Bromide Does Not Bind to the Mn 4 Ca Complex in Its S 1 State in Cl - -Depleted and Br - -Reconstituted Oxygen-Evolving Photosystem II: Evidence from X-ray Absorption Spectroscopy at the Br K-Edge ² Michael Haumann, ‡ Marcos Barra, ‡ Paola Loja, ‡ Simone Lo ¨scher, ‡ Roland Krivanek, ‡ Alexander Grundmeier, ‡ Lars-Erik Andreasson,* ,§ and Holger Dau* ,‡ Freie UniVersita ¨t Berlin, FB Physik, Arnimallee 14, D-14195 Berlin, Germany, and Department of Chemistry, DiVision of Biochemistry and Biophysics, Go ¨teborg UniVersity, P.O. Box 462, SE-405 30 Go ¨teborg, Sweden ReceiVed June 29, 2006; ReVised Manuscript ReceiVed August 15, 2006 ABSTRACT: Chloride is an important cofactor in photosynthetic water oxidation. It can be replaced by bromide with retention of the oxygen-evolving activity of photosystem II (PSII). Binding of bromide to the Mn 4 Ca complex of PSII in its dark-stable S 1 state was studied by X-ray absorption spectroscopy (XAS) at the Br K-edge in Cl - -depleted and Br - -substituted PSII membrane particles from spinach. The XAS spectra exclude the presence of metal ions in the first and second coordination spheres of Br - . EXAFS analysis provided tentative evidence of at least one metal ion, which may be manganese or calcium, at a distance of ∼5 Å to Br - . The native Cl - ion may bind at a similar distance. Accordingly, water oxidation may not require binding of a halide directly to the metal ions of the Mn complex in its S 1 state. The oxygen of the atmosphere is produced by the oxidation of two water molecules at the protein-bound manganese- calcium (Mn 4 Ca) complex of photosystem II (PSII). 1 PSII is imbedded in the thylakoid membranes of green plants, algae, and cyanobacteria (1). Intensive research on this photosynthetic water oxidation has not led to a consensus about its mechanism. Oxygen evolution activity requires the presence of four Mn ions and one calcium per PSII reaction center, and maximal activity also requires chloride (2, 3). One calcium ion per PSII is essential for oxygen evolution as in its absence, activity is eliminated (2). The Ca 2+ is ∼3.4 Å from two to three Mn ions (4-7). The location and functional role of chloride are less clear. A single Cl - ion bound to PSII seems to be involved in oxygen evolution (8). If Cl - removal is performed under conditions which lead to the partial release of the extrinsic polypeptides of PSII, activity may cease completely (refs 3 and 9 and references cited therein). However, the effect of Cl - depletion is not always that severe. By dialysis of PSII membrane particles containing 36 Cl - against Cl - -free medium, all Cl - ions were removed, as verified by radiography, and the three extrinsic polypeptides remained bound (8). Such Cl - -depleted PSII preparations still exhibited an O 2 activity of ∼35% of the control. The activity was almost fully restored within seconds by the re-addition of either chloride or bromide (8-10). These results tentatively were interpreted as suggesting that Cl - does not bind directly to Mn (10). Recent crystallographic results for PSII from cyanobacteria have not provided indications for any halides in the vicinity of the manganese and/or calcium ions (4, 5). On the other hand, X-ray absorption spectroscopy (XAS) data at the Mn K-edge of synthetic model compounds revealed that Mn- Cl interactions might contribute to the Mn XAS spectrum of PSII (ref 11 and references cited therein). However, if one of the 20-24 ligands of Mn were a halide, it would be difficult to resolve the corresponding contributions to the Mn XAS spectrum. Therefore, we employ an alternative approach. By XAS at the halide K-edge, we look for the presence of manganese and/or calcium in its first or higher coordination spheres. A variety of modes of binding of Cl - to the water- oxidizing complex has been discussed (Figure 1B-D) and crucial roles in the oxygen evolution chemistry proposed (see, e.g., refs 12-14). In most of these models, Cl - has been suggested to bind directly to the manganese and/or Ca 2+ ions. XAS at the halide K-edge should be well suited to detect this binding mode since pronounced edge shape changes would be expected, as shown for model compounds with Cl - ligated to manganese (11) or Br - ligated to vanadium (15). Halide binding in higher coordination spheres of manganese or calcium may become detectable by EXAFS analysis. In a vanadium-dependent bromoperoxidase, Br - ² Financial support from the Deutsche Forschungsgemeinschaft to H.D. and M.H. (Grants SFB498-C6 and -C8) and from the Swedish Science Council to L.-E.A. is gratefully acknowledged. * To whom correspondence should be addressed. H.D.: FU Berlin, FB Physik, Arnimallee 14, D-14195 Berlin, Germany; phone, +49 30 8385 3581; fax, +49 30 8385 6299; e-mail, holger.dau@physik.fu- berlin.de. L.-E.A.: Department of Chemistry, Division of Biochemistry and Biophysics, Go ¨teborg University, P.O. Box 462, SE-405 30 Go ¨teborg, Sweden; phone, +46 31 773 3932; fax, +46 31 773 3910; e-mail, lars-erik.andreasson@chem.gu.se. ‡ Freie Universita ¨t Berlin. § Go ¨teborg University. 1 Abbreviations: chl, chlorophyll; DCBQ, dichloro-p-benzoquinone; DF, delayed chl fluorescence; EPR, electron paramagnetic resonance spectroscopy; EXAFS, extended X-ray absorption fine structure; fwhm, full width at half-maximum; MES, 2-morpholinoethanesulfonic acid; PSII, photosystem II; S i, oxidation states in the S state cycle of water oxidation; TXRF, total reflection X-ray fluorescence analysis; XANES, X-ray absorption near-edge structure; XAS, X-ray absorption spec- troscopy. 13101 Biochemistry 2006, 45, 13101-13107 10.1021/bi061308r CCC: $33.50 © 2006 American Chemical Society Published on Web 10/04/2006