )1 in the absence and presence of 10 m M NaCl and 5 m M CaCl 2 , respectively. The activity further recovered to 142 and 243 lmol O 2 Æmg chl )1 Æh )1 in the absence and presence of 10 m M NaCl by additional reconstitution of the unmodified 23 kDa protein, though little effects were detected on the activity in the presence of 5 m M CaCl 2 by the additional reconstitution. When the NSP-modified 23 kDa proteins were Fig. 2. Reconstitution of the unmodified, NSP- or GME-modified 23 kDa protein together with the 33 kDa protein with urea/NaCl- washed PSII membranes. Urea/NaCl-washed PSII membranes were reconstituted with the unmodified, NSP- or GME-modified 23 kDa protein together with the 33 kDa protein. The reconstituted PSII membranes were again treated with 2.6 M urea, 0.2 M NaCl and their centrifuged supernatants were analyzed by SDS/PAGE to determine the amounts of the 33 and 23 kDa proteins rebound after reconstitu- tion. Lane 1, unwashed PSII-membranes; lane 2, urea/NaCl-washed PSII membranes; lane 3, urea/NaCl–washed PSII membranes recon- stituted with the 33 kDa protein; lane 4, urea/NaCl–washed PSII reconstituted with the 33 kDa protein and unmodified 23 kDa protein; lanes 5–9, urea/NaCl–washed PSII membranes reconstituted with the 33 kDa protein and the 23 kDa protein modified by 0.5 m M NSP (lane 5), 1 m M NSP (lane 6), 2 m M NSP (lane 7), 4 m M NSP (lane 8), and 6 m M NSP (lane 9); lane 10, urea/NaCl–washed PSII membranes reconstituted with the 33 kDa protein and the GME-modified 23 kDa protein. Fig. 1. Isoelectric focusing of the NSP- (A) or GME- (B) modified 23 kDa protein. (A) Lane 1, unmodified 23 kDa protein; lanes 2–6, the 23 kDa protein modified by NSP at concentrations of 0.5 m M (lane 2), 1m M (lane 3), 2 m M (lane 4), 4 m M (lane 5), 6 m M (lane 6). (B) Lane 1, unmodified 23 kDa protein; lane 2, the 23 kDa protein modified with 100 m M GME in the presence of 2 m M EDC at 25 °Cfor12h. Table 1. Reactivation of oxygen evolution by reconstitution of the NSP- or GME-modified 23 kDa protein to urea/NaCl-washed PSII membranes reconstituted with the 33 kDa protein. Values shown are the averages of three measurements. 23, 23 kDa protein; 33, 33 kDa protein. PS II membrane treatment Oxygen evolution [lmol O 2 Æ(mg chl) )1 Æh )1 ] –Ion (%) +10 mM NaCl (%) +5 mM CaCl 2 (%) Control PSII membranes 523 ± 26 (100) 525 ± 17 (100) 535 ± 18 (100) Urea/NaCl-washed PSII 0 ± 0 (0) 0 ± 0 (0) 0 ± 0 (0) + 33 0 ± 0 (0) 96 ± 7 (18) 252 ± 13 (47) + 33 + 23 142 ± 7 (27) 243 ± 10 (46) 274 ± 12 (51) + 33 + 0.5 mM NSP-modified 23 25 ± 5 (5) 120 ± 9 (23) 265 ± 11 (50) + 33 + 1.0 mM NSP-modified 23 13 ± 3 (2) 110 ± 7 (21) 267 ± 12 (50) + 33 + 2.0 mM NSP-modified 23 7 ± 2 (1) 103 ± 7 (20) 260 ± 10 (49) + 33 + 4.0 mM NSP-modified 23 0 ± 0 (0) 95 ± 5 (18) 263 ± 12 (49) + 33 + 6.0 mM NSP-modified 23 0 ± 0 (0) 94 ± 6 (18) 253 ± 10 (47) + 33 + GME-modified 23 140 ± 9 (27) 250 ± 9 (48) 252 ± 12 (47) Ó FEBS 2004 Interaction between the 23 and 33 kDa proteins (Eur. J. Biochem. 271) 965 reconstituted together with the 33 kDa protein, their reactivations in the absence and presence of 10 m M NaCl decreased with increasing NSP concentrations, and no reactivation effects were observed in PSII membranes reconstituted with the 23 kDa protein modified with NSP above 4 m M . Figure 3 shows the correlation between the amounts of rebound 23 kDa protein (Fig. 2) and reactivation of oxygen evolution in the absence (open circles) and presence (closed circles) of 10 m M NaCl (Table 1). Their good correlation indicates that loss of the reactivating capability of the NSP- modified 23 kDa protein was caused directly by loss of their rebinding, which in turn suggests that the modified protein, when rebound, are fully functional and