During the light-driven cycle of photosystem II (PSII), water oxidation proceeds through the Kok-cycle S-states at a Mn4CaO5 cluster. The last metastable state before O2 release, S3, exists in two experimentally resolved configurations in higher plants: a water-bound form with total spin S=3S=3 and a water-unbound high-spin form with S=6S=6 (arrested substrate binding). The unresolved problem is the mechanism of the final oxidation step in physiological turnover: which S3 configuration is the catalytically operative precursor of the S3→S4 transition, what structural and protonation rearrangements interconvert the two forms, whether the ensuing S4 intermediate is a Mn(IV)-oxyl radical or a five-coordinate Mn(V)-oxo species, and whether the O–O bond forms by oxyl–oxo radical coupling or by nucleophilic oxo attack. Later work (2021–2026) has constrained the problem — S3 is established as all-Mn(IV) with no oxyl or peroxo; room-temperature serial femtosecond crystallography has captured microsecond-to-millisecond structural intermediates of the S3→[S4]→S0 transition, interpreted as a possible bound peroxide but with contested assignments; and a microsecond-scale transient of possible peroxo nature has been detected by time-resolved X-ray emission spectroscopy — yet competing O–O-forming mechanisms are still being proposed as of 2026, the electronic structure of the S4 intermediate remains unsettled, and the productive precursor in higher-plant physiological turnover is unidentified.

Background

Photosystem II (PSII) is the membrane protein complex of oxygenic photosynthesis that uses light to strip electrons and protons from water, releasing the dioxygen that sustains aerobic life. Water oxidation chemistry happens at the oxygen-evolving complex (OEC), an inorganic Mn4CaO5 cluster shaped like a distorted chair, ligated by amino-acid residues. The OEC advances through a ladder of storage states S0–S4 (the Kok cycle): each absorbed photon oxidizes the cluster by one equivalent via the redox-active tyrosine Z (Yz), and after the fourth oxidation the cluster converts back to S0 while releasing O2. The states S0–S3 are metastable and experimentally observable; S4, one oxidizing equivalent above S3, is the fleeting transition state region in which the O–O bond is believed to form, and it has never been directly characterized.

A central, still-open mechanistic question is how exactly the O–O bond forms in the S3→S4→S0 sequence. Two families of proposals dominate: (i) oxyl–oxo radical coupling, in which an oxyl radical (an oxygen-centered radical, formally O•−, on manganese) couples with a nearby oxo bridge; and (ii) nucleophilic attack, in which a nucleophilic oxo/hydroxo oxygen attacks an electrophilic high-valent Mn(V)=O group. These imply different S4 electronic structures — a Mn(IV)-oxyl radical versus a five-coordinate Mn(V)-oxo — and different substrate-water identities.

An X- and Q-band electron paramagnetic resonance (EPR) study of spinach (Spinacia oleracea) PSII [1] detects paramagnetic centers and resolves the spin structure of the S3 state. Using methanol as a perturbation of substrate-water access, the study showed that the S3 state in higher plants is heterogeneous, containing two configurational components with different total spin quantum numbers SS (not to be confused with S-state labels): a previously known intermediate-spin (S=3S=3) form, assigned as a water-bound all-Mn(IV) six-coordinate oxo-hydroxo geometry, and a newly identified high-spin (S=6S=6) form, assigned as a water-unbound '3+1' geometry in which a coordinatively unsaturated five-coordinate Mn(IV) ion couples ferromagnetically to a Mn(IV)3 subunit through anisotropic exchange (direction-dependent magnetic coupling between metal centers, which was required to simulate the S=6S=6 EPR spectrum). Methanol was shown to arrest substrate-water binding without preventing manganese oxidation, implying that metal oxidation and water binding are separable events in the S2→S3 transition.

