Quantifying the ion selectivity of the Ca2+ site in photosystem II: Evidence for direct involvement of Ca2+ in O-2 formation

Calcium is an essential cofactor in the oxygen-evolving complex (OEC) of ph otosystem II (PSII). The removal of Ca2+ or its substitution by any metal i on except Sr2+ inhibits oxygen evolution. We used steady-state enzyme kinet ics to measure the rate of O-2 evolution in PSII samples treated with an ex tensive series of mono-, di-, and trivalent metal ions in order to determin e the basis for the affinity of metal ions for the Ca2+-binding site. Our r esults show that the Ca2+-binding site in PSII behaves very similarly to th e Ca2+-binding sites in other proteins, and we discuss the implications thi s has for the structure of the site in PSII, Activity measurements as a fun ction of time show that the binding site achieves equilibrium in 4 h for al l of the PSII samples investigated. The binding affinities of the metal ion s are modulated by the 17 and 23 kDa extrinsic polypeptides; their removal decreases the free energy of binding of the metal ions by 2.5 kcal/mol, but does not significantly change the time required to reach equilibrium. Mono valent ions are effectively excluded from the Ca2+-binding site, exhibiting no inhibition of O-2 evolution. Di- and trivalent metal ions with ionic ra dii similar to that of Ca2+ (0.99 Angstrom) bind competitively with Ca2+ an d have the highest binding affinity, while smaller metal ions bind more wea kly and much larger ones do not bind competitively. This is consistent with a size-selective Ca2+-binding site that has a rigid array of coordinating ligands, Despite the large number of metal ions that competitively replace Ca2+ in the OEC, only Sr2+ is capable of partially restoring activity. Comp aring the physical characteristics of the metal ions studied, we identify t he pK(a) of the aqua ion as the factor that determines the functional compe tence of the metal ion. This suggests that Ca2+ is directly involved in the chemistry of water oxidation and is not only a structural cofactor in the OEC. We propose that the role of Ca2+ is to act as a Lewis acid, binding a substrate water molecule and tuning its reactivity.