title: Probing the N-terminal sequence of spinach PsbO: evidence that essential threonine residues bind to different functional sites in eukaryotic photosystem II.
abstract: The N-terminal ¹E-⁶L domain of the manganese-stabilizing protein (PsbO) from spinach prevents non-specific binding of the subunit to photosystem II (PSII) and deletions of the ¹E-⁷T or ¹E-¹⁵T sequences from the PsbO N-terminus reduce or impair, respectively, functional binding of PsbO to PSII (Popelkova et al., Biochemistry 42:6193-6200, 2003). The work presented here provides deeper insights into the interaction of PsbO with PSII. The data show that a single mutation, ¹⁵T → A in mature PsbO from spinach reduces the stoichiometry of its functional binding from two to one subunit per PSII and decreases reconstitution of activity to about 45 % of the wild-type control. Replacement of the ¹E-⁶L domain with ⁶M in the T15A PsbO mutant has no additional negative effect on recovery of O₂ evolution activity, but it significantly weakens both functional and nonspecific binding of the truncated mutant to PSII. These results suggest that the ¹⁵T side-chain by itself is essential for binding of one of two PsbO subunits to eukaryotic PSII and that specific PSII-binding sites for PsbO are distinguishable; one PSII-binding site does not require PsbO-¹⁵T and probably interacts with the other N-terminal domain of PsbO. Identity of the latter domain is revealed by a requirement for the presence of the ¹E-⁶L sequence that is shown here to be necessary for high-affinity binding of PsbO to PSII. When combined with previous results, the data presented here lead to a more detailed model for PsbO binding in eukaryotic PSII.
authors.affiliation: Department of Molecular, Cellular, and Developmental Biology, The University of Michigan, Ann Arbor, MI 48109-1048, USA. popelka@umich.edu
authors.forename: Hana
authors.initials: H
authors.lastname: Popelka
authors.forename: Charles
authors.initials: C
authors.lastname: Yocum
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D015335:Molecular Probes; D008969:Molecular Sequence Data; D045332:Photosystem II Protein Complex / Q000378:metabolism*; D010940:Plant Proteins / Q000737:chemistry / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D018724:Spinacia oleracea / Q000737:chemistry*; D013912:Threonine / Q000378:metabolism*
chemical_list: D017931:DNA Primers; D015335:Molecular Probes; D045332:Photosystem II Protein Complex; D010940:Plant Proteins; D011994:Recombinant Proteins; C052389:oxygen-evolving enhancer protein 1, plant; D013912:Threonine