This heterogeneity matters mechanistically: the water-bound and water-unbound S3 configurations lead naturally to different O–O-forming routes (nucleophilic attack on a Mn(V)=O center versus oxyl–oxo coupling), so knowing which configuration is on the productive catalytic pathway — and what converts one into the other before the final oxidation — is prerequisite to a molecular mechanism of biological O2 formation. Subsequent work has sharpened the constraints: coupled-cluster and HERFD-XAS (high-energy-resolution-fluorescence-detected X-ray absorption spectroscopy) studies established that the active S3 configuration stores its oxidizing equivalent on manganese (all Mn(IV)), with no oxyl or peroxo present, pushing the onset of O–O chemistry to the final S3→S4 oxidation; density functional theory studies of the open/closed-cubane isomerism preceding O–O formation have been published; time-resolved Mn X-ray emission spectroscopy has detected an early microsecond-scale transient of possible peroxo character during the S3→S0 step; and room-temperature serial femtosecond X-ray crystallography has captured structural intermediates of the S3→[S4]→S0 transition — the extra oxygen Ox, inserted between calcium and Mn1 during S2→S3, disappears or relocates from about 700 microseconds after the third flash, and oxygen evolution sets in near 1,200 microseconds in what may be a bound peroxide intermediate — although these crystallographic state assignments are themselves contested. Nevertheless, as of 2026 competing O–O-forming mechanisms are still being proposed, and the operative S3 precursor, the electronic structure of S4, the coupling partners, and the quantitative factors governing the relative populations of the two S3 configurations remain experimentally unresolved.

Problem Statement

Establish the molecular mechanism by which the final, O–O-bond-forming step of water oxidation proceeds in photosystem II under physiological turnover, given the established two-configuration heterogeneity of the S3 state. Concretely: (1) determine whether the catalytically operative precursor of the S3→S4 oxidation is the water-bound (S=3S=3) or the water-unbound (S=6S=6) S3 configuration — or whether productive turnover proceeds through their interconversion; if it does, identify the sequence of water-binding, structural, and proton-relocation events that connects them before the final oxidation and quantify the associated energetic barriers; (2) determine the electronic nature of the S4 intermediate reached after the fourth oxidation (a Mn(IV)-oxyl radical versus a five-coordinate Mn(V)-oxo species, or another description); (3) determine the mechanism of O–O bond formation — oxyl–oxo radical coupling versus nucleophilic attack of an oxo/hydroxo on an electrophilic oxo — including the identity of the two oxygen atoms that couple; and (4) establish quantitatively which physiological variables (temperature, membrane and channel architecture, local protonation, ionic composition) control the relative populations of the two S3 configurations. These four required determinations jointly constitute one scientific objective — a validated, physiologically relevant mechanism of biological O–O bond formation.

