Electron donation to photosystem II by

Electron donation to photosystem 11 by diphenylcarbazide is inhibited both by the endogenous manganese complex and by exogenous manganese ions Biochem. Cell Biol. Downloaded from www.nrcresearchpress.com by Laurentian University on 03/29/13 For personal use only.

Abdur Rashid and Radovan Popovic

Abstract: Diphenylcarbazide (DPC) is an efficient electron donor to the inactive oxygen-evolvingcomplex of photosystem I1 (PSII). We investigated the role of manganese on the rate of electron donation from DPC to PSII in both Mn-depleted (Tris washed) and Mn-retaining (NaC1 washed) PSII preparations. The rate of electron donation from DPC to PSII was significantly higher in Mn-depleted than in Mn-retaining preparations, indicating a negative role of native Mn complex on DPC electron donation. The apparent Kmvalues for DPC were found to be 0.11 and 0.17 mM for Mn-depleted and Mn-retaining PSII preparations, respectively. This difference in the K,,,values also indicates an antagonistic effect of endogenous Mn cluster on electron donation from DPC, which was markedly inhibited by exogenous ~ n " . However, the magnitude of inhibition was greater in Mn-depleted than in Mn-retaining PSII preparations. This indicates a higher accessibility of DPC to PSII in the absence of native Mn complex. Our results suggest (i) that Mn, either endogenous or added, acts as an accessibility barrier for DPC to donate electrons to PSII and (ii) that the native Mn complex not only functions as an accumulator of oxidizing equivalents but may also protect PSII from exogenous reductants. Key words:photosystem 11, extrinsic polypeptides, Mn complex, electron transport, diphenylcarbazide.

R6sumC : La diphenylcarbazide (DPC) est un bon donneur d'tlectrons au complexe de production d'oxygkne inactif du photosystkme I1 (PSII). Nous avons CtudiC le r6le du mangankse (Mn) sur le taux de transfert des Clectrons de la DPC au PSII dans des preparations du PSII depourvu de Mn (lave avec le tampon Tris) et du PSII ayant conserve le Mn (lave avec le NaC1). Le taux de transfert des electrons de la DPC au PSII est significativement plus tleve dans les preparations du PSII depourvu de Mn que dans celles du PSII ayant conserve le Mn, ce qui indique un r6le nCgatif du complexe d'ions Mn endogkne sur le transfert d'electrons. Les valeurs de Km apparent envers la DPC sont 0,11 mM dans les prtparations du PSII depoumu de Mn et 0,17 mM dans les preparations du PSII ayant conservC le Mn. Cette difference entre les valeurs de Kmindique Bgalement que un effet antagoniste du noyau d'ions Mn endogkne sur le transfert d'electrons B partir de la DPC. Ce taux de transfert des electrons est inhibt de faqon importante par un apport exogkne de ~ n ~Cependant, + . l'inhibition est plus grande dans la preparation du PSII depourvu de Mn que dans celle du PSII ayant conserve le Mn. Cela indique une plus grande accessibilite de la DPC au PSII en absence du complexe d'ions Mn endogkne. Nos resultats suggkrent (i) que le mangankse, endogkne ou exogkne, agit comme une bamkre limitant l'accessibilite de la DPC au PSII pour le transfert d'electrons et (ii) que le complexe d'ions Mn endogkne n'est pas seulement un accumulateur d'tquivalents d'agents oxydants, mais il protegerait Bgalement le PSII des agents rtducteurs exogknes. Mots clis :photosyst&me11, polypeptide extrenskque, complex d'ions Mn, transportationd'tlectrons, diphenylcarbazide. [Traduit par la r15dactionI

Received December 20, 1994. Accepted June 1,1995.

Abbreviations: DPC, diphenylcarbazide; PSII, photosystem 11; OEC, oxygen-evolving complex; kDa, kilodalton(s); PMSF, phenylmethylsulfonylfluoride; MES, morpholinoethane sulfonic acid; chl, chlorophyll; DCIP, dichlorophenolindophenol.

