Hydrogencarbonate is not a tightly bound constituent of the water-oxidizing complex in photosystem II

Since the end of the 1950s hydrogencarbonate (‘bicarbonate’) is discussed as a possible cofactor of photosynthetic water-splitting, and in a recent X-ray crystallography model of photosystem II (PSII) it was displayed as a ligand of the Mn4OxCa cluster. Employing membrane-inlet mass spectrometry (MIMS) and isotope labelling we confirm the release of less than one (≈ 0.3) HCO3− per PSII upon addition of formate. The same amount of HCO3− release is observed upon formate addition to Mn-depleted PSII samples. This suggests that formate does not replace HCO3− from the donor side, but only from the non-heme iron at the acceptor side of PSII. The absence of a firmly bound HCO3− is corroborated by showing that a reductive destruction of the Mn4OxCa cluster inside the MIMS cell by NH2OH addition does not lead to any CO2/HCO3− release. We note that even after an essentially complete HCO3−/CO2 removal from the sample medium by extensive degassing in the MIMS cell the PSII samples retain ≥ 75% of their initial flash-induced O2-evolving capacity. We therefore conclude that HCO3− has only ‘indirect’ effects on water-splitting in PSII, possibly by being part of a proton relay network and/or by participating in assembly and stabilization of the water-oxidizing complex.