Photosynthesis Research 17:255-266 (1988) © Kluwer Academic Publishers, Dordrecht - Printed in the Netherlands

Regular paper

The status of cysteine residues in the extrinsic 33 kDa protein of spinach photosystem II complexes* SATOSHI TANAKA l & KEISHIRO WADA.2 1Department of Biology, Faculty of Science, Osaka University, Machikaneyama, Toyonaka, Osaka 560 Japan; 2Department of Biology, Faculty of Science, Kanazawa University, Marunouchi, Kanazawa 920 Japan; to whom correspondence should be addressed.) Received 28 October 1987; accepted in revised form 19 February 1988

Key words: disulfide (S-S) bond, extrinsic 33 kDa protein, Mn chelation, oxygen evolution Abstract. Two cysteine residues of the extrinsic 33kDa protein in the oxygen-evolving photosystemlI (PS II) complexes were found to exist as cystine residues in situ. The 33 kDa protein, when reduced by 2-mercaptoethanol in either the presence or the absence of 6 M guanidine-HC1 (Gdn-HC1), could not rebind with the CaCl2-treated PS II complexes, from which the 33 kDa protein was removed, and evolve any oxygen. Two sulfhydryl (SH) groups of the 33 kDa protein were easily reoxidized to a disulfide (S-S) bond by stirring under aerobic conditions with the concomitant regaining of both the binding ability to the CaC12-treated PS II complexes and the oxygen-evolving activity. The molecular conformation of the 33 kDa protein was examined by circular dichroic (CD) spectrometry in the UV regions to reveal that the conformation in the reduced state was completely different from those of the untreated and reoxidized states. The disulfide (S-S) bond of the 33 kDa protein is thus essential to maintain the molecular conformation required to function. Abbreviations: CD - circular dichroism, Chl - chlorophyll, DMQ - 2,5-dimethyl-p-benzoquinone, DTNB - 5,5'-dithio-bis (2-nitrobenzoic acid), E D T A - ethylendiamine-tetraacetic acid, Gdn-HC1- guanidine-hydrochloric acid, PS II - photosystem II, SDS - sodium dodecylsulfate

Introduction The oxygen-evolving photosystem II (PS II) complex is believed to consist of six intrinsic proteins (47, 43 kDa, Dr, DE and two subunits of cytochrome b559) and three extrinsic proteins (33, 23 and 18kDa) (Inoue et al. 1983, Murata and Miyao 1985). Among the three extrinsic proteins, the 18 and 23 kDa proteins are released by treatment with concentrated NaC1. This also results in a partial loss of oxygen-evolving activity (Kuwabara and Murata * This paper was presented at the U.S.-Japan Binational Seminar on Solar Energy Conversion, Okazaki, Japan, March 17-21, 1987

256 1983). All three extrinsic proteins are released by treatment with concentrated CaC12 (Ono and Inoue 1984), Tris-HC1 buffer (,~kerlund and Jansson 1981, Yamamoto et al. 1981), urea (Miyao and Murata 1984) or alkaline pH (Kuwabara and Murata 1982), resulting in complete loss of the oxygen-evolving activity. In the PS II complexes treated with 1.0 M CaCI2, four Mn atoms remain where they are, and the oxygen-evolving activity is restored considerably by the rebinding of the 33 kDa protein (Kuwabara et al. 1985, Ono and Inoue 1984). Dissociation and reconstitution experiments of the extrinsic proteins, have clearly shown that the 18 and 23 kDa proteins play a role in raising the affinity to the reaction center core complexes of chloride and calcium ions, which are essential for full oxygen-evolving activity (Akabori et al. 1984, Akerlund et al. 1982, Ghanotakis et al. 1984, Miyao and Murata 1984a). The 33 kDa protein, which is the most essential to oxygen-evolving activity among the three, functions to strongly preserve two of the four Mn atoms in the PS II complexes (Abramowicz and Dismukes 1984, Miyao and Murata 1984b, Ono and Inoue 1984a, Ono and Inoue 1984b). However, the knowledge as to how the 33 kDa protein holds Mn atoms is limited. The 33 kDa protein has two cysteine residues as described by Oh-oka et al. (1986). The sequence around the Cys-28 is very similar to the partial sequence containing an aspartic acid of a bacterial Mn-superoxide dismutase, which is considered to be one of the ligands to the Mn atom (Marres et al. 1985). This suggests that the cysteine residue can be a ligand to the Mn atom or that at least the cysteine residue and/or the region containing the cysteine residue have some interactions with Mn atoms. In this paper, we report the status of cysteine residues of 33 kDa protein, the restoration of oxygen-evolving activity upon the addition of the reduced or reoxidized 33 kDa protein to PS II complexes treated with CaCI2, and the role of the S-S bond in maintaining molecular conformation.

