Bicarbonate Accelerates Assembly of the Inorganic Core of the Water-Oxidizing Complex in Manganese-Depleted Photosystem II: A Proposed Biogeochemical Role for Atmospheric Carbon Dioxide in Oxygenic Photosynthesis

摘要

The proposed role for bicarbonate (HCO_3~-) as an intrinsic cofactor within the water-oxidizing complex (WOC) of photosystem II (PSII) [Klimov et al. (1997) Biochemistry 36, 16277-16281] was tested by investigation of its influence on the kinetics and yield of photoactivation, the light-induced assembly of the functional inorganic core (Mn_4O_yCa_1Cl_x) starting form the cofactor-depleted apo-WOC-PSII center and free Mn~2+, Ca~2+, and Cl~-. two binding sites for bicarbonate were found that stimulate photoactivation by accelerating the formation and formation and suppressing the decay, respectively, of the first lightinduced assembly intermediate, IM_1 [apo-WOC-Mn(OH)_2~+]. A high-affinity bicarbonate site (K_D <= 10 #mu#M) stimulates both the rate of recovery of O_2 evolving centers and decreases (by a factor of 1.2-3) t_lag, the time for formation of IM_1. This stimulation involves enhanced binding of the inital Mn~2+ and occurs only at concentrations of Mn~2+ at or below the stoichiometric requirements fo rwater oxidation (<=4 Mn/PSII) and disappears above 4 Mn/PSII. The absence of an effect from added bicarbonate on photoactivation kinetics and yield at saturating concentrations of Mn~2+ and Ca~2+ may be due to the availability of atmospheric bicarbonate dissolved in the buffers (～4 #mu#M at pH 6.0) sufficient for photoactivation. The second bicarbonate site also stimulates the rate of formation of IM_1 but has much lower affinity (K_D approximately millimolar) and becomes observable only at low concentrations of Ca~2+ that are limiting for photoactivation. This stimulation effect appears to occur by complexation of free Ca~2+, thereby reducing its activity in competing with Mn~2+ in the formation of IM_1. Bicarbonate had no effect on the calcium effector site responsible for the rate-limiting dark step of photoactivation (Ca~2+ binding to IM_1). Four interpretations of the high-afinity bicarbonate effect may be advanced as testable hypotheses: bicarbonate may (1) act as an integral cofactor within the WOC (possible ligand to the first Mn), (2) act as a Bronsted base to accelerate proton release during formation of either the dark precursor [apo-WOC-Mn(OH)~+] or IM_1 [apo-WOC-Mn(OH)_2~+], (3) directly deliver one or more hydroxide ions during formation of the latter two species (with release of CO_2), or (4) act as a membrane-soluble anion that electrostatically elevates the local concentration of Mn~2+ in PSII. These results support a possible biogeochemical role for bicarbonate in the evolution of hte first oxygenic photosynthetic organism. An improvement in the illumination method for photoactivation is presented in which light flashes of increasing duration are used to extend the pre-steady-state lag phase and to suppress photoinhibitionk, thereby improving the accuracy of t_lag determination.