A.

ashi id' and R. Popovic. Departement de chimie, Universitt du Quebec I? Montreal, CP 8888, Succursale A, Montreal, QC

H3C 3P8,

Canada. Author to whom all correspondenceshould be sent at the following address: Pest Management Branch, Alberta EnvironmentalCentre, Bag 4000, Vegreville, AB T9C 1T4, Canada. Biochem. Cell Biol. 73: 241-245 (1995). Printed in Canada I Imprim6 au Canada

Biochem. Cell Biol. Vol. 73, 1995

Biochem. Cell Biol. Downloaded from www.nrcresearchpress.com by Laurentian University on 03/29/13 For personal use only.

Introduction The OEC of PSII provides binding domains for three extrinsic polypeptides of molecular masses 16-17,23-24, and 33 kDa. Several inorganic cofactors, such as chloride, calcium, and manganese, are also reported to be associated with the OEC. The oxygen-evolving efficiency of PSII depends on the cooperation of extrinsic polypeptides and the inorganic cofactors (see reviews by Govindjee et al. 1985; Ghanotakis and Yocum 1985; Homann 1987; Coleman 1990). Among the cofactors, Mn in the form of a tetra-Mn complex plays a central role in the four-step oxidation of H20 to molecular oxygen. It has been established that the Mn complex accumulates successively four oxidizing equivalents generated during lightdriven electron transport from PSII and utilizes this oxidizing power to split H20 to evolve oxygen (Rutherford 1989). In PSII preparations, if the water oxidizing capacity is lost either as a result of the depletion of extrinsic polypeptides or the loss of both extrinsic polypeptides and Mn, then an artificial electron donor, such as H202, can partially restore the electron transport activity. In fact, in the latter preparation, H202donates electrons to the very tightly bound Mn (residual Mn, not removed by Tris treatment) that is photooxidized by PSII. However, H202-supported PSII electron transport activity can be completely restored if exogenous ~ n ' +is added. From the above observations, it was concluded that H202oxidation by PSII is mediated via endogenous or added Mn (Pan and Izawa 1979; Velthuys 1983; Inoue and Wada 1987; Sandusky and Yocum 1988; Rashid and Carpentier 1989). Diphenylcarbazide, another electron donor to the inactive OEC (Izawa 1980; Rashid and Popovic 1990; Purcell et a1 1991), was reported to donate electrons to Z+ (Babcock 1987; Metz et al. 1989; Blubaugh and Cheniae 1990), the tyrosine161 residue of the Dl-polypeptide of PSII (Debus et al. 1988). The position of Z+ in the photosynthetic electron transport chain is thought to be after the Mn complex and immediately before the reaction centre of PSII, P680 (Barry and Babcock 1987). In view of the separate physical locations of the Mn cluster and DPC action site (ZC)in the PSII oxygen-evolving complex, we initially expected that electron donation from DPC to PSII would be independent of the involvement of Mn. However, we provide data in this manuscript that show that electron donation from DPC to PSII is inhibited by both the endogenous Mn complex and by exogenously added Mn ion.

Materials and methods The PSII membranes were prepared from spinach (Spinacia oleracea L.) leaves following the procedure of Berthold et al. (1981) with some modifications (Rashid et al. 1994). The deveined leaves were homogenized in a Waring blender containing 25 mM HEPES-NaOH buffer (pH 7.0), 10 rnM NaCl, 5 mM MgCl,, 0.1% (wlv) ascorbate, 1 mM PMSF, and 0.4 M sorbitol. The homogenate was filtered through eight layers of cheesecloth and the filtrate was centrifuged for 5 min at 2000 x g. The pellet was suspended in the same buffer, but without sorbitol and PMSF, and then centrifuged under the same conditions. The resulting pellet was suspended in a solution containing 20 mM MES-NaOH buffer (pH 6.5), 10 mM NaC1, 5 mM MgCl,, and 0.5 M sucrose. Triton X-100 was added to the above suspension to achieve a chl to Triton X-100 ratio of 1:25. After an incubation of 20 min in the dark on ice, the mix-

