Bicarbonate-controlled reduction of oxygen by the Q_A semiquinone in Photosystem II in membranes

Photosystem II (PSII), the water/plastoquinone photo-oxidoreductase, plays a key energy input role in the biosphere. Q^•_A², 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 Q^•_A² 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 Q^•_A², with an apparent dissociation constant of 70 ± 20 μM. Unexpectedly, Q^•_A² 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 Q^•_A². These results, together with molecular dynamics simulations and hybrid quantum mechanics/molecular mechanics calculations, indicate that electron transfer from Q^•_A² 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 Q^•_A² 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 Q^•_A². This could be beneficial by oxidizing Q^•_A² and by producing superoxide, a chemical signal for the overreduced state of the electron transfer chain.