that there is apparently no nonspecific binding of the modified protein. In contrast to the NSP-modified 23 kDa protein, the GME-modified 23 kDa protein retained its capabilities to rebind with the 33 kDa protein associated with PSII and to reactivate the oxygen evolution. Figure 1B shows that the pI values were upshifted from 6.8 of unmodified protein (lane 1) to 9.2 (lane 2) by modification of carboxyl groups with GME in the presence of EDC. This change was estimated to result from modification of around three negatively charged carboxyl groups to uncharged groups, as calculated using a computer pI/Mr tool. The GME- modified 23 kDa protein completely rebound to the 33 kDa protein associated with PSII membranes (Fig. 2, lane 10) and its rebinding reactivated the oxygen evolution to extents comparable with the rebinding of the unmodified 23 kDa protein (Table 1). These results clearly indicate that surface negative charges on the 23 kDa protein do not participate in its functional binding with the 33 kDa protein associated with PSII membranes. Next, we attempted to identify the lysyl residues on the 23 kDa protein modified with NSP. Both of the modified 23 kDa proteins treated with 0.5 m M NSP and 4 m M NSP whose binding abilities were lost by about 82% and 100%, were denatured with urea and digested with Staphylococcus V8 protease followed by determination of the mass of the resultant peptide fragments with mass spectroscopy. Whether a peptide fragment can be detected by the MALDI-TOF MS depends in some cases on the matrix employed, three different matrices were used: They were, a-cyano-4-hydroxycinnamic acid (CHC), 2-mercapto- benzothiazole (MBT) and 2,5-dihydroxybenzoic acid (DHB). This led to a more complete identification of the peptide fragments resulting from the V8 protease digestion of the modified 23 kDa protein. The results were shown in Table 2 (the 23 kDa protein modified with 0.5 m M NSP) and Table 3 (the 23 kDa protein modified with 4 m M NSP). Peptide fragments yielded could be assigned to the known amino acid sequence within a 0.01% mass error, as shown in Tables 2 and 3. Modifi- cation of the amino group with each NSP molecule results in an addition of an N-propionyl group, which corres- ponds to an increase of 56.0 Da in the molecular mass. In the 23 kDa protein modified with 0.5 m M NSP, there were 31 peptides identified ranging in mass from 703.32 to 2840.49 Da (Table 2). Of these peptides, eight lysyl residues were identified to be modified with NSP, two Lys between Lys11 and Lys14; one Lys among Lys27 and Lys38; one Lys at Lys40; one Lys at Lys90 or Lys96; one Lys at Lys143 or Lys152; two Lys between Lys166 and Lys174 (Table 2). These modified lysyl residues were arranged in the amino acid sequence of the 23 kDa protein as shown in Fig. 4. This indicates that eight lysyl residues modified with 0.5 m M NSP are located in six domains, namely Lys11–Lys14, Lys27–Lys38, Lys40, Lys90–Lys96, Lys143–Lys152, Lys166–Lys174. In the 23 kDa protein modified with 4 m M NSP, 32 peptides ranging in mass from 703.33 to 2760.30 Da were identi- fied. Of these peptides, 11 lysyl residues were identified to be modified with NSP, which were two Lys between Lys11 to Lys14; two Lys between Lys27 and Lys38; one Lys at Lys40; one Lys at Lys68 or Lys69; one Lys at Lys90 or Lys96; one Lys at Lys143 or Lys152 and three Lys between Lys166 and Lys174 (Table 3). Ten residues in these modified Lys were found in the six domains that wereidentifiedtobemodifiedwith0.5m M NSP, as shown in Fig. 4. Only one domain of Lys68–Lys69 was modified uniquely with 4 m M NSP in addition to the six domains. Discussion The present results clearly demonstrated that modification of amino groups on the 23 kDa protein