Electronic structure of the $S_2$ state of the photosystem II oxygen-evolving complex: reconciling manganese oxidation-state assignments with the complete spectroscopic fingerprint

The $S_2$ state of the oxygen-evolving complex (OEC), the Mn$_4$CaO$_5$ cluster that catalyzes photosynthetic water oxidation in photosystem II, remains the most intensively studied intermediate of the Kok cycle, yet a single electronic-structural model that quantitatively reproduces its complete spectroscopic fingerprint does not exist. A 2018 computational study of low-oxidation-paradigm (LOP) models found that Mn(II)-containing formulations fit the geometric constraints but fail the ligand ($^{17}$O, $^{14}$N, $^1$H) hyperfine data, reproduce only part of the $^{55}$Mn ENDOR pattern, and cannot explain how the $S=1/2$ multiline and $S=5/2$ $g\approx 4.1$ EPR signals could arise from the same oxidation pattern if Mn(II) were present. Later experiments — flash-counting during photoassembly, Mn K$\alpha$ X-ray emission spectroscopy, and high-energy-resolution fluorescence-detected X-ray absorption spectroscopy — strongly favor the high-oxidation assignment Mn(III)Mn(IV)$_3$ for $S_2$, but the interpretation of the X-ray free-electron laser structures remains contested, a minority low-oxidation program is still active through 2025–2026, and the source paper's specific unresolved items (the two anomalously small $^{55}$Mn ENDOR couplings near 45 MHz and 7 MHz, the untested ENDOR-invisibility hypothesis, and EPR-parameter computation with explicit zero-field splitting and spin-orbit coupling) have not been taken up by any published study. The problem posed here is to determine the oxidation, protonation, and spin-coupling assignment of the $S_2$ state that quantitatively reproduces the full fingerprint, and thereby to adjudicate the residual low-oxidation claims.

Background

In oxygenic photosynthesis, photosystem II (PSII) oxidizes water to dioxygen at the oxygen-evolving complex (OEC), an inorganic Mn$_4$CaO$_5$ cluster ligated by amino acid residues and waters. The cluster advances through the five oxidation states $S_0$–$S_4$ of the Kok cycle, storing oxidizing equivalents until O$_2$ is released on the $S_4\to S_0$ transition. The one-electron-oxidized $S_2$ state is the best characterized intermediate and exhibits two EPR (electron paramagnetic resonance) signals: a multiline signal (MLS) of about 16–19 partially resolved $^{55}$Mn hyperfine lines centered at $g\approx 2$, assigned to a total spin $S=1/2$ ground state, and a broad signal at $g\approx 4.1$ assigned to an $S=5/2$ state; the two signals interconvert under near-infrared illumination, temperature, and chemical perturbations, indicating two forms of the same oxidation level in equilibrium.

Hyperfine couplings — the magnetic interactions between the electron spin and nearby nuclear spins — are the most site-specific observables available. Electron nuclear double resonance (ENDOR) resolves them for individual nuclei: the four $^{55}$Mn ions, the $^{14}$N of the histidine ligand His332, $^{17}$O-labeled oxo bridges and substrate waters, and exchangeable protons. The isotropic component $A_{iso}$ (Fermi contact) and the anisotropic dipolar part together constrain the oxidation state and spin density at each metal and ligand site.

Two competing paradigms assign the manganese oxidation states. The high-oxidation paradigm (HOP) assigns $S_2$ as one Mn(III) plus three Mn(IV) ions, and explains the two EPR signals by two interconvertible geometric isomers of this same oxidation level — the closed-cubane and open-cubane forms, differing in whether the dangling Mn approaches the cubane oxo O5 (Pantazis et al., 2012). The low-oxidation paradigm (LOP) posits two additional electrons on the cluster, lowering the mean Mn oxidation to 3.25 instead of 3.75 (for example the Mn(II)-free Mn(III)$_3$Mn(IV) or the Mn(II)-containing Mn(II)Mn(III)Mn(IV)$_2$); this program is grounded in computational chemistry and magnetic resonance analysis (Terrett et al., 2016; Jin et al., 2014). The $^{55}$Mn Davies ENDOR (a pulsed ENDOR sequence) study at 2.5 K by Jin et al. (2014) reported a hyperfine pattern including two anomalously small couplings (approximately 45 MHz and 7 MHz) alongside larger couplings — a feature that any candidate electronic structure must explain.

