Photosystem II (PSII), the water/plastoquinone photo-oxidoreductase, plays a key energy input role in the biosphere. QA•-, the reduced semiquinone form of the nonexchangeable quinone, is often considered capable of a side reaction with O2, forming superoxide, but this reaction has not yet been demonstrated experimentally. Here, using chlorophyll fluorescence in plant PSII membranes, we show that O2 does oxidize QA•- at physiological O2 concentrations with a t1/2 of 10 s. Superoxide is formed stoichiometrically, and the reaction kinetics are controlled by the accessibility of O2 to a binding site near QA•-, with an apparent dissociation constant of 70 ± 20 µM. Unexpectedly, QA•- could only reduce O2 when bicarbonate was absent from its binding site on the nonheme iron (Fe2+) and the addition of bicarbonate or formate blocked the O2-dependant decay of QA•-. These results, together with molecular dynamics simulations and hybrid quantum mechanics/molecular mechanics calculations, indicate that electron transfer from QA•- to O2 occurs when the O2 is bound to the empty bicarbonate site on Fe2+. A protective role for bicarbonate in PSII was recently reported, involving long-lived QA•- triggering bicarbonate dissociation from Fe2+ [Brinkert et al., Proc. Natl. Acad. Sci. U.S.A. 113, 12144-12149 (2016)]. The present findings extend this mechanism by showing that bicarbonate release allows O2 to bind to Fe2+ and to oxidize QA•-. This could be beneficial by oxidizing QA•- and by producing superoxide, a chemical signal for the overreduced state of the electron transfer chain. [ABSTRACT FROM AUTHOR]