In oxygenic photosynthesis, the final step of water oxidation — formation of the O–O bond and release of O2O_2 — occurs during the S3→[S4]→S0S_3\to[S_4]\to S_0 transition of the Mn4CaO5 oxygen-evolving complex (OEC) of photosystem II. Room-temperature serial femtosecond X-ray crystallography (Bhowmick et al., Nature 2023) filmed this transition and inferred a transient reduced intermediate, plausibly a bound peroxide, existing between roughly 700 and 1,200 μ\mus after the third flash, but could not determine its chemical identity, decide which two oxygen atoms form the O–O bond, or assign protonation states. The 2023–2026 follow-up literature shows: substrate-water exchange experiments now identify the central oxo bridge O5 as the slowly exchanging substrate water, an assignment that dissenting proposals contest; anomalous-diffraction structures at ~1.9 Å confirm the extra oxygen OxO_x/O6 ligated between Mn1 and Ca in S3S_3 but show no O5–Ox interaction in that state; time-resolved Mn Kα emission and infrared spectroscopy constrain the redox and proton-release sequence; yet the chemical identity and structure of the intermediate, the second (fast-exchanging) substrate oxygen, and the protonation states of the OEC ligands remain experimentally undetermined, and competing mechanistic proposals (O5–Ox, O5–W2/W3, O3–O6) are all still live. The problem — a chemically complete, experimentally grounded mechanism of the O2O_2-forming step — remains open.

Background

Photosystem II (PSII) is the membrane-protein complex that uses light to oxidize water, releasing the molecular oxygen that sustains aerobic life. Its catalytic core, the oxygen-evolving complex (OEC), is a cluster of four manganese ions and one calcium ion bridged by five oxygen atoms (Mn4CaO5\mathrm{Mn_4CaO_5}), ligated by amino-acid residues of the D1 and CP43 subunits. Water oxidation proceeds through the Kok cycle: a clock of storage states S0S_0–S3S_3 (plus the transient S4S_4) in which each of four successive photon-driven charge separations, mediated by the redox-active tyrosine YZY_Z (D1-Tyr161), removes one electron from the cluster, coupled to proton release, until the fourth oxidation triggers O–O bond formation, O2O_2 release, and reset to S0S_0. The cluster's oxygen ligands are conventionally labeled O1–O5 (O5 being the central oxygen bridge of the cluster) plus terminal waters W1–W4 (W1 and W2 on Mn4, W3 and W4 on Ca). Room-temperature serial femtosecond X-ray crystallography (SFX) at X-ray free-electron lasers (XFELs) — diffraction from thousands of microcrystals, each shot before radiation damage accumulates, with visible-laser pump flashes to advance the S-state clock — has produced time-resolved structures of the catalytic cycle. Kern et al. (Nature 563:421–425, 2018) established that an extra oxygen atom, OxO_x (also called O6), inserts as a bridge between Mn1 and Ca during the S2→S3S_2\to S_3 transition. Suga et al. (Nature 543:131–135, 2017) reported cryogenic XFEL structures whose short O5–Ox distance was read by some as evidence of an O–O (peroxide) species already in S3S_3; that reading remains contested. Bhowmick et al. (Nature 617:629–636, 2023) then filmed the entire S3→[S4]→S0S_3\to[S_4]\to S_0 transition at room temperature with microsecond-to-millisecond delays, finding that OxO_x disappears or relocates in parallel with YZY_Z reduction starting near 700 μ\mus, and that the onset of O2O_2 evolution (reported by shortening of the Mn1–Mn4 distance) occurs around 1,200 μ\mus, implying a reduced intermediate — possibly a bound peroxide — between the transient S4S_4 state and S0S_0. Complementary probes constrain the same step: substrate-water exchange kinetics measured by time-resolved membrane-inlet mass spectrometry (TR-MIMS, following H218_2^{18}O incorporation into the evolved O2O_2) resolve a fast (WfW_f) and a slow (WsW_s) exchanging substrate water; high-energy-resolution fluorescence-detected X-ray absorption spectroscopy (HERFD-XAS) resolves the Mn oxidation-state structure of each S state; time-resolved Mn Kα X-ray emission (TR-XES) and infrared (TR-IR) spectroscopy track redox and proton-release events, and 17O electron-electron double-resonance-detected NMR (17O-EDNMR) observes exchangeable oxygen ligands directly, while electron paramagnetic resonance (EPR) reports the cluster's spin states. Electron and proton transfer to and from the cluster proceed through defined channels, notably the Cl1 channel near D1-Val185 and the D1-Glu65/D2-Glu312 proton gate. Despite this arsenal, the chemical mechanism of the O2O_2-forming step — what exactly the intermediate is, which two oxygens couple, and in what protonation states — is still not determined.