Chen, Case, and Dismukes (2018) performed the most detailed computational test of LOP $S_2$ models, using broken-symmetry density functional theory (BS-DFT — DFT solutions with spin-localized, antiferromagnetically coupled metal centers) combined with spin-projection techniques to compute EPR and ENDOR parameters. They found LOP geometries consistent with X-ray diffraction and EXAFS (extended X-ray absorption fine structure) at accessible energies, but severe spectroscopic failures: all LOP models gave overly large $^{17}$O $A_{iso}$ for the oxo bridges O5/O4 (up to $-221.4$ MHz) and large $^{14}$N $A_{iso}$ for His332 ($-10.4$ to $-17.4$ MHz versus the experimental $|6.95|$ MHz); the Mn(II)-containing isomer reproduced the large and intermediate $^{55}$Mn couplings but not the two small ones, which the authors speculated might arise from a Mn(IV) ion rendered ENDOR-invisible at 2.5 K by very slow nuclear relaxation — an untested hypothesis. The computed hyperfine projections relied on the strong-exchange limit, in which the cluster's local spins are rigidly projected onto the total spin; this neglects the zero-field splitting (ZFS, the splitting of a spin multiplet in zero field caused by spin-orbit coupling and anisotropic exchange) and is invalid when exchange couplings $J$ are small ($|J|<75$ cm$^{-1}$). A large empirical scaling factor (1.53) applied to the Mn isotropic hyperfine couplings added systematic uncertainty. The paper thus left open whether any low-oxidation formulation can be reconciled with the complete spectroscopic fingerprint, and what electronic structure actually produces it.

After 2018, three independent experimental lines bore directly on the oxidation inventory: flash-counting during photoactivation (light-driven assembly of the cluster from Mn$^{2+}$) required nine flashes for the first O$_2$, supporting the HOP inventory (Cheah et al., 2020); room-temperature Mn K$\alpha$ X-ray emission spectroscopy showed Mn-centered oxidation in the observable S-state transitions up to $S_3$, excluding Mn reduction at the cluster (Schuth et al., 2018); and high-energy-resolution fluorescence-detected X-ray absorption spectroscopy of all S-states established Mn-centered storage of oxidative equivalents in every observable transition (Chrysina et al., 2023). Meanwhile, however, the interpretation of the X-ray free-electron laser (XFEL) crystallographic models nominally depicting $S_2$ was shown to be problematic (Drosou et al., 2023), and the low-oxidation program continues to publish supportive analyses through 2025–2026 (Ariafard et al., 2026; Gates et al., 2024). Within the HOP consensus, the structure and protonation of the $S=5/2$ isomer and the protonation state behind the MLS continue to be actively refined (Corry and O'Malley, 2019; Taguchi et al., 2020; Kosaki and Mino, 2023; Saito et al., 2023; Flesher et al., 2022; Lampert et al., 2026), and synthetic $[\mathrm{Mn}^{III}\mathrm{Mn}^{IV}_3\mathrm{O}_4]$ cubanes reproduce both signal types (Shiau et al., 2023). What no study has delivered is a single model that quantitatively reproduces the complete fingerprint — both EPR signals, the full $^{55}$Mn tensor set including the small couplings, and the ligand hyperfine data — under an explicit ZFS/spin-orbit treatment, which is the problem stated here.

Problem Statement

Determine the electronic structure of the $S_2$ state of the photosystem II oxygen-evolving complex — its manganese oxidation-state pattern, ligand and water protonation pattern, and exchange-coupling scheme — that quantitatively reproduces, to within the experimental uncertainty of the cited EPR and ENDOR values, the complete spectroscopic fingerprint of this state within a single model. Specifically, the model must account for: (i) the simultaneous presence and interconversion of the $S=1/2$ multiline EPR signal and the $S=5/2$ $g\approx 4.1$ signal arising from the same oxidation pattern; (ii) the full set of $^{55}$Mn hyperfine couplings measured by ENDOR, including the two anomalously small couplings of approximately 45 MHz and 7 MHz reported in the 2.5 K Davies ENDOR study; and (iii) the ligand hyperfine couplings, namely the $^{14}$N coupling of His332, the $^{17}$O couplings of the oxo bridges and substrate waters, and the exchangeable $^1$H couplings — with zero-field splitting and spin-orbit coupling treated explicitly, beyond the strong-exchange limit, wherever exchange couplings are small. Resolving the statement requires both the fingerprint-reproducing model and the low-oxidation adjudication: establish whether any Mn(II)-containing low-oxidation formulation of $S_2$ in the mean-oxidation-3.25 class defined in the Background (including via the proposed mechanism in which a Mn(IV) ion becomes ENDOR-invisible at 2.5 K through very slow nuclear relaxation) can be reconciled with this complete fingerprint, or demonstrate that the fingerprint requires the Mn(III)Mn(IV)$_3$ assignment with its two spin isomers, thereby settling the residual low-oxidation claims against the X-ray-structural and electron-counting evidence on which they rest.

