Probing the N-terminal sequence of spinach PsbO: evidence that essential threonine residues bind to different functional sites in eukaryotic photosystem II

The N-terminal 1E-?L 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-?T or 1E-1?T 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, 1?T → 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-?L domain with ?M in the T15A PsbO mutant has no additional negative effect on recovery of O? evolution activity, but it significantly weakens both functional and nonspecific binding of the truncated mutant to PSII. These results suggest that the 1?T 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-1?T 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-?L 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.