with NSP significantly affected its rebinding ability and thus the reactivating capability of oxygen evolution. In contrast, modification of carboxyl groups on the protein with GME in the presence of EDC did not affect the rebinding and reactivation capabilities. We thus conclude that the positive charges, but not the negative charges, on the 23 kDa protein, are important for its interaction with PSII and in particular, the 33 kDa protein associated with PSII. The 23 kDa protein from spinach is composed of 186 amino acid residues including 14 Asp, 10 Glu, 20 Lys, and 3 Arg [43]. In the present study, around three carboxyl groups Fig. 3. Relationship between the amounts of NSP-modified 23 kDa protein rebound and oxygen evolution restored. s, oxygen evolution in the absence of NaCl; d, oxygen evolution in the presence of 10 m M NaCl. 966 A. Tohri et al. (Eur. J. Biochem. 271) Ó FEBS 2004 were estimated to be modified with GME within the total of 24 carboxyl groups of Asp + Glu in the 23 kDa protein, when chemical modification of carboxyl groups was performed in 100 m M GME (pH 6.2) and 2 m M EDC at 25 °C for 12 h. The changes of the pI values were almost saturated within 12 h even by the modification in the presence of 4 m M and 8 m M EDC, implying that a number of the carboxyl groups on the 23 kDa protein are non- reactive with the chemical modification reagent. In spite of this extended modification with GME, no significant effects were observed on the binding and reactivating abilities of the 23 kDa protein. Thus, we conclude that the negative charges on the 23 kDa protein do not contribute to its interaction with PSII. In contrast, 1–5 or 5–10 amino groups in total of the 20 Lys of the 23 kDa protein were modified with NSP when the protein was treated with 0.5 m M or 4m M NSP at 25 °C for 90 min, respectively. This indicates that amino groups of Lys residues on the protein are much more reactive than carboxyl groups of Asp and Glu with respect to the chemical modification reagents. A loss of the rebinding of the 23 kDa protein following chemical modification can, in principle, be caused by two different mechanisms, as described previously [34]. First, chemical modification may induce a conformational change of the protein, resulting in a protein structure that is no longer able to bind to the 33 kDa protein associated with the PSII membranes. Second, the residues that are modified may participate directly in the electrostatic interaction of the 23 kDa protein with the 33 kDa protein associated with the PSII membranes. The former possibility appears rather Table 2. Assignments for peptide fragments from a Staphylococcus V8 protease digest of the extrinsic 23 kDa protein modified with 0.5 mM NSP. Deamidation (NG), deamidation of Asn22–Gly23 to Asp22–Gly23; NP, N-propionyl; Oxydation (M), oxydation of Met. Observed mass (Da) Predicted mass (Da) Change in mass (%) Peptide assignment NSP-modified domains (Lys–Lys)CHC MBT DHB CHC MBT DHB 703.32 703.34 )0.00 Ser116–Asp122 843.31 843.31 0.00 Phe18 –Asp24 Deamidation (NG) 855.37 855.40 )0.00 Ser178–Ala186 1107.57 1107.57 1107.57 1107.57 0.00 0.00 0.00 Phe42–Glu50 1222.59 1222.61 1222.60 )0.00 0.00 Phe42–Asp51 1322.72 1322.72 1322.70 1322.73 )0.00 )0.00 )0.00 Gly141–Asp153 1364.71 1364.71 1364.69 1364.71 0.00 0.00 )0.00 Lys40–Glu50 1378.71 1378.72 1378.76 )0.00 )0.00 Gly141–Asp153 + NP 143–152 1402.71 1402.72 1402.76 )0.00 )0.00 Ala5–Glu17 1420.74 1420.73 1420.71 1420.73 0.00 0.00 )0.00 Lys40–Glu50 + NP 40 1479.72 1479.74 )0.00 Lys40–Asp51 1514.81 1514.80 1514.78 1514.81 0.00 )0.00 )0.00 Ala5–Glu17 + 2 NP 11–14 1535.76 1535.76 1535.76 0.00 0.00 Lys40–Asp51 + NP 40 1578.90 1578.91 1578.90 1578.90 0.00 0.00 0.00 Lys166–Glu177 + NP 166–174 1634.93 