References

1. He Chen, David A. Case, G. Charles Dismukes, 'Reconciling Structural and Spectroscopic Fingerprints of the Oxygen-Evolving Complex of Photosystem II: A Computational Study of the S2 State', The Journal of Physical Chemistry B 122 (2018) 11868–11882. DOI: 10.1021/acs.jpcb.8b08147. https://doi.org/10.1021/acs.jpcb.8b08147
2. Lu Jin, Paul Smith, Christopher J. Noble, Rob Stranger, Graeme R. Hanson, Ron J. Pace, 'Electronic structure of the oxygen evolving complex in photosystem II, as revealed by 55Mn Davies ENDOR studies at 2.5 K', Physical Chemistry Chemical Physics 16 (2014) 7799–7812. DOI: 10.1039/c3cp55189j. https://doi.org/10.1039/c3cp55189j
3. Dimitrios A. Pantazis, William Ames, Nicholas Cox, Wolfgang Lubitz, Frank Neese, 'Two Interconvertible Structures that Explain the Spectroscopic Properties of the Oxygen-Evolving Complex of Photosystem II in the S2 State', Angewandte Chemie International Edition 51 (2012) 9935–9940. DOI: 10.1002/anie.201204705. https://doi.org/10.1002/anie.201204705
4. Richard Terrett, Simon Petrie, Rob Stranger, Ron J. Pace, 'What computational chemistry and magnetic resonance reveal concerning the oxygen evolving centre in Photosystem II', Journal of Inorganic Biochemistry 162 (2016) 178–189. DOI: 10.1016/j.jinorgbio.2016.04.009. https://doi.org/10.1016/j.jinorgbio.2016.04.009
5. Nils Schuth, Ivelina Zaharieva, Petko Chernev, Gustav Berggren, Magnus Anderlund, Stenbjörn Styring, Holger Dau, Michael Haumann, 'Kα X-ray Emission Spectroscopy on the Photosynthetic Oxygen-Evolving Complex Supports Manganese Oxidation and Water Binding in the S3 State', Inorganic Chemistry 57 (2018) 10424–10430. DOI: 10.1021/acs.inorgchem.8b01674. https://doi.org/10.1021/acs.inorgchem.8b01674
6. Mun Hon Cheah, Miao Zhang, Dmitry Shevela, Fikret Mamedov, Athina Zouni, Johannes Messinger, 'Assessment of the manganese cluster's oxidation state via photoactivation of photosystem II microcrystals', Proceedings of the National Academy of Sciences 117 (2020) 141–145. DOI: 10.1073/pnas.1915879117. https://doi.org/10.1073/pnas.1915879117
7. Maria Chrysina, Maria Drosou, Rebeca G. Castillo, Michael Reus, Frank Neese, Vera Krewald, Dimitrios A. Pantazis, Serena DeBeer, '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 (2023) 25579–25594. DOI: 10.1021/jacs.3c06046. https://doi.org/10.1021/jacs.3c06046
8. Maria Drosou, Gerard Comas-Vilà, Frank Neese, Pedro Salvador, Dimitrios A. Pantazis, 'Does Serial Femtosecond Crystallography Depict State-Specific Catalytic Intermediates of the Oxygen-Evolving Complex?', Journal of the American Chemical Society 145 (2023) 10604–10621. DOI: 10.1021/jacs.3c00489. https://doi.org/10.1021/jacs.3c00489
9. Thomas A. Corry, Patrick J. O'Malley, 'Proton Isomers Rationalize the High- and Low-Spin Forms of the S2 State Intermediate in the Water-Oxidizing Reaction of Photosystem II', The Journal of Physical Chemistry Letters 10 (2019) 5226–5230. DOI: 10.1021/acs.jpclett.9b01372. https://doi.org/10.1021/acs.jpclett.9b01372
10. Shota Taguchi, Takumi Noguchi, Hiroyuki Mino, 'Molecular Structure of the S2 State with a g = 5 Signal in the Oxygen Evolving Complex of Photosystem II', The Journal of Physical Chemistry B 124 (2020) 5531–5537. DOI: 10.1021/acs.jpcb.0c02913. https://doi.org/10.1021/acs.jpcb.0c02913
11. Shota Taguchi, Liangliang Shen, Guangye Han, Yasufumi Umena, Jian-Ren Shen, Takumi Noguchi, Hiroyuki Mino, 'Formation of the High-Spin S2 State Related to the Extrinsic Proteins in the Oxygen Evolving Complex of Photosystem II', The Journal of Physical Chemistry Letters 11 (2020) 8908–8913. DOI: 10.1021/acs.jpclett.0c02411. https://doi.org/10.1021/acs.jpclett.0c02411
