Quantification of bound bicarbonate in photosystem II
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In this study, we presented a new approach for quantification of bicarbonate (HCO3−) molecules bound to PSII. Our method, which is based on a combination of membrane-inlet mass spectrometry (MIMS) and 18O-labelling, excludes the possibility of “non-accounted” HCO3− by avoiding (1) the employment of formate for removal of HCO3− from PSII, and (2) the extremely low concentrations of HCO3−/CO2 during online MIMS measurements. By equilibration of PSII sample to ambient CO2 concentration of dissolved CO2/HCO3−, the method ensures that all physiological binding sites are saturated before analysis. With this approach, we determined that in spinach PSII membrane fragments 1.1 ± 0.1 HCO3− are bound per PSII reaction center, while none was bound to isolated PsbO protein. Our present results confirmed that PSII binds one HCO3− molecule as ligand to the non-heme iron of PSII, while unbound HCO3− optimizes the water-splitting reactions by acting as a mobile proton shuttle.
Additional key wordshydrogen carbonate inorganic carbon mass spectrometry Mn-stabilizing protein non-heme iron oxygen-evolving complex
membrane-inlet mass spectrometry
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