Materials and methods

Oxygen-evolving PS II particles were prepared from spinach chloroplasts with Triton X-100, as described by Kuwabara and Murata (1982a). They were suspended in a 25 mM MES buffer, pH 6.5, containing 1.0M CaC12 at a chlorophyll (Chl) concentration of 0.5 mg/ml and incubated for 30 rain at 4 °C under room light. The pellet was washed with the same medium and resuspended in a 25 mM MES buffer, pH 6.5, containing 300 mM sucrose and 200 mM NaC1. After centrifugation, the resulting precipitates were used as CaCl~-treated PS II complexes.

257 The 33 kDa protein was extracted from the NaCl-treated PS II complexes by 1.0 M CaCI2 treatment and purified by column chromatography with DEAE-Sepharose CL-6B as described by Kuwabara and Murata (1982b). The purified protein was dialyzed against 10mM NH4HCOa and lyophilized. The resultant powder was dissolved in a small volume of 25 mM MES buffer, pH 6.5, and the protein solution was centrifuged at 100,000 x g for 30 min to remove the insoluble components. The reduced 33 kDa protein was prepared by incubating it with about a 1000-molar excess of 2-mercaptoethanol to a cystine residue in the presence of 6 M Gdn-HCI for 3 h at 25°C and separated from the residual reagents by passing through a Sephadex G-25 column equilibrated with 10mM sodium acetate buffer, pH 4.0, containing 30 mM NaCI and 5 #M EDTA. Reoxidation of sulfhydryl (SH) groups of the reduced 33 kDa protein was performed as follows. To five volumes of reduced 33 kDa protein solution in 10 mM acetate buffer, pH 4.0, one volume of 200 mM sodium phosphate buffer, pH 8.0, was added, and the mixture was incubated at 25°C with vigorous stirring. Subsequently, an aliquot of the solution was withdrawn and used for SH titration and measurements of the oxygen-evolving activity and the rebinding activity to the CaCl2-treated PS II complexes. For measurement of CD spectra, the SH groups were oxidized by the addition of 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB). To the neutralized 33 kDa protein solution, one-tenth portions of DTNB were added at l-min intervals until the total amount of DTNB became twice that of 33 kDa protein (mole/mole). Then, reoxidized 33kDa protein was passed through a Sephadex G-25 column equilibrated with 10mM Tris-HC1, pH 7.5. SH groups were titrated by DTNB. The protein solution was mixed with an equal volume of 0.2 M Tris-HCl buffer, pH 8.0, and added to freshly prepared DTNB. The absorbance increase at 412 nm was then measured and the amount of SH groups was calculated using a millimolar extinction coefficient of 13.6 (Ellman 1958). The SH reagent, pCMB, was also used to titrate the SH groups of the 33 kDa protein within the PS II complexes. The PS II complexes were incubated with a 200-molar excess of pCMB to the reaction center of PS II, for 1 h at 0°C. The 33 kDa protein was released by CaC12 treatment and the amount of mercury bound to the 33 kDa protein was determined by dithizone according to the method of Madsen and Gurd (1956), after acid hydrolysis with 6 N HC1 for 24 h at 110°C in an evacuated and sealed tube. Oxygen-evolving activity was assayed using a Clark-type oxygen electrode (YSI). The assay medium contained 0.5 mM 2,5-dimethyl-p-benzoquinone (DMQ), 300mM sucrose, 25mM NaC1, 0.05% bovine serum albumin, CaC12-treated PS II complexes corresponding to 10#g/ml Chl concentra-

