Quantification of bound bicarbonate in photosystem II |
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Authors: | K Tikhonov D Shevela V V Klimov J Messinger |
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Institution: | 1.Institute of Basic Biological Problems,Russian Academy of Science,Pushchino,Russia;2.Department of Chemistry, Chemical Biological Centre,Ume? University,Ume?,Sweden;3.Department of Chemistry, Molecular Biomimetics,?ngstr?m Laboratory, Uppsala University,Uppsala,Sweden |
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Abstract: | 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. |
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