Photosynthesis Research 29: 149-155, 1991. © 1991 Kluwer Academic Publishers. Printed in the Netherlands. Regular paper

Effect of the manganese complex on the binding of the extrinsic proteins (17, 23 and 33 kDa) of Photosystem II K. Kavelaki & D.F. Ghanotakis*

Department of Chemistry, University of Crete, Iraklion, Crete, Greece Received 10 May 1991; accepted in revised form 11 July 1991

Key words:

Photosystem II, extrinsic proteins, manganese complex

Abstract

Selective extraction-reconstitution experiments with the extrinsic Photosystem II polypeptides (33 kDa, 23 kDa and 17 kDa) have demonstrated that the manganese complex and the 33 kDa polypeptide are both necessary structural elements for the tight binding of the water soluble 17 and 23 kDa species. When the manganese complex is intact the 33 kDa protein interacts strongly with the rest of the photosynthetic complex. Destruction of the Mn-complex has two dramatic effects: i) The binding of the 33 kDa polypeptide is weaker, since it can be removed by exposure of the PS II system to 2 M NaC1, and ii) the 17 and 23 kDa species do not rebind to Mn-depleted Photosystem I! membranes that retain the 33 kDa protein.

Abbreviations: C h l - chlorophyll; H Q - hydroquinone; M E S - 2(N-morpholino)ethanesulfonic acid; PS II - Photosystem II; Tris - 2-amino-2-hydroxymethylpropane-l,3 - diol

1. Introduction

The extrinsic polypeptides of Photosystem II (17, 23 and 33 kDa) are the subject of intense research. The 33 kDa protein is tenaciously bound to PS II, but can be removed by various treatments. Exposure of PS II membranes to high concentrations of Tris results in release of all three extrinsic proteins with concomitant destruction of the manganese complex (Yamamoto et al. 1981, Kuwabara and Murata 1982, Franzen and Andersson 1984); although this treatment was taken initially as evidence that the 33-kDa protein is required for Mn binding, it was later shown by Ono and Inoue (1983, 1984) and by Miyao and Murata (1984) that it is possible to remove the 33 kDa protein without removing the * Address for correspondence: D.F. Ghanotakis, Department of Chemistry, University of Crete, 71 409 Iraklion, Crete, Greece

manganese complex. Although after the release of the 33 kDa protein the manganese complex remains bound to the PS II membrane, it is very unstable. This observation has led to a model which attributes to the 33 kDa species a stabilizing role. The 17 and 23 kDa proteins are amenable to extraction/reconstitution experiments, and so it has been possible to demonstrate that although they are not directly involved in the catalytic mechanism of the photosynthetic water cleavage, the 23 and 17kDa proteins play an important role under physiological conditions. On one hand they act as 'concentrators' of the inorganic cofactors calcium and chloride which are essential for oxygen evolution activity (Ghanotakis et al. 1984, Andersson et al. 1984, Miyao and Murata 1985), and on the other hand they play an important role in the structural arrangement around the manganese complex (Ghanotakis et al. 1984b, Tamura et al. 1986).

150 Removal of the 17 and 23 kDa species results in exposure of the manganese complex to bulky exogenous reductants which reduce and destroy the manganese complex (Ghanotakis et al. 1984b). Using a series of selective extraction/reconstitution techniques, we have demonstrated that the manganese complex regulates the structure and properties of the 33 kDa polypeptide. Although manganese ligation is not essential for the binding of the 33 kDa protein to the PS II complex, its release has two major effects on the organization of the extrinsic proteins: First the interaction of the 33 kDa polypeptide with the rest of the photosynthetic unit is weaker, and second the 17 and 23 kDa species can no longer bind to PSII.