258 tion, 3/~g/ml designated form of the 33 kDa protein (to Chl ratio of 0.3, w/w) and 25 mM MES buffer, pH 6.5. The assay medium in the cell, controlled at 25°C by a circulating bath, was illuminated by a halogen lamp (650 W) through a 10-cm thick water layer. The Chl concentration was determined by the method of Arnon (1949). The rebinding experiments of the 33 kDa protein to the CaCl2-treated PS II complexes were carried out as described by Kuwabara et al. (1985), except that the ratio of the 33 kDa protein to Chl was 0.3 (w/w). The amount of 33 kDa protein bound to PS II complexes was analyzed densitometrically on an SDS polyacrylamide gel run by a Laemmli's buffer system (Laemmli 1970). Measurements of CD spectra were carried out with a JASCO J-500A spectropolarimeter, equipped with a JASCO DP-501 data processor. The conditions of measurements were 0.25s, time constant; 1 mdegree/cm, sensitivity; 5ms, sampling time; and 50nm/min, scanspeed at 25°C. The spectral scanning was repeated 16 times. The concentration of sample was 20/~M for a near-UV region and 6/~M for a far-UV region in 10mM Tris-HC1, pH 7.5. The spectra were recorded by using cells with 1.0 cm and 0.1 cm light path for near- and far-UV regions, respectively. The concentration of the 33kDa protein was determined spectrophotometrically by using millimolar extinction coefficients of 20 (Kuwabara and Murata 1984) and 18 at 276 nm for native and reduced forms, respectively, determined from amino acid analysis.

Results and discussion

The cysteine residues of the 33 kDa protein released from the PS II complexes with the successive treatment of NaC1 and CaC12 were titrated with DTNB. As shown in Table 1, no SH group was detected by DTNB titration in the absence of 2-mercaptoethanol. On the other hand, two SH groups per mole of protein were titrated after reduction by 2-mercaptoethanol, regardless of the presence or absence of 6 M guanidine-HCl. It was therefore concluded that two cysteine residues of the 33 kDa protein existed in the form of cystine residue, as Kuwabara and Murata (1979) had suggested from the mobility change in SDS-free gel electrophoresis in the presence of 2-mercaptoethanol. In order to confirm the presence of an S-S bond of the 33 kDa protein in situ in freshly prepared PS II complexes, the complexes were titrated with pCMB, which is considered to penetrate the interior portion of proteins or complexes and to form a mercaptide with SH groups. However, neither the binding of mercury to the 33 kDa protein, nor an effect

259 Table 1. Titration of sulfhydryl groups of the 33 kDa protein by DTNB.

Treatments

SH titrated by DTNB (moles/mole of 33 kDa protein)

None 6 M Gdn-HCP 6M Gdn-HC1 + 2-mercaptoethanol b 2-mercaptoethanoF

< 0.1 < 0. I 1.8 2.1

Assay medium contained 0.1 M Tris-HC1 buffer, pH 8.0, 50#M DTNB and 5pM 33 kDa protein in the sample cell. Reference cell contained the same medium except for the 33 kDa protein. Absorbance changes at 412 nm before and after the addition of DTNB were measured. a) Assay medium contained 6 M Gdn-HC1. b) The 33 kDa protein was incubated with 6 M Gdn-HC1 and 10% 2-mercaptoethanol for 3 hr at 25°C and desalted by passing through a Sephadex G-25 column before SH titration with DTNB. c) The 33 kDa protein was incubated as in b) except that 6 M Gdn-HC1 was omitted.

