S-State Dependence of Chloride Binding Affinities and Exchange Dynamics in the Intact and Polypeptide-Depleted O 2 Evolving Complex of Photosystem II † Hanna Wincencjusz, ‡ Charles F. Yocum, § and Hans J. van Gorkom* ,‡ Biophysics Department, Huygens Laboratory, Leiden UniVersity, P.O. Box 9504, NL-2300 RA, Leiden, The Netherlands, and Departments of Biology and Chemistry, UniVersity of Michigan, Ann Arbor, Michigan 48109-1048 ReceiVed October 27, 1997; ReVised Manuscript ReceiVed March 31, 1998 ABSTRACT: The Cl - binding properties in the successive oxidation states of the O 2 evolving complex of photosystem II were investigated by measurements of UV absorbance changes, induced by a series of saturating flashes, that monitor manganese oxidation state transitions. In dark-adapted, intact photosystem II, Cl - can be replaced by NO 3 - in minutes, in an exchange reaction that depends on the NO 3 - concentration and that is not rate-limited by dissociation of Cl - from its binding site. Preillumination of dark-adapted photosystem II by one or two flashes accelerated the NO 3 - substitution reaction by an order of magnitude. A quantitative analysis of the Cl - concentration dependence of UV absorbance changes, measured in photosystem II preparations depleted of extrinsic 17 and 23 kDa polypeptides, shows that the Cl - binding properties of photosystem II change with the oxidation state of the oxygen evolving complex. Although the affinity for the individual S-states could not be determined with precision, it is shown that the affinity is an order of magnitude lower in the S 2 state than in the S 1 state. Comparison of the results obtained using intact photosystem II and preparations depleted of the 17 and 23 kDa extrinsic polypeptides suggests that these proteins constitute a diffusion barrier, which prevents fast equilibration of the Cl - binding site with the medium, but does not change the Cl - affinity of the binding site. Photosystem II (PSII) 1 is a multisubunit, membrane-bound enzyme system that couples light absorption to the oxidation of H 2 O and the reduction of plastoquinone. The O 2 evolving reaction involves a number of components that are col- lectively called the O 2 evolving complex (OEC). 1 Suspended in a framework comprised of both intrinsic and extrinsic polypeptides, the active site of the complex is associated with a cluster of four Mn atoms along with one atom each of Ca 2+ and of Cl - (1-3). The structure of the complex is unknown, but EPR and X-ray absorption studies (4) have led to detailed proposals for the structure of the Mn cluster and its ligand environment (5). The Mn is involved in the stepwise accumulation of oxidizing equivalents, which raise the oxidation state of the complex in terms of the Kok model (6) from S 0 to S 3 . In darkness, the system relaxes to S 1 , in which the Mn valency is proposed to be III, III, IV, IV (5, 7). The S 1 fS 2 transition has been identified as a Mn III to Mn IV oxidation state advancement (reviewed in 4). S 0 fS 1 may involve Mn II to Mn III and S 2 fS 3 Mn III to Mn IV , but these assignments are more controversial, although an absorp- tion increase in the UV region as well as a shift to higher energy of the Mn K-edge in X-ray absorption strongly impli- cates Mn involvement in these S-state transitions (see 8 for a detailed discussion). Light-induced charge separation by PSII in the S 3 state, as in the lower oxidation states, causes a transient oxidation of Y Z (1), the tyrosine residue that acts as the secondary electron donor in PSII. In the S 3 state, how- ever, this is followed in a few milliseconds by simultaneous reduction, by H 2 O, both of the tyrosine and of S 3 to S 0 . Calcium and chloride are essential cofactors for O 2 evo- lution. Although the Cl - requirement for photosynthetic O 2 evolution has been studied intensively (8-11), the binding site for this cofactor has not yet been identified with certainty and its role in H 2 O oxidation is unknown. Several observa- tions suggest that Cl - binds in the vicinity of the Mn cluster. Removal of the halide results in a change in the magnetic properties of the Mn complex. Chloride-depleted PSII reac- tion centers show no EPR multiline signal (12), although it can be shown by optical and X-ray absorption spectroscopies that oxidation of Mn has taken place (13, 14, but see 15, 16). The observation that Cl - , primary amines, and other Lewis bases compete for the same binding site was taken as an indication that Cl - binds directly to a metal (17). The effectiveness of amines in the competition was proportional to their basicity, suggesting that they bind as Lewis bases to a Lewis acid, in this case Mn in an oxidation state higher than 2+. Certain EXAFS results may support this conclu- sion. The group of Klein reported (18,19) that the differences in EXAFS spectra of cyanobacteria grown on Br - or Cl - † This research was supported by Netherlands Foundation for Chemical Research (SON) of The Netherlands Organization for Scientific Research (NWO), on EU DGXII Human Capital and Mobility network grant (CHRX-CT94-0524), and by the NRICGP, Photosyn- thesis and Respiration Program, of the United States Department of Agriculture (C.F.Y.). C.F.Y. was also supported by a Fulbright Scholar Grant and an NWO Visitor’s Grant. * Corresponding author. Phone: (31) 71 5275981. FAX: (31) 71 5275819. E-mail: vangorkom@biophys.leidenuniv.nl. ‡ Leiden University. § University of Michigan. 1 Abbreviations: Chl, chlorophyll; DCBQ, 2,6-dichloro-p-benzo- quinone; MES, 2-(N-morpholino)ethanesulfonic acid; OEC, oxygen evolving complex; PSII, photosystem II; Hepes, N-(2-hydroxyethyl)- piperazine-N′-2-ethanesulfonic acid; YZ, the tyrosine residue that acts as the primary electron donor to P680. 8595 Biochemistry 1998, 37, 8595-8604 S0006-2960(97)02660-3 CCC: $15.00 © 1998 American Chemical Society Published on Web 05/22/1998