1634.92 1634.93 0.00 )0.00 Lys166–Glu177 + 2 NP 166–174 1636.92 1636.91 1636.90 1636.76 0.01 0.01 0.01 Ser99–Glu115 1718.86 1718.86 1718.85 1718.85 0.00 0.00 0.00 Tyr86–Glu100 1728.97 1728.97 1728.96 1728.96 0.00 0.00 0.00 Gly25–Glu39 1785.00 1785.00 1784.98 1784.98 0.00 0.00 0.00 Gly25–Glu39 + NP 27–28 1834.89 1834.86 0.00 Asp51–Asp67 2016.07 2016.07 0.00 Ala55–Asp73 2507.22 2507.23 2507.22 2507.22 0.00 0.00 0.00 Asp51–Asp73 2523.22 2523.22 2523.21 2523.22 0.00 0.00 )0.00 Asp79–Glu100 2554.24 2554.25 2554.26 )0.00 )0.00 Phe18–Glu39 Deamidation (NG) 2570.25 2570.25 0.00 Phe18–Glu39 Deamidation (NG); Oxidation (M) 2579.23 2579.24 )0.00 Asp79–100 Glu + NP 90–96 2610.28 2610.28 2610.27 2610.28 0.00 0.00 )0.00 Phe18–Glu39 + NP Deamidation (NG) 27–38 2626.29 2626.28 2626.24 2626.28 0.00 0.00 )0.00 Phe18–Glu39 + NP Deamidation (NG); Oxidation (M) 27–38 2684.29 2684.40 )0.00 Gly141–Asp165 + NP 143–152 2828.48 2828.45 2828.52 )0.00 )0.00 Gly154–Glu177 2840.49 2840.35 0.00 Phe80–Asp104 + NP 90–96 Ó FEBS 2004 Interaction between the 23 and 33 kDa proteins (Eur. J. Biochem. 271) 967 unlikely on the basis of the following considerations. If loss of the binding ability of the 23 kDa protein is caused by conformational changes following chemical modification, its binding ability should similarly decrease upon modifica- tion of carboxyl groups with GME, because chemical modification with GME results in an addition of a similar size of methyl ester group as that with NSP, as described in our previous paper [34]. The GME modification did not, however, affect the binding ability of the 23 kDa protein at all. These considerations indicate that the loss of the binding ability of the NSP-modified 23 kDa protein is due to neutralization of positively charged lysyl residues of the protein, though conformational changes induced by the chemical modification cannot be completely excluded. Thus, we conclude that positive charges of lysyl residues of the 23 kDa protein are important for its binding to the 33 kDa protein associated with PSII membranes, whereas negative charges of carboxyl groups of the 23 kDa protein do not participate in its binding. This conclusion is consistent with the hypothesis predicted by Bricker and Frankel [24] that negative charges on the 33 kDa protein are important for electrostatic interaction with the 23 kDa protein. The locations of lysyl residues on the 23 kDa protein that were modified with NSP were determined in the present study. It should be noted again that the modified 23 kDa protein is composed of proteins having different numbers of amino residues modified. In fact, the band of the modified protein appeared much broader than the unmodified protein upon isoelectric focusing (Fig. 1). The Table 3. Assignments for peptide fragments from a Staphylococcus V8 protease digest of the extrinsic 23 kDa protein modified with 4 mM NSP. Deamidation (NG), deamidation of Asn 22–Gly23 to Asp22–Gly23; Oxydation (M), oxydation of Met; NP, N-propionyl. Observed mass (Da) Predicted mass (Da) Change in mass (%) Peptide assignment NSP-modified domains (Lys–Lys) CHC MBT DHB CHC MBT DHB 703.33 703.34 )0.00 Ser116–Asp122 822.35 822.38 )0.00 Asp79–Asp85 843.34 843.31 0.00 Phe18–Asp24 Deamidation (NG) 855.36 855.40 )0.00 Ser178–Ala186 859.30 859.31 )0.00 Phe18–Asp24 Deamidation (NG); Oxidation (M) 1107.57 1107.58 1107.57 1107.57 0.00 0.00 0.00 Phe42–Glu50 1222.59 1222.62 1222.60 )0.00 0.00 Phe42–Asp51 1322.71 1322.69 1322.73 )0.00 )0.00 Gly141–Asp153 1364.71 1364.70 1364.70 1364.71 0.00 )0.00 )0.00 Lys40–Glu50 1378.72 1378.76 )0.00 Gly141–Asp153 + NP 143–152 1402.73 1402.69 1402.69 1402.76 )0.00 )0.00 )0.00 Ala5–Glu17 1420.73 1420.73 1420.73 1420.73 0.00 0.00 0.00 Lys40– Glu50 + NP 40 1514.83 1514.80 1514.80 