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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Abbreviations
- Chl:
-
chlorophyll
- MIMS:
-
membrane-inlet mass spectrometry
- OEC:
-
oxygen-evolving complex
- PQ:
-
plastoquinone
- PQH2 :
-
plastoquinol
- RC:
-
reaction center
- NHI:
-
non-heme iron
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This paper is dedicated to the memory of Prof. Vyacheslav V. Klimov (12 January 1945 – 9 May 2017), our dear colleague and co-author, a world leader in the field of photosynthesis research for almost five decades. Among many scientific achievements, Vyacheslav Klimov (known to many as “Slava”) was one of the discoverers of pheophytin as the primary electron acceptor in PSII, and rediscoverer of “bicarbonate effect” on the electron donor side of PSII.
Acknowledgements: The authors thank Govindjee and A. Stemler for fruitful and stimulating discussions on “bicarbonate effects” over the years, the reviewers and the editor for their valuable comments and suggestions, and T.N. Smolova for isolation of PsbO protein. This work was supported by the Russian Foundation for basic research (grant No. 17-04-01011), by the Knut and Wallenberg Foundation, and by the Swedish Science Foundation (VR, grant No. 2016-05183).
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Tikhonov, K., Shevela, D., Klimov, V.V. et al. Quantification of bound bicarbonate in photosystem II. Photosynthetica 56, 210–216 (2018). https://doi.org/10.1007/s11099-017-0758-4
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DOI: https://doi.org/10.1007/s11099-017-0758-4