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Important questions for understanding the reaction by which water is oxidized to molecular O2 during photosynthesis are (1) at which step in the catalytic sequence do the two substrate water molecules bind, and (2) how does the OO bond form? In the past, it has been experimentally difficult to get information on these questions, mainly because of the immense difficulty to distinguish the substrate water from the solvent water. However, we have recently developed special sampling techniques for mass spectrometric measurements that have enabled us to resolve the rate of isotopic incorporation from 18O-labeled water into the molecular O2 produced by plants, algae, and cyanobacteria. The results from these measurements have definitively shown that at least one of the two substrate water molecules is bound to the catalytic site at the beginning of the four-step reaction sequence and that the OO bond forms during the last step, just before the release of the O2 product. This article summarizes the special techniques developed by our group and the implications of the results for the mechanism of photosynthetic O2 production. Keywords: photosynthesis; photosystem II; oxygen evolution; water oxidation; atmospheric oxygen; oxygen evolving complex; H218O; oxygen isotopes; isotope exchange; membrane inlet mass spectrometry; gas/water reactions; stable isotopes; mass spectrometry |