1514.81 0.00 )0.00 )0.00 Ala5–Glu17 + 2 NP 11–14 1535.78 1535.77 1535.76 0.00 0.00 Lys40–Asp51 + NP 40 1634.95 1634.92 1634.94 1634.93 0.00 )0.00 0.00 Lys166–Glu177 + 2 NP 166–174 1636.93 1636.76 0.01 Ser99–Glu115 1690.98 1690.97 1690.96 1690.96 0.00 0.00 0.00 Lys166–Glu177 + 3 NP 166–174 1718.89 1718.87 1718.85 0.00 0.00 Tyr86–Glu100 1728.99 1728.98 1728.96 0.00 0.00 Gly25–Glu39 1774.92 1774.90 1774.88 0.00 0.00 Tyr86–Glu100 + NP 90–96 1834.91 1834.92 1834.86 0.00 0.00 Asp51–Asp67 1841.05 1841.03 1841.03 1841.01 0.00 0.00 0.00 Gly25–Glu39 + 2 NP 27–38 1935.03 1934.97 0.00 Ala1–Glu17 + 2 NP 11–14 2016.02 2016.06 2016.07 )0.00 )0.00 Ala55–Asp73 2507.24 2507.23 2507.22 0.00 0.00 Asp51–Asp73 2523.24 2523.22 2523.20 2523.22 0.00 0.00 )0.00 Asp79–Glu100 2563.31 2563.23 2563.27 0.00 )0.00 Asp51–Asp73 + NP 68–69 2579.29 2579.25 2579.21 2579.24 0.00 0.00 )0.00 Asp79–Glu100 + NP 90–96 2666.33 2666.27 2666.31 0.00 )0.00 Phe18–Glu39 + 2 NP Deamidation (NG) 27–38 2682.31 2682.27 2682.30 0.00 )0.00 Phe18–Glu39 + 2 NP Deamidation (NG); Oxidation (M) 27–38 2684.27 2684.40 )0.00 Gly141–Asp165 + NP 143–152 2760.30 2760.27 0.00 Ala1– Asp24 + 2 NP Deamidation (NG) 11–14 968 A. Tohri et al. (Eur. J. Biochem. 271) Ó FEBS 2004 changes of pI values were estimated to correspond to modifications of 1–5 amino groups in the 0.5 m M NSP- modified protein. However, our mass spectroscoic analysis indicated that there were eight lysyl residues that were modified (Results, Fig. 4). These facts indicate that lysyl residues of the 23 kDa protein were heterogeneously modified; some lysyl residues may be modified in a fraction of the protein by NSP but not in other fractions of the protein. The 23 kDa protein modified with 0.5 m M NSP lost about 82% of its binding and reactivating capabilities (Figs 2 and 3, Table 1). In other words, 18% of the binding and reactivating capabilities were still retained after NSP modification. This may well be attributed to the heterogeneous modification of lysyl residues. NSP modifies not only the lysyl residues required for electrostatic interaction with the 33 kDa protein but also the residues not involved in the interaction. In conclusion, we propose that the candidates for electro- static interaction of the 23 kDa protein with the 33 kDa protein associated with the PSII membranes are at least present in lysyl residues of the six domains of Lys11– Lys14, Lys27–Lys38, Lys40, Lys90–Lys96, Lys143– Lys152, Lys166–Lys174 (Fig. 4). Complete loss of the binding ability was obtained by treatment with 4 m M NSP (Figs 2 and 3) in which only one domain, Lys68–Lys69, was detected to be modified in addition to the six domains (Fig. 4). In the lysyl residues present in the six domains, 11, 13, 33, 38, 143, 166, 170 and 174 (circled K in Fig. 4) were completely conserved in the 23 kDa protein from the 12 species of higher plants currently available in databases. The N-terminal region of the 23 kDa protein has been reported to be important for its binding with PSII. Ifuku and Sato [36] showed that the binding affinity of a recombinant mutant of the 23 kDa protein, of which the N-terminal 19 residues were truncated, were apparently weaker than that of the native 23 kDa protein. Two lysyl residues, Lys11 and Lys13, in the N-terminal 19 residues were modified with NSP and thus these lysyl residues are likely to participate in the electrostatic interaction. The negative charges of Glu1, Glu32, Glu139 and/or Glu187 on the 33 kDa protein have been suggested to be important for the binding of the 23 kDa protein [24]. Our current results thus indicate that at least some of the positive charges of the lysyl residues in the six domains of the 23 kDa protein interact electrostatically with these negative charges