ture was centrifuged for 10 min at 3600 x g. The pellet was discarded and the PSII membranes were collected from the supernatant by centrifugation for 30 min at 36 000 x g. The pellet was suspended in a solution containing 20 mM MESNaOH buffer (pH 6.2), 10 mM NaC1, 5 mM MgCl,, and 1 M sucrose to obtain a PSII membrane preparation with 8 mg chll rnL. Chl was determined following the procedure of Arnon (1949). The PSII membrane preparations were stored in liquid nitrogen until use. The NaC1-washed (Mn retaining) and Tris-washed (Mn depleted) PSII membranes were prepared as follows. The PSII membranes were thawed on ice and washed once with a medium containing 20 mM MES-NaOH buffer (pH 6.5), 5 mM MgCl,, and 0.1 M sucrose. The subsequent NaCl or Tris washing of PSII membranes was carried out according to the procedure of Nakatani (1984). The washed membranes were collected by centrifugation at 36 000 x g for 20 min and were resuspended in a buffer containing 20 mM MES-NaOH (pH 6.5) and 1 M sucrose to the original concentration of chl and stored as mentioned above. The assay of DCIP photoreduction was performed spectrophotometrically following the method of Rashid and Homann (1992). The reaction medium, in a final volume of 2 mL, contained 20 mM MES-NaOH buffer (pH 6.2), 0.4 M sucrose, and 60 pM DCIP. The membrane preparations were added to the assay medium to achieve afinal concentration of 9 pg chl/mL.

Results and discussion We used three types of PSII preparations in elucidating the role of Mn, either endogenous or added, in electron donation from DPC to PSII. The first type was native PSII. The second type was NaC1-washed PSII, in which 17- and 23-kDa extrinsic polypeptides are depleted. This preparation retains only a limited electron transport activity; however, the activity can be substantially restored by adding CaC12 (Ghanotakis and Yocum 1985; Homann 1987). The activity can also be restored by adding exogenous electron donors, such as H202 or H202plus ~ n ' + or , DPC (Inoue and Wada 1987; Sandusky and Yocum 1988; Rashid and Carpentier 1989; Rashid et al. 1991). The third type of PSII preparation was Tris-washed PSII, in which all three extrinsic polypeptides (17,23, and 33 kDa) and endogenous Mn complex are depleted. This preparation generally does not retain electron transport activity, and the addition of CaC1, does not restore it. The activity can only be restored by adding exogenous electron donors as mentioned above. Chloride, calcium, and manganese are essential cofactors of photosynthetic water oxidation. The involvement of these cofactors in the catalysis of water is well recognized (Govindjee et al. 1985; Ghanotakis and Yocum 1985; Homann 1987; Coleman 1990). Table 1 shows that the addition of 10 mM C1(NaCl) or ca2+(CaCl,) to the native PSII preparation did not change the electron transport activity. However, addition of a similar concentration of ~ n ' +(MnC1,-Mn(NO,),) was inhibitory. DPC (0.5 mM) slightly stimulated the electron flow. A combination of C1- or ca2+with DPC did not change the stimulatory effect of DPC. However, when ~ n ' +was combined with DPC, the electron transport activity was significantly inhibited. In the native PSII membranes where water-oxidizing ability is completely present, it was difficult to predict or interpret the complex interaction of two donors, H20 and

Rashid and Popovic

Table 1. Photoreduction of DCIP in three different types of PSII preparations in the absence or presence of various additives. pmol DCIP/(mg chl - h) Additive

