Quantifying the Ion Selectivity of the Ca²⁺ Site in Photosystem II:  Evidence for Direct Involvement of Ca²⁺ in O₂ Formation

Abstract

Calcium is an essential cofactor in the oxygen-evolving complex (OEC) of photosystem II (PSII). The removal of Ca2+ or its substitution by any metal ion except Sr2+ inhibits oxygen evolution. We used steady-state enzyme kinetics to measure the rate of O2 evolution in PSII samples treated with an extensive series of mono-, di-, and trivalent metal ions in order to determine the basis for the affinity of metal ions for the Ca2+-binding site. Our results show that the Ca2+-binding site in PSII behaves very similarly to the Ca2+-binding sites in other proteins, and we discuss the implications this has for the structure of the site in PSII. Activity measurements as a function of time show that the binding site achieves equilibrium in 4 h for all of the PSII samples investigated. The binding affinities of the metal ions 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. Monovalent ions are effectively excluded from the Ca2+-binding site, exhibiting no inhibition of O2 evolution. Di- and trivalent metal ions with ionic radii similar to that of Ca2+ (0.99 A) bind competitively with Ca2+ and have the highest binding affinity, while smaller metal ions bind more weakly 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. Comparing the physical characteristics of the metal ions studied, we identify the pK(a) of the aqua ion as the factor that determines the functional competence 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.

Substrate Water Exchange in Photosystem II Depends on the Peripheral Proteins

“…This observation is consistent with a number of nuclear magnetic resonance studies showing that water bound within proteins normally exchanges rapidly with the solvent water (55)(56)(57). Thus, counter to the earlier mechanistic proposals (47)(48)(49), the current data suggest that accessibility is not a major factor in regulating the 18 O exchange rates in PSII. Consequently, we consider the Ca-OH 2 species to be an unlikely intermediate in the water oxidation sequence for the S 3 state (42).…”

Calcium controls the assembly of the photosynthetic water-oxidizing complex: a cadmium(II) inorganic mutant of the Mn 4 Ca core

“…Our IC 50 value for Cd 2C inactivation of Y SS (120 mM) during photoactivation compares well with its IC 50 reported for O 2 evolution from intact functional PSII membrane complexes (K D Z144 mM; Vrettos et al 2001). These authors report a higher binding affinity for Ca 2C to the intact functional PSII complex (K D Z 69 mM) compared with other monovalent (Na, K) and divalent (Mn, Ni, Cu, Co, Cd, Sr, Ba) ions.…”

Photoassembly of the water-oxidizing complex in photosystem II

“…If Ca 2+ is left out of the solution, binding and photooxidation of many more Mn 2+ occurs (up to 20) to the apo-WOC-PSII protein and no O 2 evolution activity is observable [45,58]. The relative binding affinities of various divalent metals (Mg 2+ , Ca 2+ , Mn 2+ , Sr 2+ ), and the oxo-cation UO 2 2+ have been characterized [11,59,60]. At low Ca/RC concentrations measurements of the kinetics of photoactivation reveal that the Ca 2+ binding affinity increases following photooxidation of the first Mn 2+ → Mn 3+ , during which 1 Ca 2+ binds per PSII [55].…”