2. Materials and methods

Photosystem II membranes were prepared as described by Ghanotakis et al. (1984b). Extraction of the 17 kDa and the 23 kDa polypeptides was carried out by exposure of intact PS II membranes to 2 M NaC1 for 45min; the 33kDa species was removed by exposure of the (17, 23 kDa)-depleted PS II membranes to 1 M CaC12 for 45 min. The polypeptide depleted PS II membranes were collected by centrifugation (20 min, 45 000 × g). Selective reconstitution with the various extrinsic proteins was carried out as follows: The 33 kDa and the 17kDa + 23 kDa extracts were transferred, by dialysis, into a solution containing 50mM MES, pH 6.0, and 200mM NaC1; rebinding of the appropriate protein(s) was carried out by resuspending the PS II membranes in the solution containing the extracted protein(s) and further decreasing the ionic strength by dialysis against a solution containing 50mM MES, pH 6.0, and 10mM NaC1. Gel electrophoresis was carried out as described by Ghanotakis et al. (1984b). The relative amount of the various polypeptides was determined by a Hoefer densitometer. The oxygen evolution activity of the various PS II preparations was measured by a YSI Clark type oxygen electrode. The manganese content of the various preparations was determined by atomic absorption spectroscopy (Perkin-Elmer, model 4000).

3. Results

Exposure of intact Photosystem II membranes to 2 M NaC1 results in release of the water soluble 17 and 23 kDa polypeptides (Fig. 1, lane 2) (Akerlund et al. 1982, Ghanotakis et al. 1984c); subsequent exposure of the (17, 23kDa)depleted system to 1 M CaC12 results in removal of the 33 kDa species (Fig. 2, lane 1). However, the Mn-complex remains intact as long as a high concentration of NaC1 is maintained in the system (Ono and Inoue 1985). The manganese complex of both of the above PS II systems is easily destroyed by exposure to an exogenous reductant such as hydroquinone (Ghanotakis et al. 1984b); under these conditions destruction of the manganese complex is not accompanied by removal of the 33 kDa protein (Fig. 1). The PS II system which retains both the 33 kDa polypeptide and the manganese complex has completely different properties when compared to the 33 kDa-retaining but Mn-depleted PSII system. The interaction of the 33 kDa polypeptide with the rest of the PS II system is weaker when the Mn-complex has been destroyed as demonstrated by the fact that exposure of this system to 2 M NaC1 results in partial removal of the 33kDa extrinsic protein; exposure of the (33 kDa/(Mn)x-retaining) PS II to 2 M NaC1 has no significant effect on the 33 kDa species (data summarized in Table 1). In addition, although the 17 and 23 kDa species easily rebind to the (33 kDa/(Mn)x-retaining) PS II (Fig. 1, lane 4) no rebinding of these polypeptides is observed in the Mn-depleted system which retains the 33 kDa species (Fig. 1, lane 5). The above results could be explained by two models. In the first, the release of manganese affects the interaction of all three extrinsic polypeptides with the rest of the PS II membrane; in other words the Mn-complex is required for the binding of the 17 and 23 kDa species and the high affinity binding of the 33 kDa polypeptide. According to the second model, the Mn-complex regulates the structure of the 33 kDa protein and thus its interaction with the PS II membrane and the 17 and the 23kDa species as well; when manganese is released the structure of the 33 kDa protein is altered in such a way that its

151

Fig. 1. Gel electrophoresis patterns of various PS II preparations. Lane 1, control PS II; lane 2, 2 M NaCl-treated PS II; lane 3, as lane 2, then treated with 2 mM HQ; lane 4, as lane 2, then incubated with 17, 23 kDa; lane 5, as lane 3, then incubated with 17, 23 kDa; lanes 6 & 7, as lane 5 (different experiments).

