During water oxidation in photosystem II (PSII), the light-driven oxidation steps of the Mn4CaO5 oxygen-evolving complex are coupled to proton release to the lumenal bulk — on average one proton per electron transferred, with the accepted per-transition release stoichiometry at functional pH being 1, 0, 1, and 2 protons for S0→_0\toS1_1, S1→_1\toS2_2, S2→_2\toS3_3, and S3→_3\to[S4_4]→\toS0_0 — but the exit routes and their gating are not settled. Three water-filled channels — O1, O4, and Cl1 — connect the cluster to the protein surface. A QM/MM study (Allgöwer et al., 2024) proposed that oxidation of the redox-active tyrosine Yz lowers the barrier for proton transfer from the putative substrate water W3/Wx to D2-Glu310 along the Cl1 channel, with D1-Glu65 acting as a gate; competing proposals assign early deprotonation to the O4 water chain, other proton donors, or other channels. The open problem, at its natural generality: for each S-state transition of the Kok cycle, identify the deprotonating donor(s), the proton-release route(s) and gating residues, and the coupling to electron transfer, without presuming any particular channel or substrate assignment.

Background

Photosystem II (PSII) is the membrane-protein complex of oxygenic photosynthesis that splits water: in each catalytic turnover it removes four electrons and four protons from two water molecules and releases one molecule of O2_2, supplying the electrons (and the proton gradient) that power the rest of photosynthesis. The catalyst is the oxygen-evolving complex (OEC), an oxo-bridged inorganic cluster, Mn4_4CaO5_5, buried roughly 15–20 Å below the lumenal (thylakoid-interior) protein surface. Light-driven charge separation creates the strong oxidant P680+^+ at the reaction center; P680+^+ is re-reduced by the redox-active tyrosine D1-Tyr161, called Yz (or TyrZ), which in its oxidized radical form Yz∙^\bullet oxidizes the OEC one equivalent at a time. The cluster thus steps through the oxidation states S0_0–S4_4 of the Kok cycle — the 'clock' of water oxidation, with O2_2 released on the S3→_3\to[S4_4]→\toS0_0 transition. Because each oxidation would otherwise accumulate positive charge on the cluster, oxidation is coupled to proton release (on average one proton per electron transferred, with the per-transition release pattern depending on pH and protein buffering: at functional pH the accepted stoichiometry is 1 for S0→_0\toS1_1, 0 for S1→_1\toS2_2, 1 for S2→_2\toS3_3, and 2 for the combined S3→_3\to[S4_4]→\toS0_0 transition, i.e., no net release to the bulk on S1→_1\toS2_2 and two sequential protons on the O2_2-releasing step), and this proton-coupled electron transfer (PCET) — the joint movement of an electron from the cluster to Yz/P680 and a proton from the cluster to the bulk — largely sets the kinetics of the cycle.

To leave the protein, protons must travel through one of the water-filled channels that connect the OEC to the lumenal bulk. Structural and simulation studies describe three: the O1 channel (also called the large channel), running from the O1 oxo position of the cluster past D1-Asn298 outward, generally assigned to substrate-water intake; the O4 channel (narrow channel), leading from the O4 μ-oxo bridge through a chain of strongly hydrogen-bonded water molecules; and the Cl1 channel (broad channel), passing the chloride-binding site Cl1 near the D1-Asp61/D2-Lys317 ion pair and a cluster of conserved carboxylates (D1-Glu65, D2-Glu310, D2-Glu312, and PsbO-Asp224) toward the lumen. (Some authors use an older large/narrow/broad channel nomenclature that maps onto these three.)

