Binding of Manganese Stabilizing Protein to Photosystem II: Identification of Essential N-Terminal Threonine Residues and Domains that Prevent Nonspecific Binding † Hana Popelkova, ‡ Michael M. Im, ‡ and Charles F. Yocum* ,‡,# Department of Molecular, Cellular and DeVelopmental Biology, and Department of Chemistry, UniVersity of Michigan, Ann Arbor, Michigan 48109-1048 ReceiVed December 23, 2002 ABSTRACT: The N-terminus of spinach photosystem II manganese stabilizing protein (MSP) contains two amino acid sequences, 4 KRLTYD 10 E and 15 TYL 18 E, that are necessary for binding of two copies of this subunit to the enzyme [Popelkova et al. (2002) Biochemistry 41, 10038-10045]. To better understand the basis of MSP-photosystem II interactions, the role of threonine residues in the highly conserved motifs T(Y/F)DE and TY has been characterized. Deletion mutants lacking the first 5, 6, 7, and 15 amino acid residues at the N-terminus of the protein were examined for their ability to reconstitute activity in MSP-depleted photosystem II. The results reported here show that truncations of five and six amino acid residues (mutants ΔR5M and ΔL6M mutants) have no negative effect on recovery of oxygen evolution activity or on binding of MSP to photosystem II. Deletion of seven residues (mutant ΔT7M) decreases reconstitution activity to 40% of the control value and reduces functional binding of the mutant protein to photosystem II from two to one copy. Deletion of 15 amino acid residues (mutant ΔT15M) severely impairs functional binding of MSP, and lowers O 2 evolution activity to less than 20% of the control. ΔT7M is the only mutant that exhibited neither nonspecific binding to photosystem II nor changes in tertiary structure. These, and previous results, show that the highly conserved Thr7 and Thr15 residues of MSP are required for functional binding of two copies of the eukaryotic protein to photosystem II. Although the N-terminal domains, 1 EGGKR 6 L, 8 YDEIQS 14 K, and 16 YL 18 E of spinach MSP are unnecessary for specific, functional binding interactions, these sequences are necessary to prevent nonspecific binding of the protein to photosystem II. Chlorophyll-containing organisms produce molecular oxy- gen as a byproduct of water oxidation, which takes place in the oxygen evolving complex (OEC) 1 of photosystem II (PSII). In eukaryotes, three extrinsic proteins (17, 23, and 33 kDa) provide a structural framework for retention and stabilization of the essential inorganic cofactors (Ca + , Cl - , and a cluster of four Mn atoms (1)) that form the active site of the OEC. The 33-kDa protein, also known as manganese stabilizing protein (MSP), functions to stabilize binding of the tetranuclear manganese cluster to the OEC, and to accelerate the rate of O 2 evolution activity (2-6). It has also been suggested that MSP regulates the Mn-Cl - interaction by facilitating Cl - binding to the OEC (7), and the presence of MSP is required for binding of the 23-kDa protein to PSII (8). A direct role for MSP in Mn redox reactions (deproto- nation of glutamate and/or aspartate residues during water oxidation) has been proposed as well (9). The 17, 23, and 33 kDa proteins can be selectively extracted from intact PSII membranes (BBY). Washing intact PSII preparations in 2 M NaCl releases the 17- and 23-kDa proteins along with Ca 2+ and Cl - ; addition of these cofactors restores substantial amounts of activity (10). A subsequent exposure of the salt-washed preparation to 2-3 M urea specifically extracts MSP, which lowers O 2 evolution activity and destabilizes binding of the Mn cluster to PSII (3, 11). When the psbO gene encoding MSP is deleted in the cyanobacterium Synechocystis sp. PCC6803, oxygen evolu- tion activity is also lowered (12, 13). In the green alga Chlamydomonas, however, deletion of the psbO gene produces cells that are unable to grow photoautotrophically and to accumulate other PSII subunits (14). Interactions of MSP with the E loop of the CP47 subunit of PSII have been documented (15-19). Although results of three-dimensional cryoelectron microscopy of spinach PSII † This research was supported by a grant to C.F.Y. from the National Science Foundation (MCB-0110455). * Correspondence should be addressed to this author at the Depart- ment 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. # Department of Chemistry. 1 Abbreviations: CD, circular dichroism; Chl, chlorophyll; EDC, 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide; IPTG, isopropyl-- D-thiogalactopyranoside; MSP, manganese stabilizing protein; OEC, O2 evolving complex; PAGE, polyacrylamide gel electrophoresis; PCR, polymerase chain reaction; TOPO, plasmid for gene expression with T7 RNA polymerase; PS, photosystem; psbO, gene encoding precursor MSP; SDS, sodium dodecyl sulfate; sw-PSII, NaCl-washed photosystem II membranes depleted of 23 and 17 kDa extrinsic proteins; TMACl, tetramethylamonium chloride; usw-PSII, urea salt-washed photosystem II membranes depleted of 33, 23, and 17 kDa extrinsic proteins; UV, ultraviolet; Δ, represents missing amino acid residues; 2°, secondary structure of protein; 3°, tertiary structure of protein. 6193 Biochemistry 2003, 42, 6193-6200 10.1021/bi0207115 CCC: $25.00 © 2003 American Chemical Society Published on Web 05/03/2003