Native PSI1

NaC1-washed PSI1

Tris-washed PSI1

319(6.8) 336(4.5) 315(5.7) 315(7.0) 273(4.8) 269(5.4) 354(8.2) 357(6.5) 350(4.1) 354(8.7) 254(5.9) 240(5.6)

30(1.6) 106(3.1) 40(2.1) 242(6.2) 1lg(3.4) lOO(3.7) 303(7.5) 300(6.8) 297(5.4) 309(6.3) 139(5.0) 136(5.1)

0 0 0 0 70(4.0) 76(3.9) SOS(9.0) 495(8.9) 485(8.8) 278(5.0) 116(4.6) 121(4.6)


-

None NaCl NaNO, CaCI, MnCl, Mn(NO,), DPC DPC+NaCl DPC+NaNO, DPC+CaCl, DPC+MnCI, DPC+Mn(NO,),

Note: the concentrations of additives used were 10 m M salts and 0.5 mM dPC. Standard errors are in parentheses.

DPC, with the ~ n ~Thus, + . to obtain a clear understanding as to how DPC interacts with Mn (either endogenous or added), we used NaC1-washed PSII, in which water-oxidizing ability was significantly inactivated but the Mn complex was present. Table 1 further shows that the activity of this preparation was partially restored by NaCl and substantially restored by CaCl,, an observation similar to that of many others (see review by Homann 1987). NaN03 had little effect, indicating that the stimulating effects were attributed to C1- and ca2+. Both MnCl, and Mn(N03), partially stimulated the electron flow, an observation opposite to that observed with native PSII. It seemed that exogenous Mn acted as a weak electron donor in this preparation. On the other hand, DPC significantly restored the electron flow. A combination of NaC1-NaNO, or CaCl, with DPC did not affect the electron donation from DPC. However, when either MnC1, or Mn(N03)2 (both showing a similar effect, indicating that Ivln2+was the responsible agent) were combined with DPC, the electron transport from DPC was significantly inhibited. This was verified by the more conclusive experiment shown in Fig. 1A. In the absence of added ~ n ~DPC + ,restored the activity in a concentration-dependent manner. The maximal activity was obtained at more than 1 rnM DPC. The apparent K, for DPC, calculated from Fig. lA, was 0.17 mM. As will be seen later, this K,,, value for DPC is much higher than that obtained in Tris-washed PSII, in which the Mn complex is absent. When 10 mM Mn(NO,), was added to the reaction medium, the stimulatory effect of DPC was completely eliminated (Fig. 1A). This is a clear indication of inhibition of DPC electron donation by exogenous ~ n in~ NaC1-washed PSII preparations. The results presented above indicate that in NaC1-washed PSII, where the native Mn complex was present, DPC significantly restored the activity and exogenous ~ n almost ~ + completely inhibited the electron donation from DPC. We extended this investigation to use Tris-washed PSII, in which the water oxidizing ability was completely absent and Mn complex was removed. Tris-washed PSII did not retain any electron transport activity and activity was not restored by the addition of either C1- or ca2+(Table 1). This is in agreement

Fig. 1. DPC concentration dependence of photoreduction of DCIP in (A) NaC1-washed PSII (where 17- and 23-kDa extrinsic polypeptides are depleted); (B) Tris-washed PSII (where, in addition to 17-, 23-, and 33-kDa polypeptides, the Mn complex is depleted). The concentration of Mn(NO,),, wherever present, was 10 mM. The 100% activities of NaC1-PSII and Tris-PSI1 were 303 and 505 pmol DCIP/(mg chl.h), respectively. Other conditions are described in Materials and methods.

