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J. Biol. Chem., Vol. 283, Issue 19, 13330-13340, May 9, 2008

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Biosynthetic Exchange of Bromide for Chloride and Strontium for Calcium in the Photosystem II Oxygen-evolving Enzymes^*

Naoko Ishida¹, Miwa Sugiura, Fabrice Rappaport^¶, Thanh-Lan Lai, A. William Rutherford, and Alain Boussac²

From the Institut de Biologie et Technologie de Saclay, URA CNRS 2096, Commissariat à l'Energie Atomique Saclay, 91191 Gif-sur-Yvette, France, the Department of Plant Biosciences, School of Life and Environmental Sciences, Osaka Prefecture University, 1-1 Gakuen-cho, Naka-ku, Sakai, Osaka 599-8531, Japan, and the ^¶Institut de Biologie Physico-Chimique, Université Pierre et Marie Curie/CNRS UMR 7141, 13 rue Pierre et Marie Curie, 75005 Paris, France

The active site for water oxidation in photosystem II goes through five sequential oxidation states (S₀ to S₄) before O₂ is evolved. It consists of a Mn₄Ca cluster close to a redox-active tyrosine residue (Tyr_Z). Cl^- is also required for enzyme activity. To study the role of Ca²⁺ and Cl^- in PSII, these ions were biosynthetically substituted by Sr²⁺ and Br^-, respectively, in the thermophilic cyanobacterium Thermosynechococcus elongatus. Irrespective of the combination of the non-native ions used (Ca/Br, Sr/Cl, Sr/Br), the enzyme could be isolated in a state that was fully intact but kinetically limited. The electron transfer steps affected by the exchanges were identified and then investigated by using time-resolved UV-visible absorption spectroscopy, time-resolved O₂ polarography, and thermoluminescence spectroscopy. The effect of the Ca²⁺/Sr²⁺ and Cl^-/Br^- exchanges was additive, and the magnitude of the effect varied in the following order: Ca/Cl < Ca/Br < Sr/Cl < Sr/Br. In all cases, the rate of O₂ release was similar to that of the S₃Tyr_Z^. to S₀Tyr_Z transition, with the slowest kinetics (i.e. the Sr/Br enzyme) being 6-7 slower than in the native Ca/Cl enzyme. This slowdown in the kinetics was reflected in a decrease in the free energy level of the S₃ state as manifest by thermoluminescence. These observations indicate that Cl^- is involved in the water oxidation mechanism. The possibility that Cl^- is close to the active site is discussed in terms of recent structural models.

Received for publication, December 31, 2007 , and in revised form, February 28, 2008.

^* This work was supported in part by the Japan Society for the Promotion of Science (JSPS) and the CNRS under the Japan-France Research Cooperative Program and Solar-H, a Space Technologies Research Program from the European Community. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Equations 1-6, Figs. S1-S6, and Table ST1.

¹ Supported in part by the Bio-Hydrogen Program of the Commissariat à l'Energie Atomique.

² To whom correspondence should be addressed. Tel.: 33-1-69-08-72-06; E-mail: alain.boussac{at}cea.fr.

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