of the 33 kDa protein. Some of the positive charges on the 23 kDa protein may also be important for its interaction with PSII intrinsic proteins. Which residues in these modified lysyl residues directly participate in the electrostatic interaction with the 33 kDa protein (and PSII intrinsic proteins) cannot be identified at present. The present study, however, pro- vides important clues for site-directed mutagenesis studies to identify the lysyl residues that directly participate in the electrostatic interaction. Acknowledgements We thank Prof Jian-Ren Shen of Okayama University for his critical reading of the manuscript. Fig. 4. The amino acid sequence of spinach 23 kDa protein. Boxes indicate domains containing lysyl residues modified by NSP at both concen- trations of 0.5 m M and 4 m M . Dashed box is the domain modified only by 4 m M NSP but not by 0.5 m M NSP. The number of lysyl residues modified with 0.5 m M and 4 m M NSP in each domain are shown below each box. The circled amino acids indicate lysyl residues that are completely conserved in 12 species of higher plants currently available in data bases. The sequences were obtained from SwissProt and TrEMBL databases. Arrows show the cleavage sites with Staphyrococcus V8 protease. Ó FEBS 2004 Interaction between the 23 and 33 kDa proteins (Eur. J. Biochem. 271) 969 References 1. Seidler, A. (1996) The extrinsic polypeptides of Photosystem II. Biochim. Biophys. Acta 1277, 35–60. 2. Miyao, M. & Murata, N. (1989) The mode of binding of three extrinsic proteins of 33 kDa, 23 kDa and 18 kDa in the photo- system II complex of spinach. Biochim. Biophys. Acta. 977, 315–321. 3. Miyao, M. & Murata, N. (1983) Partial disintegration and reconstitution of the photosynthetic oxygen evolution complex. Biochim. Biophys. Acta. 725, 87–93. 4. Suzuki, T., Minagawa, J., Tomo, T., Sonoike, K., Ohta, H. & Enami, I. (2003) Binding and functional properties of the extrinsic proteins in oxygen-evolving photosystem II particle from a green alga, Chlamydomonas reinhardtii having His-tagged CP47. Plant Cell Physiol. 44, 76–84. 5. Shen, J R. & Inoue, Y. (1993) Binding and functional properties of two new extrinsic components, cyctochrome c 550 and a 12 kDa protein, in cyanobacterial photosystem II. Biochememistry 32, 1825–1832. 6. Enami, I., Murayama, H., Ohta, H., Kamo, M., Nakazato, K. & Shen, J R. (1995) Isolation and characterization of a Photosystem II complex from the red alga Cyanidium caldarium: association of cytochrome c-550 and a 12 kDa protein with the complex. Biochim. Biophys. Acta 1232, 208–216. 7. Enami, I., Kikuchi, S., Fukuda, T., Ohta, H. & Shen, J R. (1998) Binding and functional properties of four extrinsic proteins of photosystem II from a red alga, Cyanidium caldarium,asstudied by release-reconstitution experiments. Biochemistry 37, 2787– 2793. 8. Miyao, M. & Murata, N. (1984) Role of the 33 kDa polypeptide in preserving Mn in the photosynthetic oxygen-evolution. FEBS Lett. 170, 350–354. 9. Ono, T. & Inoue, Y. (1984) Ca 2+ -dependent restoration of O 2 -evolving activity in CaCl 2 -washed PSII particles depleted of 33, 24 and 16 kDa polypeptides. FEBS Lett. 168, 281–286. 10. Shutova, T., Irrgang, K D., Shubin, V., Klimov, V.V. & Renger, G. (1997) Analysis of pH-induced structural changes of the isolated extrinsic 33 kilodalton protein of photosystem II. Biochemistry 36, 6350–6358. 11. Shutova, T., Villarejo, A., Zietz, B., Klimov, V., Gillbro, T., Samuelsson, G. & Renger, G. (2003) Comparative studies on the properties of the extrinsic manganese-stabilizing protein from higher plants and of a synthetic peptide of its C-terminus. Biochim. Biophys. Acta 1604, 95–104. 