Inorganic Cofactor Stabilization and Retention: The Unique Functions of the Two PsbO Subunits of Eukaryotic Photosystem II

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.

An Intrinsically Disordered Photosystem II Subunit, PsbO, Provides a Structural Template and a Sensor of the Hydrogen-bonding Network in Photosynthetic Water Oxidation

“…These results are consistent with previous studies of other spinach PSII preparations (20,64,66,72,77,79,80). The V235A mutation employed here does not affect the solution structure, binding, or activity at room temperature (24).…”

“…60 and 61, for examples) have identified domains that are important in structure, function, and assembly. Although one copy of PsbO is bound per reaction center in thermophilic cyanobacteria (6,7), plant PSII binds two copies of PsbO (19,23,25,32,41,50,(62)(63)(64).…”

Probing the topography of the photosystem II oxygen evolving complex: PsbO is required for efficient calcium protection of the manganese cluster against dark-inhibition by an artificial reductant

“…binding near the Mn cluster can hinder its slow deactivation. Based on previous results (Popelkova et al 2008), this deactivation consists principally of loss of Mn from PSII centers. The greatest effect of Ca 2?…”

“…retention under continuous illumination and that the Ca 2? K D in the steady state is about 0.15 mM for PSII samples reconstituted with mutated PsbO (Popelkova et al 2006(Popelkova et al , 2008(Popelkova et al , 2009). The latter observation indicates that despite a fast exchange of Ca 2?…”

“…In the dark, Ca 2? at a high (10 mM) concentration in the incubation buffer protects the Mn cluster against inhibition by DMHA in the absence of PsbO (Popelkova et al 2008). However, there are no experimental data to show that this would be the case at significantly lower concentrations of Ca 2?…”