J. Photochem. Photobiol. B: Biol., 13 (1992) 323-326 323

Preliminary Note

Requirement of manganese for the photooxidation hydroxylamine by photosystem II

Abdur Rashid+ and Radovan Popovic

of

Depament of Chem&y, University of Quebec at Montreal, CP 8888, Succ. A, Montreal, Que. H3C 3P8 (Canada)

(Received October 23, 1991; accepted November 15, 1991)

Abstract

The relation of hydroxylamine photooxidation by photosystem II (PSII) and the presence of manganese was studied in PSI1 membranes either native or depleted in extrinsic polypeptides (17, 23 and 33 kDa) or depleted in both polypeptides and manganese. It was observed that the PSI1 catalyzed oxidation of hydroxylamine was inhibited if manganese was absent. We showed with various types of PSI1 preparations that the presence of manganese, endogenous or added, was essential for the photooxidation of hydroxylamine by photosystem II.

Keywords: Photosystem II, oxygen-evolving complex, hydroxylamine, manganese, electron transport.

1. Introduction

In photosynthesis, the tetramanganese complex is involved in the four electron oxidation processes of two water molecules which results in the liberation of molecular oxygen [l]. In the oxygen evolving complex (OEC), manganese atoms function as accumulators of oxidizing equivalents generated through the water oxidation by PSI1 [2]. When the water oxidation process is inhibited, specific PSI1 artificial electron donors can substitute for water [3-61. However, the role of manganese in such reactions is unclear. It has been shown, for example, that hydrogen peroxide, a substrate analog of water as the electron donor, cannot undergo oxidation by manganese depleted chloroplasts. The addition of exogenous manganese with hydrogen peroxide can stimulate the PSI1 electron transport activity [3, 5, 61. On the other hand, it has been shown that diphenylcarbazide (DPC) can be an efficient donor to PSI1 only when the OEC is depleted of endogenous manganese [4].

In studies on PSI1 activity, hydroxylamine has been frequently used as an electron donor to PSI1 [7-91. Whether or not the function of hydroxylamine as an electron

+Author to whom correspondence should be addressed at Institute of Molecular Biophysics, Florida State University, Tallahassee, FL 32306-3015 B-165, USA.

loll-1344/92/$5.00 0 1992 - Elsevier Sequoia. All rights reserved

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donor to PSI1 is dependent on manganese participation is not known. It was assumed that thylakoid bound manganese was probably not needed for the photooxidation of hydroxylamine by PSI1 [7]. However, this study shows that hydroxylamine photooxidation by PSI1 is mediated by endogenous or added (when the OEC is depleted of endogenous manganese ions) manganese ions.

2. Materials and methods

The experiments were carried out with (1) native PSI1 membranes, prepared from spinach, following [lo] with a modification as in [6]; (2) NaCl-washed membranes, with inhibited (70-80%) oxygen evolving activity, depleted in two extrinsic polypeptides (17 and 23 kDa), and retaining the entire manganese complex [6, 111; (3) CaCl*- washed membranes, with completely inhibited oxygen evolving activity, depleted in three extrinsic polypeptides (17, 23 and 33 kDa), and mostly retaining the endogenous manganese ions [6, 121; and (4) tris-washed membranes, with completely inhibited oxygen evolving activity, and depleted in three extrinsic polypeptides with endogenous manganese [6, 111. The native and the treated PSI1 membranes were suspended in a buffer containing 20 mM Mes-NaOH (pH 6.3) at a chlorophyll concentration of 1.5 mg ml-l.

Dichlorophenolindophenol (DCIP) photoreduction was estimated according to [13] with PSI1 membranes containing 6 pg chl ml- ‘. The measuring medium contained 20 mM Mes-NaOH (pH 6.3) and 30 PM DCIP.

3. Results and discussion

It was observed that hydroxylamine photooxidation by the various types of PSI1 membranes, described in Section 2, was dependent on the presence of either endogenous or added manganese ions (Table 1). In native PSI1 membranes, the presence of hydroxylamine (1 mM), alone or in combination with MnClz (0.5 mM), did not significantly change the PSI1 electron transport activity. However, in NaCl- and CaC12- washed PSI1 membranes, where the water oxidation capacity was inhibited, and the endogenous manganese ions were mostly retained, hydroxylamine without addition of manganese stimulated PSI1 activity. The electron transport activity was completely recovered when MnClz was added together with hydroxylamine. However, it was noticed that in tris-washed PSI1 membranes, where oxygen evolving capacity was completely inhibited and endogenous manganese ions were mostly depleted, the presence of hydroxylamine without the addition of manganese did not stimulate PSI1 activity. The highest PSI1 activity was recorded when MnClz was added together with hydroxylamine in tris-washed PSI1 membranes. The results showed that in tris-washed PSI1 membranes, where the endogenous manganese was mostly lost, hydroxylamine could restore the PSI1 activity bnly in presence of exogenous manganese ions. CaCl, or NaCl were unable to substitute for MnClz (results not shown). This clearly suggests that manganese ions, either endogenous or added, are essential for hydroxylamine photooxidation by PSII.

The dependence of hydroxylamine photooxidation by PSI1 on manganese con- centration was further studied in tris-washed PSI1 membranes. The concentration of 20 PM MnClz was enough to induce a maximum photooxidation activity of hydroxylamine by PSII. However, MnCl* itself could not stimulate the PSI1 activity (Fig. 1). These

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TABLE 1

Effects of additives on DCIP photoreduction of various types of PSI1 membranes. NHrOH at 1 mM and MnC12 at 0.5 mM were used (other conditions are described in Section 2)

Type of PSI1 membranes

Addition DCIP photoreduction pmol (mg chl)-’ h-r

Intact None 160 NI-I,OH 155 NHrOH + MnCl, 170 MnClz 160

NaCl-washed

CaCl,-washed

None 40 NH,OH 72 NH20H+ MnCl, 176 MnCl* 50

None 8 NH,OH 48 NH,OH + MnClz 176 MnCl* 8

Tris-washed None 0 NH,OH 8 NHrOH + MnCI, 184 MnClz 0

0 0.02 0.04 006

MnClt [mM]

Fig. 1. Measurement of DCIP photoreduction in tris-washed PSI1 membranes as a function of MnCI, concentrations: (0) no addition, (0) 1 mM NHrOH added. The 100% activity represents 184 pmol DCIP (mg chl)-’ h-r.

results indicate that exogenous manganese could not reconstitute the process of water oxidation, but that it could reconstitute the process of hydroxylamine oxidation in tris- washed PSI1 membranes. Part of these results is consistent with the previous report indicating that manganese reconstitution of water oxidation in tris/GQ-extracted PSI1 required the reassembly of 33 kDa polypeptide in OEC [14].

Since manganese ligation in the OEC, which was depleted in extrinsic polypeptides and manganese, required illumination [14], our results suggest that during the mea-

326

surements of DCIP photoreduction with continuous illumination the manganese ions underwent simultaneous photoligation in the polynuclear manganese complex of NaCI-, CaC12- and tris-washed PSI1 membranes. And as a consequence, hydroxylamine could efficiently utilize the tetramanganese complex for its photooxidation by PSII. Thus our results demonstrate that manganese is required for electron donation to PSI1 by hydroxylamine.

Acknowledgments

This work was supported by the Natural Science and Engineering Research Council of Canada through the grant #G-1929, G-1930 and A-3047 to Radovan Popovic. The authors thank Linda Robinson for helping in the production of this manuscript.

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