on the oxygen evolution o f PS I I complexes was observed (data not shown). These results, also r e p o r t e d b y Barr a n d C r a n e (1982), suggest that PS II complexes have no iron-sulfur cluster, unlike P S I complexes. As s h o w n in T a b l e 2, the addition o f 33 k D a protein to C a C I 2-treated PS I I complexes resulted in the rebinding o f the protein and restored oxygenevolving activity. T h e oxygen-evolving activity o f CaC12-treated PS II c o m plexes did n o t b e c o m e zero, because C a C l 2 t r e a t m e n t could not completely release the 33 k D a protein. T h e recovery o f oxygen evolution by the untreated 33 k D a protein was 34%, as in Exp. 3 o f Table 2. This figure was rather low c o m p a r e d with the oxygen-evolving activity o f the original PS II complexes. A recovery o f o v e r 80% can be o b t a i n e d when the a p p r o p r i a t e a m o u n t s o f calcium a n d chloride ions are a d d e d to assay medium. O n the other hand, the reduced 33 k D a protein could not rebind to the CaCI 2treated PS I I complexes or cause oxygen-evolving activity to resume (Exp. 4 in T a b l e 2). The reoxidized 33 k D a protein behaved in the same m a n n e r Table 2. Abilities of rebinding and oxygen evolution of the treated 33 kDa protein.

Exp.

Treatments

Oxygen evolution (%)

Bindingb ability (%)

1 2 3 4 5

Untreated CaC12-treated 2 + untreated 33 kDa 2 + reduced 33 kDa 2 + reoxidized 33 kDa

100a 10 34 11 33

100 12 82 15 76

a) Corresponding to 300/~moles 02 evolution/mg Chl/hr; b)The amount of the 33 kDa protein bound to the CaC12-treated PS II complexes was determined densitometrically on SDS-polyacrylamide gel.

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Fig. /A. Reoxidation of the SH groups and recovery of rebinding and oxygen evolving activities. Reduced 33 kDa protein solution was incubated under aerobic conditions at pH 8.0 for a certain period and an aliquot of solution was withdrawn to titrate the SH groups and measure the oxygen evolving activity and rebinding abilities of the CaC12-treated PS II complexes.

as the original protein that was not subjected to the reduction-reoxidation process. The S-S bond of the 33 kDa protein is thus essential for rebinding to the CaCl2-treated PS II particles and for oxygen-evolving activity. Figure 1A shows that the reduction of the S-S bond is a reversible reaction. The reduced S-S bond is easily reoxidized within a few hours under aerobic conditions with the concomitant restoration of binding and oxygenevolving abilities. Most of the reoxidized 33 kDa protein existed in a monomeric form, judging from SDS-polyacrylamide gel electrophoresis without 2-mercaptoethanol, but two very faint bands were observed at the positions ofdimer and trimer on the gel, as shown in Fig. 1B, lane 1. They disappeared in the presence of 2-mercaptoethanol (lane 2), The incomplete recovery in the experiments shown in Table 2 seems to be partially due to the formation of a dimer and trimer of the 33 kDa protein upon reoxidation. The more reducing reagent used and the longer the incubation period, the more effective was the reduction of the S-S bond. The presence of denaturant also aided the reduction. These phenomena suggest that the S-S bond is not completely exposed 'to the molecular surface, but is close enough to the surface of the molecule to be accessible to the reductant added outside.

261

Fig. lB. SDS-polyacrylamidegel electrophoresisof the reoxidized 33 kDa protein. Electro-

phoresis was performedby using 12.5% gel containing 5 M urea in the presenceS(lane2) and absence (lane 1) of 2-mercaptoethanolin the sample buffer. Arrowheadson lane i show the positions of dimer and trimer of the 33 kDa protein. Therefore, it is feasible that the S-S bond, or a portion surrounding the S-S bond, has a direct interaction with Mn atom(s). A recent paper has postulated the possibility of an interaction between the S or S-S and Mn with different valences (Kessissoglou et al. 1987). The conformational changes with reduction and reoxidation of the 33 kDa protein were investigated by comparing CD spectra. The spectrum in the near-UV region of the protein showed two clear peaks at 294 and 287 nm, corresponding to tryptophan and tyrosine residues, respectively, and a doublet peak at 264 and 257 nm, corresponding to phenylalanine residues, as shown in Fig. 2. The CD band of a tryptophan residue located near the carboxyl-terminus of the polypeptide chain was very remarkable. The signals of tryptophan and tyrosine residues disappeared completely upon reduction of the S-S bond (Fig. 2B). Upon reoxidation of the two SH groups with DTNB used as an oxidizing reagent, these signals were recovered nearly to the original level.

