Inorganic Cofactor Stabilization and Retention: The Unique Functions of the Two PsbO Subunits of Eukaryotic Photosystem II † Hana Popelkova, ‡ Alan Commet, ‡ Thomas Kuntzleman, ‡,§ and Charles F. Yocum* ,‡,| Department of Molecular, Cellular and DeVelopmental Biology and Department of Chemistry, The UniVersity of Michigan, Ann Arbor, Michigan 48109-1048 ReceiVed August 11, 2008; ReVised Manuscript ReceiVed October 6, 2008 ABSTRACT: Eukaryotic PsbO, the photosystem II (PSII) manganese-stabilizing protein, has two N-terminal sequences that are required for binding of two copies of the protein to PSII [Popelkova, H., et al. (2002) Biochemistry 41, 10038-10045; Popelkova, H., et al. (2003) Biochemistry 42, 6193-6200]. In the work reported here, a set of selected N-terminal truncation mutants of PsbO that affect subunit binding to PSII were used to determine the effects of PsbO stoichiometry on the Mn, Ca 2+ , and Cl - cofactors and to characterize the roles of each of the PsbO subunits in PSII function. Results of the experiments with the PsbO-depleted PSII membranes reconstituted with the PsbO deletion mutants showed that the presence of PsbO does not affect Ca 2+ retention by PSII in steady-state assays of activity, nor is it required for Ca 2+ to protect the Mn cluster against reductive inhibition in darkness. In contrast to the results with Ca 2+ , PsbO increases the affinity of Cl - for the active site of the O 2 -evolving complex (OEC) as expected. These results together with other data on activity retention suggest that PsbO can stabilize the Mn cluster by facilitating retention of Cl - in the OEC. The data presented here indicate that each of two copies of PsbO has a distinctive function in PSII. Binding of the first PsbO subunit fully stabilizes the Mn cluster and enhances Cl - retention, while binding of the second subunit optimizes Cl - retention, which in turn maximizes O 2 evolution activity. Nonspecific binding of some PsbO truncation mutants to PSII has no functional significance. The photosystem II (PSII) 1 O 2 -evolving complex (OEC), the catalyst of the H 2 O oxidation reaction, consists of three inorganic cofactors (one Ca 2+ , one Cl - , and four Mn atoms) that are shielded from the thylakoid lumen by three extrinsic polypeptides attached to the intrinsic subunits of PSII. The largest extrinsic protein, called PsbO (33 kDa) or manganese- stabilizing protein, is present in all O 2 -evolving photosyn- thetic organisms. The two smaller extrinsic polypeptides in higher plants and algae, PsbP (23 kDa) and PsbQ (17 kDa), are replaced by PsbV (cyt 550) and PsbU (12 kDa) in cyanobacteria (1). Cyanobacterial homologues of PsbP and PsbQ have also been discovered in Synechocystis sp. PCC 6803 (2); their function is still under investigation. Binding of the PsbP and PsbQ polypeptides in eukaryotic PSII facilitates retention of Ca 2+ and Cl - by the OEC (3-5). Removal of these polypeptides from PSII inhibits O 2 evolution activity; addition of Ca 2+ and Cl - restores up to 75% of the intact PSII activity (3). In contrast, the largest extrinsic polypeptide, PsbO, is essential for both O 2 evolution activity and Mn cluster stability. After depletion of PsbO from PSII in in vitro experiments using CaCl 2 (6) or urea (7), O 2 evolution activity is restored to only ∼20% of the control value (intact PSII), and even this low rate requires concentrations of Ca 2+ and Cl - higher than those needed by PSII samples depleted of the PsbP and PsbQ polypeptides (8). It has been shown that incubation of PsbO-depleted PSII at low Cl - concentrations causes the loss of two of four Mn atoms from the OEC, but this loss can be prevented by addition of a high concentration of Cl - (7, 9). A recent study of Arg mutants of spinach PsbO showed that the protein is important for retention of Cl - in the OEC (10). Other results indicate that PsbO also plays a role in retention of Ca 2+ .A ¨ delroth et al. (11) used 45 Ca 2+ to show that removal of PsbO either eliminates the Ca 2+ binding site in PSII or removes a barrier that prevents rapid exchange of Ca 2+ between the OEC and the surrounding medium. Although PsbO was proposed to be a Ca 2+ binding protein (12, 13), it is acidic (pI 5.2), and therefore, the negative charges on the protein at physiological pH (between 6 and 7) would allow PsbO to bind divalent metal ions nonspecifically. † This research was supported by a grant to H.P. and C.F.Y. from the National Science Foundation (MCB-0716541). * To whom correspondence should be addressed: Department of Molecular, Cellular and Developmental Biology, The University of Michigan, Ann Arbor, MI 48109-1048. Telephone: (734) 647-0887. Fax: (734) 647-0884. E-mail: cyocum@umich.edu. ‡ Department of Molecular, Cellular and Developmental Biology. § Current address: Spring Arbor University, 106 E. Main St., Spring Arbor, MI 49283-9799. | Department of Chemistry. 1 Abbreviations: BSA, bovine serum albumin; Chl, chlorophyll; DCBQ, 2,6-dichloro-1,4-benzoquinone; DMHA, N,N-dimethylhydroxy- lamine; EPR, electron paramagnetic resonance; MES, 2-(N-morpholi- no)ethanesulfonic acid; PAGE, polyacrylamide gel electrophoresis; PsbO, manganese-stabilizing protein; OEC, O 2 -evolving complex; PS, photosystem; PSII, photosystem II; RC, reaction center; SDS, sodium dodecyl sulfate; SW-PSII, NaCl-washed photosystem II membranes depleted of 23 and 17 kDa extrinsic proteins; TMACl, tetramethylam- monium chloride; UW-PSII, urea- and NaCl-washed photosystem II membranes depleted of PsbO, PsbP, and PsbQ (33, 23, and 17 kDa, respectively) extrinsic proteins. Biochemistry 2008, 47, 12593–12600 12593 10.1021/bi801512s CCC: $40.75  2008 American Chemical Society Published on Web 11/04/2008