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Photosystem II (PSII) catalyzes light-driven water oxidation that releases dioxygen into our atmosphere and provides the electrons needed for the synthesis of biomass. The catalysis occurs in the oxygen-evolving oxo-manganese-calcium (Mn 4 O 5 Ca) cluster that drives the oxidation and deprotonation of substrate water molecules leading to the O 2 formation. However, despite recent advances, the mechanism of these reactions remains unclear and much debated. Here, we show that the light-driven Tyr161 D1 (Y z ) oxidation adjacent to the Mn 4 O 5 Ca cluster, decreases the barrier for proton transfer from the putative substrate water molecule (W3/W x ) to Glu310 D2 , accessible to the luminal bulk. By combining hybrid quantum/classical (QM/MM) free energy calculations with atomistic molecular dynamics simulations, we probe the energetics of the proton transfer along the Cl1 pathway. We demonstrate that the proton transfer occurs via water molecules and a cluster of conserved carboxylates, driven by redox-triggered electric fields directed along the pathway. Glu65 D1 establishes a local molecular gate that controls the proton transfer to the luminal bulk, while Glu312 D2 acts as a local proton storage site. The identified gating region could be important in preventing backflow of protons to the Mn 4 O 5 Ca cluster. The structural changes, derived here based on the dark-state PSII structure, strongly support recent time-resolved X-ray free electron laser data of the S 3 → S 4 transition (Bhowmick et al . Nature 617 , 2023) and reveal the mechanistic basis underlying deprotonation of the substrate water molecules. Our findings provide insight into the water oxidation mechanism of PSII and show how the interplay between redox-triggered electric fields, ion-pairs, and hydration effects control proton transport reactions., Competing Interests: Competing interests statement:The authors declare no competing interest.

Photosystem II (PSII) catalyzes light-driven water oxidization, releasing O 2 into the atmosphere and transferring the electrons for the synthesis of biomass. However, despite decades of structural and functional studies, the water oxidation mechanism of PSII has remained puzzling and a major challenge for modern chemical research. Here, we show that PSII catalyzes redox-triggered proton transfer between its oxygen-evolving Mn 4 O 5 Ca cluster and a nearby cluster of conserved buried ion-pairs, which are connected to the bulk solvent via a proton pathway. By using multi-scale quantum and classical simulations, we find that oxidation of a redox-active Tyr z (Tyr161) lowers the reaction barrier for the water-mediated proton transfer from a Ca 2+ -bound water molecule (W3) to Asp61 via conformational changes in a nearby ion-pair (Asp61/Lys317). Deprotonation of this W3 substrate water triggers its migration toward Mn1 to a position identified in recent X-ray free-electron laser (XFEL) experiments [Ibrahim et al. Proc. Natl. Acad. Sci. USA 2020, 117, 12,624-12,635]. Further oxidation of the Mn 4 O 5 Ca cluster lowers the proton transfer barrier through the water ligand sphere of the Mn 4 O 5 Ca cluster to Asp61 via a similar ion-pair dissociation process, while the resulting Mn-bound oxo/oxyl species leads to O 2 formation by a radical coupling mechanism. The proposed redox-coupled protonation mechanism shows a striking resemblance to functional motifs in other enzymes involved in biological energy conversion, with an interplay between hydration changes, ion-pair dynamics, and electric fields that modulate the catalytic barriers.

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 O 2 , forming superoxide, but this reaction has not yet been demonstrated experimentally. Here, using chlorophyll fluorescence in plant PSII membranes, we show that O 2 does oxidize [Formula: see text] at physiological O 2 concentrations with a t 1/2 of 10 s. Superoxide is formed stoichiometrically, and the reaction kinetics are controlled by the accessibility of O 2 to a binding site near [Formula: see text], with an apparent dissociation constant of 70 ± 20 µM. Unexpectedly, [Formula: see text] could only reduce O 2 when bicarbonate was absent from its binding site on the nonheme iron (Fe 2+ ) and the addition of bicarbonate or formate blocked the O 2 -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 O 2 occurs when the O 2 is bound to the empty bicarbonate site on Fe 2+ A protective role for bicarbonate in PSII was recently reported, involving long-lived [Formula: see text] triggering bicarbonate dissociation from Fe 2+ [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 O 2 to bind to Fe 2+ 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., Competing Interests: The authors declare no competing interest., (Copyright © 2022 the Author(s). Published by PNAS.)

In Photosystem II (PSII), the Mn 4 CaO 5 -cluster of the active site advances through five sequential oxidation states (S 0 to S 4 ) before water is oxidized and O 2 is generated. Here, we have studied the transition between the low spin (LS) and high spin (HS) configurations of S 2 using EPR spectroscopy, quantum chemical calculations using Density Functional Theory (DFT), and time-resolved UV-visible absorption spectroscopy. The EPR experiments show that the equilibrium between S 2 LS and S 2 HS is pH dependent, with a pK a  ≈ 8.3 (n ≈ 4) for the native Mn 4 CaO 5 and pK a  ≈ 7.5 (n ≈ 1) for Mn 4 SrO 5 . The DFT results suggest that exchanging Ca with Sr modifies the electronic structure of several titratable groups within the active site, including groups that are not direct ligands to Ca/Sr, e.g., W1/W2, Asp61, His332 and His337. This is consistent with the complex modification of the pK a upon the Ca/Sr exchange. EPR also showed that NH 3 addition reversed the effect of high pH, NH 3 -S 2 LS being present at all pH values studied. Absorption spectroscopy indicates that NH 3 is no longer bound in the S 3 Tyr Z state, consistent with EPR data showing minor or no NH 3 -induced modification of S 3 and S 0 . In both Ca-PSII and Sr-PSII, S 2 HS was capable of advancing to S 3 at low temperature (198 K). This is an experimental demonstration that the S 2 LS is formed first and advances to S 3 via the S 2 HS state without detectable intermediates. We discuss the nature of the changes occurring in the S 2 LS to S 2 HS transition which allow the S 2 HS to S 3 transition to occur below 200 K. This work also provides a protocol for generating S 3 in concentrated samples without the need for saturating flashes., (Copyright © 2018 Elsevier B.V. All rights reserved.)