12. Shinya Kosaki, Hiroyuki Mino, 'Molecular Structure Related to an S = 5/2 High-Spin S2 State Manganese Cluster of Photosystem II Investigated by Q-Band Pulse EPR Spectroscopy', The Journal of Physical Chemistry B 127 (2023) 6441–6448. DOI: 10.1021/acs.jpcb.3c01656. https://doi.org/10.1021/acs.jpcb.3c01656
13. Keisuke Saito, Shunya Nishio, Mizue Asada, Hiroyuki Mino, Hiroshi Ishikita, 'Insights into the protonation state and spin structure for the g = 2 multiline electron paramagnetic resonance signal of the oxygen-evolving complex', PNAS Nexus 2 (2023) pgad244. DOI: 10.1093/pnasnexus/pgad244. https://doi.org/10.1093/pnasnexus/pgad244
14. Bernard Baituti, Leonard Akofang, 'The Two Alternative Oxidation State Assignments of Manganese Ions: What S2 CW-EPR Multiline (ML) Signal Simulations Reveal?', Applied Magnetic Resonance 51 (2020) 389–407. DOI: 10.1007/s00723-020-01190-5. https://doi.org/10.1007/s00723-020-01190-5
15. Bernard Baituti, Sebusi Odisitse, 'A Computational Study of the S2 State in the Oxygen-Evolving Complex of Photosystem II by Electron Paramagnetic Resonance Spectroscopy', Molecules 26 (2021) 2699. DOI: 10.3390/molecules26092699. https://doi.org/10.3390/molecules26092699
16. Dimitrios A. Pantazis, 'Evaluation of new low-valent computational models for the oxygen-evolving complex of photosystem II', Chemical Physics Letters 753 (2020) 137629. DOI: 10.1016/j.cplett.2020.137629. https://doi.org/10.1016/j.cplett.2020.137629
17. David A. Flesher, Jinchan Liu, Jessica M. Wiwczar, Krystle Reiss, Ke R. Yang, Jimin Wang, Mikhail Askerka, Christopher J. Gisriel, Victor S. Batista, Gary W. Brudvig, 'Glycerol binding at the narrow channel of photosystem II stabilizes the low-spin S2 state of the oxygen-evolving complex', Photosynthesis Research 152 (2022) 167–175. DOI: 10.1007/s11120-022-00911-0. https://doi.org/10.1007/s11120-022-00911-0
18. Julianne S. Lampert, Gourab Banerjee, Ipsita Ghosh, Jinchan Liu, Krystle M. Reiss, Richard J. Debus, Victor S. Batista, Gary W. Brudvig, 'Binding of Acetate in the S2 State of the Oxygen-Evolving Complex in Photosystem II', Plants 15 (2026) 2291. DOI: 10.3390/plants15152291. https://doi.org/10.3390/plants15152291
19. Angela A. Shiau, Heui Beom Lee, Paul H. Oyala, Theodor Agapie, 'Coordination Number in High-Spin–Low-Spin Equilibrium in Cluster Models of the S2 State of the Oxygen Evolving Complex', Journal of the American Chemical Society 145 (2023) 14592–14598. DOI: 10.1021/jacs.3c04464. https://doi.org/10.1021/jacs.3c04464
20. Alireza Ariafard, Matthew Longhurst, Gerhard F. Swiegers, Robert Stranger, '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 (2026), issue 2. DOI: 10.1002/chem.202503024. https://doi.org/10.1002/chem.202503024
21. Colin Gates, Gennady Ananyev, Shatabdi Roy-Chowdhury, Petra Fromme, G. Charles Dismukes, 'Converting the 4-Flash Photosynthetic O2 Evolution Cycle to a 2-Flash Catalytic Cycle with a Simple Cocatalyst: Counting Electrons and Holes Directly and Transparently', ACS Catalysis 14 (2024) 15073–15087. DOI: 10.1021/acscatal.4c03896. https://doi.org/10.1021/acscatal.4c03896
22. Yulia Pushkar, 'New Water Oxidation Mechanism in Photosystem II Resolves Major Experimental Controversies', Angewandte Chemie International Edition 65 (2026). DOI: 10.1002/anie.1930324. https://doi.org/10.1002/anie.1930324