Bicarbonate Is a Native Cofactor for Assembly of the Manganese Cluster of the Photosynthetic Water Oxidizing Complex. Kinetics of Reconstitution of O ₂ Evolution by Photoactivation

Assembly of the inorganic core (Mn₄O_xCa ₁Cl_y) 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.