Assembly of the inorganic core (Mn4OxCa 1Cly) of the water oxidizing enzyme of oxygenic photosynthesis generates O2 evolution capacity via the photodriven binding and photooxidation of the free inorganic cofactors within the cofactor-depleted enzyme (apo-WOC-PSII) by a process called photoactivation. Using in vitro photoactivation of spinach PSII membranes, we identify a new lower affinity site for bicarbonate interaction in the WOC. Bicarbonate addition causes a 300% stimulation of the rate and a 50% increase in yield of photoassembled PSII centers when using Mn2+ and Ca2+ concentrations that are 10-50-fold larger range than previously examined. Maintenance of a fixed Mn2+/ Ca2+ ratio (1:500) produces the fastest rates and highest yields of photoactivation, which has implications for intracellular cofactor homeostasis. A two-step (biexponential) model is shown to accurately fit the assembly kinetics over a 200-fold range of Mn 2+ concentrations. The first step, the binding and photooxidation of Mn2+ to Mn3+, is specifically stimulated via formation of a ternary complex between Mn2+, bicarbonate, and apo-WOC-PSII, having a proposed stoichiometry of [Mn2+(HCO3 -)]. This low-affinity bicarbonate complex is thermodynamically easier to oxidize than the aqua precursor, [Mn2+-(OH2)]. The photooxidized intermediate, [Mn3+(HCO3-)], is longer lived and increases the photoactivation yield by suppressing irreversible photodamage to the cofactor-free apo-WOC-PSII (photoinhibition). Bicarbonate does not affect the second (rate-limiting) dark step of photoactivation, attributed to a protein conformational change. Together with the previously characterized high-affinity site, these results reveal that bicarbonate is a multifunctional "native" cofactor important for photoactivation and photoprotection of the WOC-PSII complex.
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