Bicarbonate is a native cofactor for assembly of the manganese cluster of the photosynthetic water oxidizing complex.: Kinetics of reconstitution of O2 evolution by photoactivation

Assembly of the inorganic core (Mn4OxCa1Cly) of the water oxidizing enzyme of oxygenic photosynthesis generates O-2 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 Mn2+ 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.