Effects of Sr 2+ -Substitution on the Reduction Rates of Y z • in PSII MembranessEvidence for Concerted Hydrogen-Atom Transfer in Oxygen Evolution ² Kristi L. Westphal, ‡ Nikos Lydakis-Simantiris, § Robert I. Cukier, and Gerald T. Babcock* Department of Chemistry, Michigan State UniVersity, East Lansing, Michigan 48824 ReceiVed August 1, 2000; ReVised Manuscript ReceiVed October 20, 2000 ABSTRACT: Several groups have recently investigated the kinetic effects of biochemical treatments, site- directed mutagenesis, or substitution of essential cofactors on the stepwise, water-oxidizing chemistry catalyzed by Photosystem II. Consistently, these studies show evidence for a slowing of the final, oxygen- releasing step, S 3 f S 0 , of the catalytic cycle. To a degree, some of this work also shows a slowing of the earlier S-state transitions. To study these processes in more detail, we have investigated the effect of replacing Ca 2+ with Sr 2+ on the rates of the S-state transitions by using time-resolved electron paramagnetic resonance. The results show a slowdown of the last transition in the cycle, consistent with a report from Boussac et al. [Boussac, A., Se ´tif, P., and Rutherford, A. W. (1992) Biochemistry 31, 1224-1234], and of the earlier S-state transitions as well, which suggests that a common molecular mechanism is at work and that Sr 2+ is less effective than Ca 2+ in supporting it. While the oxidation of Y z by P 680 + has been extensively studied and can be understood within the context of nonadiabatic electron tunneling combined with rapid, non-rate-limiting proton transfer in the holo-system [Tommos, C., and Babcock, G. T. (2000) Biochim. Biophys. Acta 1458, 199], the reduction of Y z • by the Mn cluster cannot be described effectively by a nonadiabatic electron-transfer formalism. This indicates that this reaction is rate limited by processes other than electron tunneling. We discuss our results for Y z • reduction and those of others for the activation parameters (E a , A, KIE, and rates) associated with this process, in terms of both sequential and concerted proton-coupled, electron transfer. Our analysis indicates that concerted hydrogen-atom transfer processes best explain the observed characteristics of the S-state advances. Oxygen evolution in photosynthesis results from light- driven water oxidation that is catalyzed by Photosystem II (PSII). 1 The catalytic site is composed of a tetrameric manganese cluster and a redox-active tyrosine, Y z , that has been identified as tyrosine 161 of the D1 protein (for reviews see refs 1-3). A histidine at the D1 190 position is the initial acceptor of the phenol proton released upon Y Z oxidation by the reaction center chlorophyll complex, P 680 (4-9). In close proximity to the catalytic center are two ions, Ca 2+ and Cl - , that are needed for efficient oxygen evolution (10- 12), but their exact roles are still under investigation. Bound to PSII on the inside of the thylakoid membrane are three extrinsic polypeptides with molecular masses of 17, 23, and 33 kDa. They have been implicated in the prevention of Ca 2+ and Cl - migration out of the catalytic site and also as stabilizers of the structure of the Mn complex. Although progress has been made recently on obtaining structural information for two- and three-dimensional crystals of PSII, these have not yet been solved to high resolution (13). Catalysis is initiated by the absorption of a photon of light by P 680 . In its excited state, P 680 ultimately transfers an electron to a quinone, Q A , thereby forming the charge- separated state, P 680 + Q A - . The subsequent reduction of P 680 + is carried out by Y z , which is, in turn, reduced by the substrate water/Mn cluster. This process occurs with the absorption of each photon as the Mn cluster accumulates the four oxidizing equivalents necessary to split water. The intermediates at the substrate water/Mn cluster that result from this photochemistry are designated by the S n notation, where n is the number of stored oxidizing equivalents (14, 15). After formation of S 4 ,O 2 is released and the system resets to S 0 . The rates at which these oxidation steps occur were first measured by Babcock et al. by using time-resolved electron paramagnetic resonance (EPR) spectroscopy and were shown to vary with the oxidation state of the Mn cluster (16). More recent studies by Razeghifard et al. have shown that these rates can be altered when the OEC is chemically, biochemi- cally, or genetically modified (17-19). The exact mechanism ² This work was supported by USDA CRGO (G.T.B.), by NIH GM37300 (G.T.B.), and by NIH GM47274 (R.I.C.). * To whom correspondence should be addressed. ‡ Current address: Department 9N6, Building AP20, Abbott Labo- ratories, 100 Abbott Park Road, Abbott Park, IL 60064. § Current Address: Mediterranean Agronomic Institute of Chania, Crete, Greece. 1 Abbreviations: A, preexponential factor; Ea, activation energy; EGTA, ethylene glycol bis(-aminoethyl ether)-N,N,N′,N′-tetraacetic acid; EPR, electron paramagnetic resonance; ET/PT, electron transfer followed by proton transfer; ETPT, concerted electron and proton transfer; KIE, kinetic isotope effect; MES, morpholinoethanesulfonic acid; (Mn) 4, tetramanganese cluster; MSP, manganese stabilizing protein; OEC, oxygen-evolving complex; PPBQ, phenyl-p-benzo- quinone; P680, photoactive chlorophyll of PSII; PSII, Photosystem II; PT/ET, proton transfer followed by electron transfer; QA, primary plastoquinone acceptor; Sn, redox state of the oxygen evolving complex; TS, transition state; Yz, tyrosine 161 of the D1 polypeptide. 16220 Biochemistry 2000, 39, 16220-16229 10.1021/bi0018077 CCC: $19.00 © 2000 American Chemical Society Published on Web 11/30/2000