(A) NaCI-PSII

= 5

5

m-

0

0

3

u

0.2

0.4

2 a

g

0.8

0.8

1

t2

0.8

1

1.2

DPC (mM) 1PO

C

(B) TRIS-PSI1

P

'

I

1

0

AP

0.4

0.6

DPC (mM)

Biochem. Cell Biol. Vol. 73, 1995 Fig. 2. Photoreduction of DCIP as a function of ~ n concentra~ ' tions in (A) NaC1-washed PSII and (B) Tris-washedPSII. The concentration of DPC, wherever present, was 0.5 mM. 420

(A)NaCI-PSI1


1Wf

L

(B) TRIS-PSI1 0

with previous reports (Rashid and Carpentier 1989; Rashid and Popovic 1990). Mn2+ slightly stimulated the electron transport, similar to NaC1-washed membranes, confirming that it acted as a weak electron donor to PSII in which H20 oxidation capacity was absent (Velthuys 1983). In this preparation, the rate of electron transport obtained in the presence of DPC was about twice that obtained in the presence of DPC with NaC1-washed PSII. This result indicates that DPC electron donation to PSII is strongly favored by the absence of native Mn complex or that native Mn complex prevented the electron donation from DPC. This became more obvious from subsequent experiments. When Mn2+was combined with DPC in this preparation, the electron transport rate was severely inhibited (Table 1). We further investigated the concentration dependence of the stimulation of electron transport by DPC in Tris-washed PSII (Fig. 1B). In the absence of exogenous Mn2+,DPC stimulated electron transport much more than in NaC1-washed PSII. The maximum stimulation of activity by DPC occurred at 0.5 mM, a concentration half of that needed in NaC1-washed PSII. This is consistent with a higher accessibility of DPC to its site of action in the absence of the native Mn complex. The apparent K, for DPC (0.11 mM), calculated from Fig. lB, was also much lower than that obtained in NaC1washed PSII (0.17 mM). Figure 1B further shows that when exogenous Mn2+was present in this preparation, the stimulatory effect of DPC was totally eliminated, confirming that

DPC electron donation to PSII was inhibited by Mn2+.For reasons not clear at this point, ca2+,but not C1-, also inhibited the DPC electron donation in this preparation (Table 1). We confirmed the above observations by measuring the concentration dependence of the stimulation of electron transport by Mn2+in NaC1- and Tris-washed PSII membranes (Fig. 2). In the absence of DPC, the stimulation of electron transport by Mn2+was biphasic in NaC1-washed PSII. There was an initial rapid stimulation phase requiring up to 1 mM Mn2+,followed by a very slow stimulation phase continuing up to 20 mM, which was the highest concentration used in this experiment (Fig. 2A). Conversely, in Tris-washed PSII only the initial rapid stimulation phase, requiring a similar Mn2+ concentration to NaC1-washed PSII, was observed. The electron transport rate between 1 and 20 rnM of Mn2+was similar (Fig. 2B). This differential effect of exogenous MnZfin stimulating electron transport between NaC1- and Tris-washed PSII membranes is not understood at this time. However, the important observation was that the presence of 0.5 mM DPC with increasing concentration of Mn2' did not affect the stimulation of electron [ransport by ~n'+ in either NaCl- or Triswashed PSI1 (Fig. 2). This confirms that ~ n "was responsible for the inhibition of eIectron donation by DPC. Since ~ a ' +significantly inhibited the electron donation from DPC to Triswashed PSII only, it might be possible that in the absence of endogenous Mn complex, the inhibition of electron donation from DPC by divalent cations was nonspecific in nature. Finally, our results suggest that Mn, either endogenous or added, acts as a barrier for DPC to donate electrons to Z+. This is clear from the significant stimulation of electron transport by DPC in Mn-depleted PSII and the marked inhibition of electron donation from DPC to PSII by exogenous Mn2+,in both Mn-depleted and Mn-retaining PSII preparations. We conclude that the native Mn complex not only functions as an accumulator of oxidizing equivalents but may also protect the PSII from exogenous reductants.

Acknowledgement This work was supported by the Natural Sciences and Engineering Research Council of Canada by grants EQP-0156546 and DGP-0093404.