12. A ˚ rklund, H.E., Jansson, C. & Andersson, B. (1982) Reconstitu- tion of photosynthetic water splitting in inside-out thylakoid vesicles and identification of a participating polypeptide. Biochim. Biophys. Acta 681, 1–10. 13. Kuwabara, T. & Murata, N. (1982) Inactivation of photosynthetic oxygen evolution and concomitant release of three polypeptides in the photosystem II particles of spinach chloroplasts. Plant Cell Physiol. 24, 741–747. 14. Ghanotakis, D.F., Topper, J.N., Babcock, G.T. & Yocum, C.F. (1984) Water-soluble 17 kDa and 23 kDa polypeptides restore oxygen evolution activity by creating a high affinity binding-site for Ca 2+ on the oxidizing side of Photosystem II. FEBS Lett. 170, 169–173. 15. Shen, J R., Ikeuchi, M. & Inoue, Y. (1997) Analysis of the psbU gene encoding the 12-kDa extrinsic protein of photosystem II and studies on its role by deletion mutagenesis in Synechocystis sp. PCC 6803. J. Biol. Chem. 272, 17821–17826. 16. Enami,I.,Yoshihara,S.,Tohri,A.,Okumura,A.,Ohta,H.& Shen, J R. (2000) Cross-reconstitution of various extrinsic pro- teins and photosystem II complexes from cyanobacteria, red algae and higher plants. Plant Cell Physiol. 41, 1354–1364. 17. Okumura, A., Ohta, H., Inoue, Y. & Enami, I. (2001) Identifica- tion of functional domains of the extrinsic 12 kDa protein in red algal PSII by limited proteolysis and directed mutagenesis. Plant Cell Physiol. 42, 1331–1337. 18. 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. 19. Kamiya, N. & Shen, J R. (2003) Crystal structure of oxygen- evolving photosystem II from Thermosynechococcus vulcanus at 3.7-A ˚ resolution. Proc. Natl Acad. Sci. USA 100, 98–103. 20. Adir, N. (1999) Crystallization of the oxygen-evolving reaction centre of photosystem II in nine different detergent mixtures. Acta Crystal. 55, 891–894. 21. Nakazato, K., Toyoshima, C., Enami, I. & Inoue, Y. (1996) Two-dimensional crystallization and cryo-electron microscopy of photosystem II. J. Mol. Biol. 257, 225–232. 22. Hankamer, B., Morris, E., Nield, J., Gerle, C. & Barber, J. (2001) Three-dimensional structure of the photosystem II core dimer of higher plants determined by electron microscopy. J. Struct. Biol. 135, 262–269. 23. Suzuki, T., Akiyama, A., Iwai, M., Tohri, A., Tomo, T., Ohta, H., Shen, J R. & Enami, I. (2001) Reconstitution of the extrinsic 23 and 17 kDa proteins with spinach PSII which had been exchanged for the 33 kDa protein from different species. In Proceeding of the 12th International Congress on Photosynthesis.(Osmond,B.ed.). CSIRO Publishing, Australia. 24. Bricker, T.M. & Frankel, L.K. (2003) Carboxylate groups on the manganese-stabilizing protein are required for efficient binding of the 24 kDa extrinsic protein to Photosystem II. Biochemistry 42, 2056–2061. 25. Enami, I., Mochizuki, Y., Takahashi, S., Kakuno, T., Horio, T., Satoh, K. & Katoh, S. (1990) Evidence from crosslinking for nearest-neighbor relationships among the three extrinsic proteins of spinach Photosystem II complexes that are associated with oxygen evolution. Plant Cell Physiol. 31, 725– 729. 26. Enami, I., Saoth, K. & Katoh, S. (1987) Crosslinking between the 33 kDa extrinsic protein and the 47 kDa chlorophyll-carrying protein of the PSII reaction center core complex. FEBS Lett. 226, 161–165. 27. Bricker,T.M.,Odom,W.R.&Queirolo,C.B.(1988)Closeasso- ciation of the 33 kDa extrinsic protein with the apoprotein of CPa-1 in Photosystem II. FEBS Lett. 231, 111–117. 28. Enami, I., Kaneko, M., Kitamura, N., Koike, H., Sonoike, K., Inoue, Y. & Katoh, S. (1991) Total immobilization of the extrinsic 33 kDa protein in spinach Photosystem II membrane prepara- tions. Protein stoichiometry and stabilization of oxygen evolution. Biochim. Biophys. Acta. 1060, 224–232. 29. Mei, R., Green, J.P., Sayre, R.T. & Frasch, W.D. (1989) Man- ganese-binding proteins of the oxygen-evolving complex. Biochemistry 28, 5560–5567. 