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^,

Abstract

Assembly of the inorganic core (Mn(4)O(x)Ca(1)Cl(y)) 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 Mn(2+) and Ca(2+) concentrations that are 10-50-fold larger range than previously examined. Maintenance of a fixed Mn(2+)/Ca(2+) 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 Mn(2+) to Mn(3+), is specifically stimulated via formation of a ternary complex between Mn(2+), bicarbonate, and apo-WOC-PSII, having a proposed stoichiometry of [Mn(2+)(HCO(3)(-))]. This low-affinity bicarbonate complex is thermodynamically easier to oxidize than the aqua precursor, [Mn(2+)(OH(2))]. The photooxidized intermediate, [Mn(3+)(HCO(3)(-))], 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.

Spectroscopic Evidence for Ca2+Involvement in the Assembly of the Mn4Ca Cluster in the Photosynthetic Water-Oxidizing Complex

“…Under saturating continuous illumination, the oxygen evolution rate of the PSII membranes was 400-500 µmol of O 2 (mg of Chl) -1 h -1 in the presence of 1 mM K 3 Fe(CN) 6 and 0.25 mM 2,5-dichloro-p-benzoquinone (DCBQ) as electron acceptors. PSII membranes depleted of Mn 2+ , Ca 2+ , and the three extrinsic proteins (apo-WOC-PSII) were prepared by a brief incubation in high-pH buffer in the presence of 200 mM MgCl 2 , as recently described (25). The resulting apo-WOC-PSII membranes were devoid of the three extrinsic proteins as confirmed by SDS-PAGE (not shown) and exhibited no residual O 2 evolution activity (i.e., less than 0.5% of the activity of the BBY membranes).…”

“…The resulting apo-WOC-PSII membranes were devoid of the three extrinsic proteins as confirmed by SDS-PAGE (not shown) and exhibited no residual O 2 evolution activity (i.e., less than 0.5% of the activity of the BBY membranes). These apo-WOC-PSII particles were capable of reconstituting the water-splitting system of PSII (upon in Vitro photoactivation in the presence of Mn 2+ , Ca 2+ , and an electron acceptor) with a yield of 60-70% as calibrated versus the extrinsic protein-depleted PSII (25).…”

“…Maintenance of an optimal concentration ratio of Mn 2+ and Ca 2+ in the photoactivation medium was shown to be essential to avoid excessive photoinhibition during photoactivation and thus to achieve a high yield of reconstituted WOC-PSII (22,28). Furthermore, at this optimal Mn 2+ / Ca 2+ ratio, the yield of reconstituted WOC-PSII was observed to increase in parallel with the rate of the first photo-oxidation step as the concentration of Mn 2+ is increased (25). It was inferred that the majority of photoinhibition damage occurs during the initial k 1 step and that enhancement in the quantum efficiency during this step helps to reduce the photoinhibition damage.…”

“…Bicarbonate ions play an important physiological role in accelerating the rate and increasing the yield of the first intermediate in photoactivation (24)(25)(26). The second step (k 2 ) is rate-limiting and occurs in the dark, and its overall slowness suggests that it involves a protein conformational change.…”

Multifrequency pulsed EPR studies of biologically relevant manganese(II) complexes

“…The cycle repeats as additional equivalents of Mn(II) as well as Ca 2+ and Cl − cofactors bind to yield the fully functional OEC. Interestingly, O 2 evolution measurements indicate that the presence of bicarbonate anion enhances the rate of photoassembly [80]. This effect can be rationalized as carboxylate coordination of the Mn(H) ion is known to stabilize the corresponding Mn(II) → Mn(III) oxidation potential from 1.5 to 0.5 V when bound to PSII [81].…”