Problem Statement

Determine the chemical mechanism of O–O bond formation and O2O_2 release during the S3→[S4]→S0S_3\to[S_4]\to S_0 transition of the Mn4CaO5 oxygen-evolving complex of photosystem II — the last and least-understood step of the Kok cycle — to a level where the following jointly constitute one coherent, experimentally grounded mechanism: (1) the atomic identity, geometry, bonding mode, and electronic structure of the transient reduced intermediate (inferred between roughly 700 and 1,200 μ\mus after the third flash, within the 500–1,200 μ\mus window in which the source brackets O–O bond formation itself, plausibly a bound peroxide-like species); (2) which two oxygen atoms of the cluster and its waters form the O–O bond — the O5–OxO_x pairing currently favored, versus O5 with the terminal waters W2 or W3, versus mechanisms not involving O5 (or, per recent dissenting proposals, couplings involving O3) — where O5 is the cluster's central oxygen bridge, OxO_x (also called O6) is the extra oxygen inserted between Mn1 and Ca in S3S_3, and O3 is an oxo bridge of the cubane core; (3) the protonation states of the OEC oxygen ligands (including whether O5 is hydroxide or oxide at the relevant moments) and of the channel waters and residues throughout the transition, and their coupling to the observed sequence of proton release and electron transfer; and (4) the fate of the extra oxygen OxO_x during the transition — physical relocation versus chemical transformation, i.e., whether it is a substrate, a refill for the O5 site, or a spectator. A complete answer must be consistent with the time-resolved structural data and with the spectroscopic and isotopic-exchange observables (substrate-water exchange kinetics of the fast and slow substrates, HERFD-XAS and TR-XES redox markers, TR-IR proton-release kinetics, EPR spin states), and must hold across PSII preparations, species, and conditions to the extent tested. A submission resolves the problem only if it commits to one definite mechanism across all four components and adjudicates the competing proposals named in (2), citing the listed observables where they discriminate and otherwise the observation that would; a mechanism that is merely compatible with the observables does not suffice. These components are scientifically inseparable: the identity of the intermediate, the substrate pair, and the protonation choreography are three views of a single bond-forming event, and a mechanism that resolves one while leaving the others undetermined would not close the problem.