262

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WAVELENGTH ( n m ) Fig. 2. CD spectra in the near-UV region of the untreated (A), reduced (B) and reoxidized (C) 33 kDa protein. The proteins were dissolved in 10mM Tris-HCl buffer, pH 7.5.

The CD spectrum of the 33 kDa protein in the far-UV region, which generally reflects the secondary structure of the polypeptide, was anomalously small compared with other proteins investigated so far (Fig. 3A). When the protein was reduced, the CD spectrum was similar to that of a random conformation (Fig. 3B) as was also observed in 4 M guanidine-HC1 (data not shown). The CD spectrum of the reoxidized 33 kDa protein recovered tO almost completely that of the untreated one (Fig. 3C). As shown in Fig. 3A, the negative molar elipticity of this protein was anomalously low. A reverse contribution in the far-UV region due to the positive CD band of aromatic amino acids and S-S bond, as suggested by Beychek

263

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WAVELENGTH (rim) Fig. 3. CD spectra in the far-UV region of the untreated (A), reduced (B) and reoxidized (C) 33 kDa protein. The measuring conditions were the same as in Fig. 2.

(1966), might be responsible. Actually the CD spectrum of the reduced form in the far-UV region became deeper than that of the untreated form. As discussed by Oh-oka et al. (1986), the 33 kDa protein behaves as a larger protein than is calculated from the primary structure (27 kDa) in SDSpolyacrylamide gel electrophoresis and gel filtration. These facts lead us t o speculate that this protein does not have a compact structure but rather a somewhat loose and expansive structure, which may relate to the abnormal CD strength in the far-UV region. The S-S bond was easily regenerated from the reduced form by stirring the protein solution under aerobic conditions (Fig. 1). A small amount of cupric

264 ion markedly accelerated the reoxidation. However, CD spectra of the 33 kDa protein reoxidized by these treatments were recovered to only half the extent of the untreated one. As judged by the far-UV CD spectra, the secondary structures of these reoxidized 33 kDa proteins, in the presence of cupric ion, were not fully restored, though they recovered oxygen-evolving activity and rebinding ability to near the original level. This partial recovery of the CD spectrum upon reoxidation might be due to the oligomers formed by intermolecular S-S bonds (Fig. 1B), although there were so few that their effect on the activity measurement was negligible. In fact there was no band corresponding to a dimer or trimer on the SDS-polyacrylamide gel when the protein was reoxidized by DTNB (data not shown) and the CD spectrum was restored to nearly the original level (Figs 2C and 3C). Further experiments will be needed to verify this. The CD spectrum of the reduced 33 kDa protein was close to that of a random form. The CD signal of the tryptophan residue located in the near carboxyl-terminus was strongly affected by a reduction of the S-S bond between Cys-28 and Cys-51, which is near the amino-terminus. These results suggest that conformational change in the reduction of the S-S bond occurred not only around the S-S bond, but in all parts of the protein. This structure was restored concomitantly with the reoxidation of SH groups. When the protein was denatured in guanidine-HCl followed by the removal of the denaturant, as long as the S-S bond was not reduced, its original CD spectrum was perfectly recovered (data not shown). These results indicate that the S-S bond of the 33 kDa protein is absolutely essential to maintain the unique structure of this protein. The S-S bond of the 33 kDa protein also contributes to its stability, because once it was reduced, the protein was easily aggregated and became insoluble during dialysis or concentration. Recent two-sequence studies revealed that the cysteine residues in spinach 33 kDa protein have been conserved in the corresponding proteins of pea (Watanabe et al. 1987) and the cyanobacterium, Anacystis nidurans R2 (Kuwabara et al. 1987), suggesting the importance of the S-S bond, though the presence of the S-S bond has not yet been confirmed. However, a partial sequence around one of the two cysteine residues was found to be dissimilar to that in bacterial Mn-superoxide dismutase, unlike that in the report by Oh-oka et al. (1986). Further investigations of the 33kDa protein are required to establish its mode of interaction with Mn atom(s) and PS II core proteins.

