Reduction-Induced Inhibition and Mn(II) Release from the Photosystem II Oxygen-Evolving Complex by Hydroquinone or NH 2 OH Are Consistent with a Mn(III)/Mn(III)/Mn(IV)/Mn(IV) Oxidation State for the Dark-Adapted Enzyme † Thomas Kuntzleman and Charles F. Yocum* Department of Molecular, Cellular and DeVelopmental Biology and Department of Chemistry, UniVersity of Michigan, Ann Arbor, Michigan 48109-1048 ReceiVed July 19, 2004; ReVised Manuscript ReceiVed NoVember 24, 2004 ABSTRACT: Hydroxylamine and hydroquinone were used to probe the oxidation states of Mn in the oxygen- evolving complex of dark-adapted intact (hydroxylamine) and salt-washed (hydroquinone) photosystem II. These preparations were incubated in the dark for 24 h in the presence of increasing reductant/ photosystem II ratios, and the loss of oxygen evolution activity and of Mn(II) was determined for each incubation mixture. Monte Carlo simulations of these data yielded models that provide insight into the structure, reactivity, and oxidation states of the manganese in the oxygen-evolving complex. Specifically, the data support oxidation states of Mn(III) 2 /Mn(IV) 2 for the dark stable S 1 state of the O 2 -evolving complex. Activity and Mn(II) loss data were best modeled by assuming an S 1 f S -1 conversion of intermediate probability, a S -1 f S -3 reaction of high probability, and subsequent step(s) of low probability. This model predicts that photosystem II Mn clusters that have undergone an initial reduction step become more reactive toward a second reduction, followed by a slower third reduction step. Analysis of the Mn- (II) release parameters used to model the data suggests that the photosystem II manganese cluster consists of three Mn atoms that exhibit a facile reactivity with both reductants, and a single Mn that is reducible but sterically trapped at or near its binding site. Activity assays indicate that intact photosystem II centers reduced to S -1 can evolve oxygen upon illumination, but that these centers are inactive in preparations depleted of the extrinsic 23 and 17 kDa polypeptides. Finally, it was found that a substantial population of the tyrosine D radical is reduced by hydroxylamine, but a smaller population reacts with hydroquinone over the course of a 24 h exposure to the reductant. Photosystem II (PSII), a membrane-bound pigment- protein complex, uses light energy to catalyze water oxidation (1, 2): 2H 2 O f 4H + + 4e - + O 2 Photons absorbed by PSII initiate a series of redox reactions that culminate in the sequential oxidation of the oxygen-evolving complex (OEC). 1 The OEC consists, in part, of a cluster of inorganic ions (4Mn, Ca 2+ , and Cl - ) that cycle through 5 oxidation states, as described by the Kok model (3, 4): Once the S 4 state is reached, O 2 is released and the cluster is reduced to the S 0 state. It is generally agreed that one oxidizing equivalent separates the S 0 /S 1 and the S 1 /S 2 states (2), and that these S-state transitions consist of oxidations of the Mn atoms in the cluster. However, generation of S 3 has been postulated to occur by oxidation either of the Mn cluster, of a substrate ligand, or of a redox-active tyrosine residue (Y Z )(2). Upon long-term (> 30 min) dark incubation, all PSII centers relax to the S 1 state (3, 4). Calcium and chloride cofactors, as well as Y Z , are required for the redox reactions leading to water oxidation (1, 2). Binding of these cofactors is regulated by extrinsic 23 and 17 kDa polypeptides (5-7), and perhaps also by intrinsic polypeptides of the PSII reaction center (8). These polypep- tides and Y Z are also components of the OEC. It is generally agreed that water binds directly to the Mn atoms and perhaps Ca 2+ in the OEC, but the S-state(s) to which water(s) bind is still under investigation. In addition to Y Z , there is a second redox active tyrosine (Y D ) that does not participate in oxygen † This research was supported by grants to C.F.Y. from the United States Department of Agriculture National Research Initiative Competi- tive Research Grants Program and the Molecular Biochemistry Program of NSF. T.K. was supported by a Molecular Biophysics Training Grant from the NIH. * To whom correspondence should be addressed: Department of Molecular, Cellular and Developmental Biology, University of Michi- gan, Ann Arbor, MI 48109-1048. Tel: 1-734-647-0897. Fax: 1-734- 647-0884. 1 Abbreviations: Chl, chlorophyll; DCBQ, 2,6-dichloro-p-benzo- quinone; EDTA, ethylenediamine tetraacetic acid; H2Q, hydroquinone; MES, 2-(N-morpholino)ethanesulfonic acid; OEC, O2-evolving com- plex; PS, photosystem; MC, buffer composed of 50 mM MES (pH 6) and 10 mM CaCl2; SMN, buffer composed of sucrose (0.4 M), MES (50 mM, pH 6), 10 mM NaCl); SWPSII, photosystem II preparation treated with 2 M NaCl to extract Ca 2+ and the 23 and 17 kDa extrinsic polypeptides; XANES, X-ray absorption near edge spectroscopy, YD • , a dark-stable tyrosine radical (Y160 of the PsbD protein) of photosystem II. 2129 Biochemistry 2005, 44, 2129-2142 10.1021/bi048460i CCC: $30.25 © 2005 American Chemical Society Published on Web 01/19/2005