Quantification of bound bicarbonate in photosystem II
2018
Tikhonov, K. | Shevela, D. | Klimov, V. V. | Messinger, J.
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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