265

Acknowledgements This work was supported in part by Grant-in-Aid for Scientific Research from the Japanese Ministry of Education, Science and Culture to KW (#61304004 and #62621004). The authors are grateful to Prof H. Matsubara for his generous support and interest, Prof K. Hamaguchi and Dr Y. Kawata, Department of Biology, Faculty of Science, Osaka University and Prof T. Takagi and Dr K. Yutani, Institute for Protein Research, Osaka University, for the use of their facilities to measure CD spectra and to Ms Y. Minami, Mr H. Oh-oka and Mr Y. Takahashi for their useful advice.

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266 Kuwabara T and Murata N (1983) Quantitative analysis of the inactivation of photosynthetic oxygen evolution and the release of polypeptides and manganese in the photosystem II particles of spinach chloroplasts. Plant Cell Physiol 24:741-747 Kuwabara T and Murata N (1984) Chemical and physicochemical characterization of the proteins involved in the oxygen evolution system. In: Sybesma C (ed.) Advances in Photosynthesis Research Vol. 1, pp 371-374. The Hague: Martinus Nijhoff/Dr W. Junk Kuwabara T, Miyao M, Murata T and Murata N (1985) The function of 33 kDa protein in photosynthetic oxygen evolution system studied by reconstitution experiments. Biochim Biophys Acta 806:283-289 Kuwabara T, Reddy KJ and Sherman LA (1987) Nucleotide sequence of the gene from the cyanobacterium Anacystis nidulans R22 encoding the Mn-stabilizing protein involved in PS II water oxidation. Proc Natl Acad Sci USA 84:8230-8234 Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680-685 Madsen NB and Gurd FRN (1956) The interaction of muscle phosphorylase with pCMB. J. Biol. Chem. 223:1075-1087 Marres CA, Van Loon APGM, Oudshoon R, Van Steeg H, Grivell VA and Slater EC (1985) Nucleotide sequence analysis of the nuclear gene coding for manganese superoxide dismutase of yeast mitochondria, a gene previously assumed to code for the Rieske iron-sulfur protein. Eur J Biochem 147:153-161 Miyao M and Murata N (1984a) Calcium ions can be substituted for the 24 kDa polypeptide in the photosynthetic oxygen evolution. FEBS Lett 168:118-120 Miyao M and Murata N (1984b) Role of the 33 kDa polypeptide in preserving Mn in the photosynthetic oxygen-evolution system and its replacement by chloride ions. FEBS Lett 170:350-354 Murata N and Miyao M (1985) Extrinsic membrane proteins in the photosynthetic oxygenevolving complex. Trends Biochem Sci 10:122-124 Oh-oka H, Tanaka S, Wada K, Kuwabara T and Murata N (1986) Complete amino acid sequence of 33 kDa protein isolated from spinach photosystem II particles. FEBS Lett 197: 63-66 Ono T and Inoue Y (1984a) Mn preserving extraction of 33, 24 and 16 kDa proteins from O2-evolving photosystem II particles by divalent salt washing. FEBS Lett 164:255-260 Ono T and Inoue Y (1984b) Reconstitution of photosynthetic oxygen-evolving activity by rebinding of 33 kDa protein to CaC12-extracted PS II particles. FEBS Lett 166:381-384 Watanabe A, Minami E, Murase M, Shinohara K and Kuwabara T (1987) Biogenesis of photosystem II complex in spinach chloroplasts. In: Biggins J (ed.) Progress in Photosynthetic Research Vol. IV pp 629-636. Dordrecht: Martinus Nijhoff/Dr W. Junk Yamamoto T, Doi M, Tamura N and Nishimura M (1981) Release of polypeptides from highly active O2-evolving photosystem 2 preparation by Tris treatment. FEBS Lett 133: 265-268