References

  1. Bhowmick A, Hussein R, Bogacz I, Simon PS, Ibrahim M, Chatterjee R, Doyle MD, Cheah MH, Fransson T, Chernev P, Kim IS, Makita H, Dasgupta M, Kaminsky CJ, Zhang M, Gätcke J, Haupt S, Nangca II, Keable SM, Aydin AO, Tono K, Owada S, Gee LB, Fuller FD, Batyuk A, Alonso-Mori R, Holton JM, Paley DW, Moriarty NW, Mamedov F, Adams PD, Brewster AS, Dobbek H, Sauter NK, Bergmann U, Zouni A, Messinger J, Kern J, Yano J, Yachandra VK (2023). Structural evidence for intermediates during O2 formation in photosystem II. Nature 617:629–636. doi:10.1038/s41586-023-06038-z, https://doi.org/10.1038/s41586-023-06038-z
  2. de Lichtenberg C, Rapatskiy L, Reus M, Heyno E, Schnegg A, Nowaczyk MM, Lubitz W, Messinger J, Cox N (2024). Assignment of the slowly exchanging substrate water of nature's water-splitting cofactor. Proceedings of the National Academy of Sciences 121(11):e2319374121. doi:10.1073/pnas.2319374121, https://doi.org/10.1073/pnas.2319374121
  3. Chrysina M, Drosou M, Castillo RG, Reus M, Neese F, Krewald V, Pantazis DA, 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
  4. Maximova O, Bhowmick S, Tuncer H, Bury G, Henning R, Kosheleva I, Kim IS, Mohammadi O, Levantino M, Cammarata M, Pushkar Y (2026). Time-resolved Mn Kα emission reveals early redox dynamics in the S3 to S0 transition of the photosystem II Kok cycle. Journal of Biological Chemistry 302(3):111215. doi:10.1016/j.jbc.2026.111215, https://doi.org/10.1016/j.jbc.2026.111215
  5. Ariafard A, Longhurst M, Swiegers GF, Stranger R (2025). XFEL-Detected Peroxide Formation From the S3 State Supports the Low Oxidation State over the High Oxidation State Paradigm in Photosystem II. Chemistry – A European Journal 32(2):e03024. doi:10.1002/chem.202503024, https://doi.org/10.1002/chem.202503024
  6. Chernev P, Aydin AO, Messinger J (2024). On the simulation and interpretation of substrate-water exchange experiments in photosynthetic water oxidation. Photosynthesis Research 162(2–3):413–426. doi:10.1007/s11120-024-01084-8, https://doi.org/10.1007/s11120-024-01084-8
  7. Yano J, Kern J, Yachandra VK (2024). Structure Function Studies of Photosystem II Using X-Ray Free Electron Lasers. Annual Review of Biophysics 53:343–365. doi:10.1146/annurev-biophys-071723-102519, https://doi.org/10.1146/annurev-biophys-071723-102519
  8. Murray JW (2026). Recent advances in the structural biology of photosystem II. The Plant Journal 126(4):e70925. doi:10.1111/tpj.70925, https://doi.org/10.1111/tpj.70925
  9. Ishikita H, Saito K (2025). Current perspectives on proton and electron transfer pathways in photosystem II water oxidation. Plant and Cell Physiology 66(11):1666–1676. doi:10.1093/pcp/pcaf107, https://doi.org/10.1093/pcp/pcaf107
  10. Ishikita H, Saito K (2025). Photosystem II: Probing Protons and Breaking Barriers. Biochemistry 64(9):1895–1906. doi:10.1021/acs.biochem.5c00112, https://doi.org/10.1021/acs.biochem.5c00112
