Photosystem II (PSII), the water/plastoquinone photo-oxidoreductase, plays a key energy input role in the biosphere. [Formula: see text], 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 [Formula: see text] at physiological O₂ concentrations with a t_1/2 of 10 s. Superoxide is formed stoichiometrically, and the reaction kinetics are controlled by the accessibility of O₂ to a binding site near [Formula: see text], with an apparent dissociation constant of 70 ± 20 µM. Unexpectedly, [Formula: see text] 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 [Formula: see text] These results, together with molecular dynamics simulations and hybrid quantum mechanics/molecular mechanics calculations, indicate that electron transfer from [Formula: see text] 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 [Formula: see text] 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 [Formula: see text] This could be beneficial by oxidizing [Formula: see text] and by producing superoxide, a chemical signal for the overreduced state of the electron transfer chain.

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