The size and charge density requirements for metal ion binding to the high-affinity Mn2+ site of the ape-water oxidizing complex (WOC) of spinach photosystem II (PSII) were studied by comparing the relative binding affinities of alkali metal cations, divalent metals (Mg2+, Ca2+, Mn2+, Sr2+), and the oxo-cation UO22+. Cation binding to the apo-WOC-PSII protein was measured by: (1) inhibition of the rate and yield of photoactivation, the light-induced recovery of O-2 evolution by assembly of the functional Mn4Ca1Clx, core from its constituent inorganic cofactors (Mn2+, Ca2+, and Cl-); and by (2) inhibition of the PSII-mediated light-induced electron transfer from Mn2+ to an electron acceptor (DCIP), Together, these methods enable discrimination between inhibition at the high- and low-affinity Mn2+ sites and the Ca2+ site of the apo-WOC-PSII. Unexpectedly strong binding: of large alkali cations (Cs+ >> Rb+ > K+ > Na+ >Li+) was found to smoothly correlate with decreasing cation charge density, exhibiting one of the largest Cs+/Li+ selectivities (greater than or equal to 5000) for any known chelator, Both photoactivation and electron-transfer measurements at selected Mn2+ and Ca2+ concentrations reveal that Cs+ binds to the high-affinity Mn2+ site with a slightly greater affinity (2-3-fold at pH 6.0) than Mn2+, while binding about 10(4)-fold more weakly to the Ca2+-specific site required for reassembly of functional O-2 evolving centers. In contrast to Cs+, divalent cations larger than Mn2+ bind considerably more weakly to the high-affinity Mn2+ site (Mn2+ >> Ca2+ > Sr2+). Their affinities correlate with the hydrolysis constant for formation of the metal hydroxide by hydrolysis of water: Me-aq(2+) --> [MeOH](+)(aq) + H-aq(+) Along with the strong stimulation of the rate of photoactivation by alkaline pH, these metal cation trends support the interpretation that [MnOH](+) is the active species that forms upon binding of Mn-aq(2+) to apo-WOC. Further support for this interpretation is found by the unusually strong inhibition of Mn2+ photooxidation by the linear uranyl cation (UO22+), The intrinsic binding constant for [MnOH](+) to apo-WOC was determined using a thermodynamic cycle to be K = 4.0 x 10(15) M-1 (at pH 6.0), consistent with a high-affinity, preorganized, multidentate coordination site. We propose that the selectivity for binding [MnOH](+), a linear low charge-density monocation, vs symmetrical Me2+ dications is functionally important for assembly of the WOC by enabling: (1) discrimination against higher charge density alkaline earth cations (Mg2+ and Ca2+) and smaller alkali metal cations (Na+ and K+) that are present in considerably greater abundance in vivo, and thus would suppress photoactivation; and (2) higher affinity binding of the one Ca2+ ion or the remaining three Mn2+ ions via coordination to form mu-hydroxo-bridged intermediates, apo-WOC-[Mn(mu-OH)(2)Mn](3+) or apo-WOC-[Mn(mu-OH)Ca](3+), during subsequent assembly steps of the native Mn4Ca1Clx core. In contrast to more acidic Me2+ divalent ion inhibitors of the high-affinity Mn2+ site, like Ca2+ and Sr2+, Cs+ does not accelerate the decay of the first light-induced intermediate, IM1, formed during photoactivation (attributed to apo-WOC-[Mn(OH)(2)](+)). The inability of Cs+ to promote decay of IM1, despite having comparable affinity as Mn2+, is consistent with its considerably weaker Lewis acidity, resulting in the reprotonation of IM1 by water becoming the rate-limiting step for decay Drier to displacement of Mn2+. All four different lines of evidence provide a self-consistent picture indicating that the initial step in assembly of the WOC involves high-affinity binding of [MnOH](+).