References

  1. Zahariou G., Ioannidis N., Sanakis Y., Pantazis D. A. (2021). Arrested Substrate Binding Resolves Catalytic Intermediates in Higher-Plant Water Oxidation. Angewandte Chemie International Edition 60(6), 3156–3162. DOI: 10.1002/anie.202012304. https://doi.org/10.1002/anie.202012304
  2. Drosou M., Pantazis D. A. (2021). Redox Isomerism in the S3 State of the Oxygen-Evolving Complex Resolved by Coupled Cluster Theory. Chemistry – A European Journal 27(50), 12815–12825. DOI: 10.1002/chem.202101567. https://doi.org/10.1002/chem.202101567
  3. Guo Y., Messinger J., Kloo L., Sun L. (2022). Reversible Structural Isomerization of Nature's Water Oxidation Catalyst Prior to O–O Bond Formation. Journal of the American Chemical Society 144(26), 11736–11747. DOI: 10.1021/jacs.2c03528. https://doi.org/10.1021/jacs.2c03528
  4. Bhowmick A., Hussein R., Bogacz I., Simon P. S., Ibrahim M., Chatterjee R., Doyle M. D., Cheah M. H., Fransson T., Chernov P., Kim I.-S., Makita H., Dasgupta M., Kaminsky C. J., Zhang M., Gätcke J., Haupt S., Nangca I. I., Keable S. M., Aydin A. O., Tono K., Owada S., Gee L. B., Fuller F. D., Batyuk A., Alonso-Mori R., Holton J. M., Paley D. W., Moriarty N. W., Mamedov F., Adams P. D., Brewster A. S., Dobbek H., Sauter N. K., Bergmann U., Zouni A., Messinger J., Kern J., Yano J., Yachandra V. K. (2023). Structural evidence for intermediates during O2 formation in photosystem II. Nature 617(7961), 629–636. DOI: 10.1038/s41586-023-06038-z. https://doi.org/10.1038/s41586-023-06038-z
  5. Drosou M., Comas-Vilà G., Neese F., Salvador P., Pantazis D. A. (2023). Does Serial Femtosecond Crystallography Depict State-Specific Catalytic Intermediates of the Oxygen-Evolving Complex? Journal of the American Chemical Society 145(19), 10604–10621. DOI: 10.1021/jacs.3c00489. https://doi.org/10.1021/jacs.3c00489
  6. Chrysina M., Drosou M., Castillo R. G., Reus M., Neese F., Krewald V., Pantazis D. A., DeBeer S. (2023). Nature of S-States in the Oxygen-Evolving Complex Resolved by High-Energy Resolution Fluorescence Detected X-ray Absorption Spectroscopy. Journal of the American Chemical Society 145(47), 25579–25594. DOI: 10.1021/jacs.3c06046. https://doi.org/10.1021/jacs.3c06046
  7. Guo Y., Messinger J., Kloo L., Sun L. (2023). Alternative Mechanism for O2 Formation in Natural Photosynthesis via Nucleophilic Oxo–Oxo Coupling. Journal of the American Chemical Society 145(7), 4129–4141. DOI: 10.1021/jacs.2c12174. https://doi.org/10.1021/jacs.2c12174
  8. Song Y.-T., Li X.-C., Siegbahn P. E. M. (2023). Is There a Different Mechanism for Water Oxidation in Higher Plants? The Journal of Physical Chemistry B 127(30), 6643–6647. DOI: 10.1021/acs.jpcb.3c03029. https://doi.org/10.1021/acs.jpcb.3c03029
  9. Pavlou A., Styring S., Mamedov F. (2024). The S1 to S2 and S2 to S3 state transitions in plant photosystem II: relevance to the functional and structural heterogeneity of the water oxidizing complex. Photosynthesis Research 162(2–3), 401–411. DOI: 10.1007/s11120-024-01096-4. https://doi.org/10.1007/s11120-024-01096-4
  10. Zahariou G. (2025). Charge separation events in photosystem II during the S2 to S3 transition as revealed by EPR spectroscopy. FEBS Letters 600(4), 493–503. DOI: 10.1002/1873-3468.70229. https://doi.org/10.1002/1873-3468.70229
  11. Jensen S., Sullivan B. T., Hartzler D. A., Kosheleva I., Henning R. W., Page A., Pushkar Y. (2025). Challenges of observing O–O bond formation in the Mn4Ca cluster of photosystem II. Chem 11(7), 102448. DOI: 10.1016/j.chempr.2025.102448. https://doi.org/10.1016/j.chempr.2025.102448
  12. Flesher D. A., Debus R. J., Brudvig G. W. (2025). Structure of the D1-Val185Asn mutated photosystem II complex with slow O–O bond formation reveals changes in the Cl1 water channel. Proceedings of the National Academy of Sciences 122(47), e2522652122. DOI: 10.1073/pnas.2522652122. https://doi.org/10.1073/pnas.2522652122
  13. Ariafard A., Longhurst M., Swiegers G. F., Stranger R. (2025). The Low Oxidation State Paradigm is More Consistent with XFEL Observations of the S3 → [S4] → S0 Transition in Photosystem II. Chemistry – A European Journal 31(38), e202501010. DOI: 10.1002/chem.202501010. https://doi.org/10.1002/chem.202501010
  14. Pushkar Y. (2026). New Water Oxidation Mechanism in Photosystem II Resolves Major Experimental Controversies. Angewandte Chemie International Edition 65(35), e1930324. DOI: 10.1002/anie.1930324. https://doi.org/10.1002/anie.1930324