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Rashid and Popovic Debus, R.J., Barry, B.A., Sithole, I, Babcock, G.T., and McIntosh, L. 1988. Directed mutagenesis indicates that the donor side to P',, in photosystem I1 is Tyrosine-161 of the Dl polypeptide. Biochemistry, 27: 907 1-9074. Ghanotakis, D.F., and Yocum, C.F. 1985. Polypeptides of photosystem I1 and their role in oxygen evolution. Photosynth. Res. 7: 97114. Govindjee, Kambara, T., and Coleman, W. 1985. The electron donor side of photosystem 11: the oxygen evolving complex. Photochem. Photobiol. 42: 187-210. Homann, P.H. 1987. The relations between the chloride, calcium, and polypeptide requirements of photosynthetic water oxidation. J. Bioenerg. Biomemb. 19: 105-123. Inoue, H., and Wada, T. 1987. Requirement of manganese for electron donation of hydrogen peroxide in photosystem I1 reaction centre complex. Plant Cell Physiol. 28: 767-773. Inoue, H., Akahori, H., and Noguchi, M. 1987. Activation of electron.donation from hydrogen peroxide by manganese in non-oxygen evolving photosystem 11 particles. Plant Cell Physiol. 28: 1339-1343. Izawa, S. 1980. Acceptors and donors for chloroplast electron transport. Methods Enzymol. 69: 413-434. Metz, J.G., Nixon, P.J., Ronger, M., Brudvig, G.M., and Diner, B.A. 1989. Directed alteration of the Dl polypeptide of photosystem 11--evidence that tyrosine-161 is the redox component, Z, connecting the oxygen-evolving complex to the primary electron donor, P680. Biochemistry, 28: 6960-6969. Nakatani, H.Y. 1984. Photosynthetic oxygen evolution does not require the participation of polypeptides of 16 and 24 kilodaltons. Biochem. Biophys. Res. Commun. 120: 299-304.

Pan, R.L., and Izawa, S. 1979. Photosystem 11 energy coupling in chloroplasts with H,O, as electron donor. Biochim. Biophys. Acta, 547: 311-319. Purcell, M., Leroux, G.D., and Carpentier, R. 1991. Interaction of electron donor diphenylcarbazide with the herbicide-binding niche of photosystemII. Biochim. Biophys. Acta, 1058:374-378. Rashid, A., and Carpentier, R. 1989. CaClz inhibition of H202electron donation to photosystem I1 in submembrane preparations depleted in extrinsic polypeptides. FEBS Lett. 258: 331-334. Rashid, A,, and Homann, P.H. 1992. Properties of iodide-activated photosynthetic water-oxidizing complexes. Biochim. Biophys. Acta, 1101: 303-310. Rashid, A., and Popovic, R. 1990. Protective role of CaCl, against Pb++inhibition in photosystem 11. FEBS Lett. 271: 181-184. Rashid, A., Bernier, M., Pazdernick, L., and Carpentier, R. 1991. Interaction of Znt+ with the donor side of photosystem II. Photosynth. Res. 30: 123-130. Rashid, A., Camm, E.L., and Ekramoddoullah, A.K.M. 1994. Molecular mechanism of action of Pb++and Zn++on water oxidizing complex of photosystem 11. FEBS Lett. 350: 296-298. Rutherford, A.W. 1989. Photosystem 11, the water splitting enzyme. Trends Biochem. Sci. 14: 227-232. Sandusky, P.O., and Yocum, C.F. 1988. Hydrogen peroxide oxidation catalized by chloride-depleted thylakoid membranes. Biochim. Biophys. Acta, 936: 149-156. Velthuys, B. 1983. Spectrophotometric methods of probing the donor side of photosystem II. In The oxygen evolving system of photosynthesis. Edited by Y. Inoue, A. Crofts, Govindjee, N. Murata, G. Renger, and K. Satoh. Academic Press, Japan, Inc., Tokyo. pp. 83-90.