The size and charge density requirements for metal ion binding to the high-affinity Mn2+ site of the apo-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 UO2 2+. 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 O2 evolution by assembly of the functional Mn4Ca1Cl(x), 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 (≥5000) for any known chelator. Both photoactivation and electrontransfer 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 104-fold more weakly to the Ca2+-specific site required for reassembly of functional O2 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: Me2+(aq) → [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 Mn2+(aq) to apo-WOC. Further support for this interpretation is found by the unusually strong inhibition of Mn2+ photooxidation by the linear uranyl cation (UO2 2+). The intrinsic binding constant for [MnOH]+ to apo-WOC was determined using a thermodynamic cycle to be K = 4.0 x 1015 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 from μ-hydroxo- bridged intermediates, apo-WOC-[Mn(μ-OH)2Mn]3+ or apo-WOC-[Mn(μ- OH)Ca]3+, during subsequent assembly steps of the native Mn4Ca1Cl(x) 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 prior 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]+.
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