Key prior results define the current frontier. Saito, Rutherford and Ishikita (2015) showed by quantum mechanics/molecular mechanics (QM/MM) calculations that in the S1_1 state the O4 μ-oxo bridge can release a proton into a chain of eight strongly hydrogen-bonded waters, making the O4 channel a candidate for the initial deprotonation. Time-resolved serial femtosecond X-ray crystallography (XFEL) structures of the S2→_2\toS3_3 transition (Hussein et al., 2021) found structural rearrangements consistent with water intake via O1 and proton release via Cl1, with D1-Glu65 suggested as a gate; time-resolved infrared experiments on the D1-N298A mutant and on nitrate-for-chloride substitution (Okamoto et al., 2021) reached the same O1-water/Cl1-proton assignment for that transition, and mutagenesis with pH-dependent oxygen-evolution and kinetic solvent isotope measurements (Ghosh et al., 2019) implicated D1-Asp61 and D2-Lys317 in proton transfer. XFEL data on the S3→_3\to[S4_4]→\toS0_0 transition (Bhowmick et al., 2023) revealed 'controlled proton release through the hydrogen-bonding network of the Cl1 channel' and an extra oxygen (Ox/O6) inserted between Ca and Mn1 during S2→_2\toS3_3, whose substrate status is itself debated. On the theory side, Allgöwer et al. (2024) combined microsecond molecular dynamics with QM/MM free-energy simulations and found that the Cl1 channel supports a water-mediated, carboxylate-chain proton pathway from the cluster to the bulk, that oxidation of Yz strengthens an electric field along this pathway and lowers the transfer barriers by a few kcal/mol, that D1-Glu65 can act as a local molecular gate, and that D2-Glu312 can store a proton. A subsequent multiscale study (Liu et al., 2026) reconciled conflicting kinetic interpretations of the S2→_2\toS3_3 transition and again favored Yz-driven deprotonation via Cl1 (through the D1-Glu65/D2-Glu312 pair, with W2 or W1 as the donor), while explicitly noting that the underlying coupling mechanism remains unresolved; time-resolved infrared work on the O2_2-evolving S3→_3\toS0_0 transition (Sugie et al., 2025) proposes yet another donor — proton release from W1 promoted by Yz oxidation via the Cl1 channel — so even among studies favoring Cl1 the donor identity is contested. Cryo-EM structures of channel-perturbing mutants (Flesher et al., 2025, on D2-Lys317Ala and D1-Val185Asn) show that disturbing the Cl1 hydrogen-bond/water network slows O–O bond formation and alters proton egress. Nevertheless, network analyses (Kaur et al., 2021) conclude that the role of each pathway is not yet established, and 2025 reviews (Ishikita and Saito) state that the deprotonation pathways and the protonation states of the cluster ligands remain open questions, with the additional caveat that efficient proton transfer requires pre-formed hydrogen-bond networks that are absent near the O5/O6 region emphasized in some substrate-assignment proposals.

Problem Statement

Determine the proton-release mechanism of PSII water oxidation across the full Kok cycle. For each S-state transition — S0→_0\toS1_1, S1→_1\toS2_2, S2→_2\toS3_3, and S3→_3\to[S4_4]→\toS0_0 — establish:

(i) which oxygen ligand or water of the Mn4_4CaO5_5 cluster donates each proton released on that transition (for example W1, W2, or W3, a μ-oxo bridge, or the S3_3 'extra' water/oxygen Wx/O6); where the established stoichiometry gives two sequential releases (S3→_3\to[S4_4]→\toS0_0 at functional pH), identify the donor of each proton, and where it gives no net release to the bulk (S1→_1\toS2_2 at functional pH), account for any internal proton rearrangement instead;

(ii) along which channel or channels (O1, O4, Cl1, or another route) each proton exits to the lumenal bulk, and which residues and cofactors (for example D1-Asp61, D1-Glu65, D2-Glu310, D2-Glu312, D2-Lys317, D1-Asn298, the Cl1 chloride site) gate, store, or relay it;

(iii) how proton release is energetically and temporally coupled to electron transfer from the cluster via Yz (D1-Tyr161) to P680+^+ — whether Yz oxidation gates proton release, whether proton release precedes or follows Yz oxidation, whether the two are concerted, or whether they are uncoupled — giving the evidence for the coupling mode of each transition.

A complete answer must be consistent with the established proton-release stoichiometry (per-transition release of 1, 0, 1, and 2 protons at functional pH, acknowledging its dependence on pH and protein buffering); with the measured S-state kinetics and H/D kinetic isotope effects, addressing — rather than silently presupposing one side of — the conflicting time constants reported by different techniques; with the effects of channel-perturbing mutations (D1-D61A, D2-K317A, D1-N298A, D1-V185N and analogous substitutions) and chloride/nitrate substitution; and with the available time-resolved structural data, stating which interpretation of contested features (for example the substrate status of the S3_3 extra oxygen O6/Ox) it adopts and why. The problem must be answered without presuming any particular channel, donor, or substrate-water assignment for any transition; if different transitions — or the sequential protons within one transition — use different routes or donors, the answer must say so and give the evidence for each. Each of (i)–(iii) must be answered for every transition — and, where the stoichiometry gives two sequential protons, for each proton — and a resolution is a per-transition assignment of donor, exit route and gating residues supported by cited evidence and consistent with the constraints above; quantitative barriers and timescales are required only where the literature reports them.

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