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

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

Cryo-EM structures of complex I from mouse heart mitochondria in two biochemically defined states.

Complex I (NADH:ubiquinone oxidoreductase) uses the reducing potential of NADH to drive protons across the energy-transducing inner membrane and power oxidative phosphorylation in mammalian mitochondria. Recent cryo-EM analyses have produced near-complete models of all 45 subunits in the bovine, ovine and porcine complexes and have identified two states relevant to complex I in ischemia-reperfusion injury. Here, we describe the 3.3-Å structure of complex I from mouse heart mitochondria, a biomedically relevant model system, in the 'active' state. We reveal a nucleotide bound in subunit NDUFA10, a nucleoside kinase homolog, and define mechanistically critical elements in the mammalian enzyme. By comparisons with a 3.9-Å structure of the 'deactive' state and with known bacterial structures, we identify differences in helical geometry in the membrane domain that occur upon activation or that alter the positions of catalytically important charged residues. Our results demonstrate the capability of cryo-EM analyses to challenge and develop mechanistic models for mammalian complex I