Fig. 2. Gel electrophoresis patterns of various PS II preparations. Lane 1, PS II membranes treated first with 2 M NaC1 and then with 1M CaCI2; lane 2, as lane 1, then treated with 2 m M HQ and subsequently incubated with 17, 23 kDa; lane 3, PS II membranes treated first with 2 M NaC1 and then with 0 . 6 M CaC12; lane 4, as lane 1, then treated with 2raM H Q and subsequently incubated with 33 kDa; lane 5, as lane 4, then incubated with 17, 23 kDa; lane 6, as lane 1, then incubated with 17, 23 kDa [in addition to the 17 and 23 kDa species, the 2 M NaC1 extract contains also a small fraction of the 33 kDa (see Table 1) which rebinds to the PS II at this point]; lane 7, as lane 1, then incubated first with 33 kDa and subsequently with 17, 23 kDa; lane 8, as lane 5; lane 9, as lane 1, then incubated with 33 kDa; lane 10, Control PS II.

152 Table i. Effect of the various treatments on the manganese and polypeptide content of PS II membranes

Treatment a

None 2M NaCI i) 2M NaC1 ii) 2 m M H Q i) 2M NaC1 ii) 2M NaC1 i) 2M NaC1 ii) 2mM HQ iii) 2M NaC1 i) 6 mM NH2OH ii) 100 m M NaC1 100 m M NaC1

Mn-content b

Polypeptide-content c 17 kDa

23 kDa

33 kDa

100% 0 0

100% 5% 5%

100% 90% 90%

100%

0

0

90%

20%

0

0

40%

15%

0

5%

100%

100%

70%

90%

100%

100% d 100% 25%

i-iii denote successive treatments. b The manganese content was determined by atomic absorption spectroscopy. The polypeptide content of the various preparations was determined from coommasie-stained gels by densitometry. d 4 Mn atoms/PS II reaction center.

interaction with the rest of the photosynthetic unit becomes weaker and, in addition, rebinding of the 17 and 23 kDa species is not possible. To further test the above two models we carried out a series of extraction-reconstitution experiments with all three extrinsic proteins. The results (Fig. 2) can be summarized as follows: When the 33 kDa polypeptide is present, rebinding of the 17 and 23 kDa species is possible, but only when the Mn-complex is intact. After release of the 33 kDa polypeptide, the 17 and 23 kDa polypeptides do not rebind to PS II even in the presence of the manganese complex (Fig. 2, lane 6). Since the rebinding of the various extrinsic polypeptides was carried out by resuspension of the PS II membranes to be reconstituted in a solution containing the extrinsic polypeptides and 200mM NaC1, followed by gradual decrease of the salt concentration to 10 mM NaC1, one could argue that in the case of the 33 kDa-depleted/Mn-retaining PSII membranes the decrease in NaCI concentration resulted in a destruction of the Mn-complex and this prevented the 17 and 23 kDa species (in the absence of the 33 kDa species the Mn-complex is stable only in the presence of 100mM NaC1 (Ono and Inoue 1985)). To address this question we repeated the reconstitution experiment with the (33 kDa-depleted/Mn-retaining) PS II under

conditions where the system was never exposed to NaC1 concentrations lower than 100 mM; even under these conditions we observed no binding of the 17 and 23kDa polypeptides (data not shown). The above data suggest that of the two possible models the one which proposes that the Mn-complex regulates the properties of the 33kDa polypeptides, and thus its interaction with the 17 and 23 kDa species, seems to be more likely. It was previously shown that manganese removal by hydroxylamine results in partial loss of the 23 and 17 kDa polypeptides (Tamura and Cheniae 1985). As shown in Fig. 3, under our conditions a concentration of hydroxylamine as high as 6 mM is required for destruction of oxygen evolution capacity. The effect of such a treatment on the polypeptide content of PSII membranes is shown in Fig. 4. Treatment of PS II with 6 mM NH2OH results in almost total release of the 17 kDa species and partial release of the 23 kDa polypeptide. Subsequent washing of the NH2OH-treated PSII with a medium containing 100 mM NaC1 removes the remaining traces of the 17 kDa, and most of the 23 kDa species. Treatment of intact PS II membranes with a medium of the same ionic strength (100 mM NaC1) removes a fraction of the 17 kDa polypeptide, but has no significant effect on the

153

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