Coupled Activation of the Donor and the Acceptor Side of Photosystem II during Photoactivation of the Oxygen Evolving Cluster † Maria Rova, ‡,§ Fikret Mamedov, ‡ Ann Magnuson, ‡ Per-Olof Fredriksson, § and Stenbjo ¨rn Styring* ,‡ Department of Biochemistry, Chemical Center, Lund UniVersity, Box 124, S-221 00 Lund, Sweden, and Department of Chemistry, UniVersity of Karlstad, S-651 88 Karlstad, Sweden ReceiVed February 18, 1998; ReVised Manuscript ReceiVed April 8, 1998 ABSTRACT: Photoactivation of photosystem II has been studied in the FUD 39 mutant of Chlamydomonas reinhardtii that lacks the 23 kDa extrinsic subunit of photosystem II. We have taken advantage of the slow photoactivation rate of FUD 39, earlier demonstrated in Rova, E. M., et al. [(1996) J. Biol. Chem. 271, 28918-28924], to study events in photosystem II during intermediate stages of the process. By measuring the EPR multiline signal, the decay of the variable fluorescence after single flashes, and electron transfer from water to the Q B site, we found a good correlation between the building of a tetrameric Mn cluster, longer recombination times between Q A - and the donor side of photosystem II, and the achievement of water splitting ability. An increased rate of electron transfer from Q A - to the Q B site on the acceptor side of photosystem II, mainly due to enhanced efficiency of binding of Q B to its site, was found to precede the building of the Mn cluster. We also showed that Tyr D was oxidized simultaneously with this increase in electron-transfer rate. Thus, it appears that photoactivation is sequential, with an increased rate of electron transfer on the acceptor side occurring together with the oxidation of Tyr D in the first step, followed by the assembly of the Mn cluster. We suggest that a conformational change of photosystem II is induced early in the photoactivation process facilitating electron transfer from the primary donor to the acceptor side. As a consequence, Tyr D , an auxiliary electron donor to P 680 + /Tyr Z • , is oxidized. That this occurs before the Mn cluster is fully functional serves to protect photosystem II against donor side induced photodamage. Photosystem II (PS II) 1 is a large multisubunit complex that catalyzes the light-driven oxidation of water in plants, green algae, and cyanobacteria (1, 2). The primary photo- chemical reaction in PS II produces the P 680 + Pheo - radical pair. This charge separation is stabilized by reduction of the primary plastoquinone electron acceptor, Q A , on the acceptor side and by oxidation of a tyrosine residue in the D1 protein, Tyr Z , which abstracts electrons from water molecules on the donor side of PS II. Q A - reduces the secondary plastoquinone electron accep- tor, Q B , first to the semiquinone and after another charge separation to the quinol form. When Q B is fully reduced, it leaves its site on the D1 protein as Q B H 2 , and the empty site is reoccupied by another quinone molecule from the plas- toquinone pool. If this forward reaction is inhibited, a recombination reaction between Q A - or Q B - and oxidized species on the donor side (i.e., the Mn cluster, Tyr Z • , or P 680 + ) occurs. To achieve water splitting, oxidizing power is stored in the water oxidizing center which contains a (Mn) 4 cluster that cycles through five different redox states, the S-states (S 0 to S 4 )(3, 4). When the PS II complex is assembled, the Mn cluster is created in a light-dependent process known as photoactivation. This process has been shown to be a low quantum yield process comprising two independent photoacts spaced by a dark period (5, 6). Each photoevent probably corresponds to the oxidation of a Mn 2+ ion (7, 8) which leads to a stronger binding of the ion to the protein matrix. After the two photoacts, two more Mn ions bind in a light- independent way to form the tetrameric Mn cluster. For maximal water oxidizing capacity, the cofactors Ca 2+ and Cl - are needed, and they are thought to be essential also for the photoactivation process (9-12). During photoactivation, highly oxidizing species such as P 680 + and Tyr Z • are formed. This is a potentially dangerous † This work was supported by the Swedish Natural Science Research Council, the Knut and Alice Wallenberg Foundation, and the Crafoord Foundation. A.M. gratefully acknowledges support by the Nordic Energy Research Program * To whom correspondence should be addressed. Telephone: +46 46 222 0108. Fax: +46 46 222 4534. E-mail: stenbjorn.styring@ biokem.lu.se. ‡ Lund University. § University of Karlstad. 1 Abbreviations: Chl, chlorophyll; cyt b559, cytochrome b559; D1, one of the PS II reaction center proteins carrying some of the redox components in the electron transport chain through photosystem II; D2, the other redox component carrying protein of the reaction center; DCMU, 3-(3,4-dichlorophenyl)-1,1-dimethylurea; DCPIP, 2,6-dichlo- rophenolindophenol; DPC, 2,2′-diphenylcarbonic dihydrazide; E, ein- stein(s); EDTA, ethylenediaminetetraacetate; EPR, electron paramag- netic resonance; FUD 39, mutant strain of C. reinhardtii lacking the 23 kDa extrinsic subunit of PS II; Fv and Fo, variable and constant fluorescence respectively; HEPES, 4-(2-hydroxyethyl)-1-piperazineethane- sulfonic acid; P 680, primary electron donor chlorophyll(s) of photosystem II; Pheo, pheophytin; PS II, photosystem II; QA, first quinone acceptor in photosystem II; QB, second quinone acceptor in photosystem II; Tris, tris(hydroxymethyl)aminomethane; TyrD, the redox active tyrosine residue on the D2 reaction center protein; TyrZ, the redox active tyrosine residue on the D1 reaction center protein. 11039 Biochemistry 1998, 37, 11039-11045 S0006-2960(98)00381-X CCC: $15.00 © 1998 American Chemical Society Published on Web 07/17/1998