30. Enami, I., Ohta, S., Mitsuhashi, S., Takahashi, S., Ikeuchi, M. & Katoh, S. (1992) Evidence from crosslinking for a close associa- tion of the extrinsic 33 kDa protein with the 9.4 kDa subunit of cytochrome b 559 and the 4.8 kDa product of the psbIgenein oxygen-evolving photosystem II complexes from spinach. Plant Cell Physiol. 33, 291–297. 31. Enami,I.,Tohri,A.,Kamo,M.,Ohta,H.&Shen,J R.(1997) Identification of domains on the 43 kDa chlorophyll-carrying protein (CP43) that are shielded from tryptic attack by binding of the extrinsic 33 kDa protein with Photosystem II complex. Biochim. Biophys. Acta 1320, 17–26. 32. Hoare, D.G. & Koshland, D.E. (1966) A procedure for the selective modification of carboxyl groups in protein. J. Am. Chem. Soc. 88, 2057–2058. 970 A. Tohri et al. (Eur. J. Biochem. 271) Ó FEBS 2004 33. Lindsay, D.G. & Shall, S. (1971) The acetylation of insulin. Biochem. J. 121, 737–745. 34. Miura, T., Shen, J R., Takahashi, S., Kamo, M., Nakamura, E., Ohta, H., Kamei, A., Inoue, Y., Dohmae, N., Takio, K., Nakazato, K., Inoue, Y. & Enami, I. (1997) Identification of domains on the extrinsic 33-kDa protein possibly involved in electrostatic interaction with Photosystem II complex by means of chemical modification. J. Biol. Chem. 272, 3788–3798. 35. Frankel, L.K. & Bricker, T.M. (1995) Interaction of the 33-kDa extrinsic protein with photosystem II: identification of domains on the 33-kDa protein that are shielded from NHS-biotinylation by photosystem II. Biochemistry 34, 7492–7497. 36. Ifuku, K. & Sato, F. (2002) A truncated mutant of the extrinsic 23 kDa protein that absolutely requires the extrinsic 17 kDa protein for Ca 2+ retention in Photosystem II. Plant Cell Physiol. 43, 1244– 1249. 37. Berthold, D.A., Babcock, G.T. & Yocum, C.F. (1981) A highly resolved oxygen-evolving Photosystem II preparation from spi- nach thylakoid membranes. FEBS Lett. 134, 231–234. 38. Kuwabara, T., Murata, T., Miyao, M. & Murata, N. (1986) Partial degradation of the 18-kDa protein of the photosynthetic oxygen-evolvingcomplex:astudyofabindingsite.Biochim. Biophys. Acta 850, 146–155. 39. Eaton-Rye, J.J. & Murata, N. (1989) Evidence that the amino- terminus of the 33 kDa extrinsic protein is required for binding to the Photosystem II complex. Biochim. Biophys. Acta 977, 219–226. 40. Ikeuchi, M. & Inoue, Y. (1988) A new photosystem II reaction center component (4.8 kDa protein) encoded by chloroplast gen- ome. FEBS Lett. 241, 99–104. 41. Porra, E.J., Thompson, W.A. & Kriedemann, P.E. (1989) Determination of accurate extinction coefficients and simulta- neous equations for assaying chlorophyll a and b extracted with four different solvents; verification of the concentration of chlo- rophyll standards by atomic absorption spectroscopy. Biochim. Biophys. Acta 975, 384–394. 42. Skoog, B. & Wichman, A. (1986) Calculation of the isoelectric points of polypeptides from the amino acid composition. Trends Anal. Chem. 5, 82–83. 43. Jansen, T., Rother, C., Steppuhn, J., Reinke, H., Beyreuther, K., Jasson, C., Andersson, B. & Herrmann, R.G. (1987) Nucleo- tide sequence of cDNA clones encoding the complete 23 kDa and 16 kDa precursor proteins associated with the photosynthe- tic oxygen-evolving complex from spinach. FEBS Lett. 216, 234–240. Ó FEBS 2004 Interaction between the 23 and 33 kDa proteins (Eur. J. Biochem. 271) 971 . Identification of domains on the extrinsic 23 kDa protein possibly involved in electrostatic interaction with the extrinsic 33 kDa protein in spinach photosystem. region of the 23 kDa protein is important for its binding with the 33 kDa protein. In the present study, the domains on the 23 kDa protein possibly involved