  11. Bhowmick A, Zhang M, Simon PS, Makita H, Nangca II, Szilagyi E, Kretzschmar M, Doyle MD, Minnetian NM, Hussein R, Hart O, Chatterjee K, Aydin AO, Shevela D, Cheah MH, Croy N, Chernev P, Fransson T, Tiwari V, Sanchez H, Schleissner P, Lemons R, Gate G, Henstridge M, Glownia JM, Poitevin F, Rosenberg DJ, Dehe S, Gee LB, Tono K, Owada S, Oggenfuss R, Ozerov D, Sander M, Mankowsky R, Lemke HT, Young ID, Holton JM, Mittan-Moreau DW, Paley DW, Afonine PV, Moriarty NW, Adams PD, Mamedov F, Dobbek H, Zouni A, Alonso-Mori R, Bergmann U, Brewster AS, Sauter NK, Messinger J, Kern JF, Yachandra VK, Yano J (2026). An additional water is introduced into the manganese cluster during the formation of the S3 state of photosystem II. Nature Communications 17:8818. doi:10.1038/s41467-026-76805-9, https://doi.org/10.1038/s41467-026-76805-9
  12. Flesher DA, Debus RJ, Brudvig GW (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. Sugie K, Kato Y, Nagao R, Noguchi T (2025). Mechanism of the Oxygen-Evolving Process in the Water-Oxidizing Complex of Photosystem II, as Revealed by Time-Resolved Infrared Spectroscopy. Journal of Physical Chemistry B 129(25):6172–6187. doi:10.1021/acs.jpcb.5c02806, https://doi.org/10.1021/acs.jpcb.5c02806
  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
  15. Greife P, Schönborn M, Capone M, Assunção R, Narzi D, Guidoni L, Dau H (2023). The electron–proton bottleneck of photosynthetic oxygen evolution. Nature 617:623–628. doi:10.1038/s41586-023-06008-5, https://doi.org/10.1038/s41586-023-06008-5
  16. Mäusle SM, Parisse G, Assunção R, De Santis C, Simon PS, Narzi D, Guidoni L, Debus RJ, Dau H (2025). Three rate-determining protein roles in photosynthetic O2-evolution addressed by time-resolved experiments on genetically modified photosystems. Nature Communications 16:9515. doi:10.1038/s41467-025-64513-9, https://doi.org/10.1038/s41467-025-64513-9
  17. Kern J, Chatterjee R, Young ID, Fuller FD, Lassalle L, Ibrahim M, Gul S, Fransson T, Brewster AS, Alonso-Mori R, Hussein R, Zhang M, Douthit L, de Lichtenberg C, Cheah MH, Shevela D, Wersig J, Seuffert I, Sokaras D, Pastor E, Weninger C, Kroll T, Sierra RG, Aller P, Butryn A, Orville AM, Liang M, Batyuk A, Koglin JE, Carbajo S, Boutet S, Moriarty NW, Holton JM, Dobbek H, Adams PD, Bergmann U, Sauter NK, Zouni A, Messinger J, Yano J, Yachandra VK (2018). Structures of the intermediates of Kok's photosynthetic water oxidation clock. Nature 563:421–425. doi:10.1038/s41586-018-0681-2, https://doi.org/10.1038/s41586-018-0681-2
  18. Suga M, Akita F, Sugahara M, Kubo M, Nakajima Y, Nakane T, Yamashita K, Umena Y, Nakabayashi M, Yamane T, Nakano T, Suzuki M, Masuda T, Inoue S, Kimura T, Nomura T, Yonekura S, Yu LJ, Sakamoto T, Motomura T, Chen JH, Kato Y, Noguchi T, Tono K, Joti Y, Kameshima T, Hatsui T, Nango E, Tanaka R, Naitow H, Matsuura Y, Yamashita A, Yamamoto M, Nureki O, Yabashi M, Ishikawa T, Iwata S, Shen JR (2017). Light-induced structural changes and the site of O=O bond formation in PSII caught by XFEL. Nature 543:131–135. doi:10.1038/nature21400, https://doi.org/10.1038/nature21400
  19. Author Correction: Bhowmick A, Hussein R, Bogacz I, Simon PS, Ibrahim M, Chatterjee R, Doyle MD, Cheah MH, Fransson T, Chernev P, Kim IS, Makita H, Dasgupta M, Kaminsky CJ, Zhang M, Gätcke J, Haupt S, Nangca II, Keable SM, Aydin AO, Tono K, Owada S, Gee LB, Fuller FD, Batyuk A, Alonso-Mori R, Holton JM, Paley DW, Moriarty NW, Mamedov F, Adams PD, Brewster AS, Dobbek H, Sauter NK, Bergmann U, Zouni A, Messinger J, Kern J, Yano J, Yachandra VK (2024). Structural evidence for intermediates during O2 formation in photosystem II. Nature 626:E12. doi:10.1038/s41586-024-07099-4, https://doi.org/10.1038/s41586-024-07099-4