REVIEW 3 major objections 5 minor 4 references
Thermodynamics of the S2-to-S3 State Transition of the Oxygen-Evolving Complex of Photosystem II
T0 review · 3 major / 5 minor · reviewed 2026-08-14 · deepseek-v4-flash
Pith's one-line read This paper claims that the S2-to-S3 transition of Photosystem II's oxygen-evolving complex is gated by a switch from the S2 g=2 to the S2 g=4.1 redox isomer, after which a hydroxide from W3 inserts at Mn4 and a second hydroxide can…
desk verdict A plausible and honestly presented computational model of the S2-to-S3 transition, but the central g=2 vs g=4.1 water-binding claim lacks an anchored bulk reference and needs sensitivity analysis before it can be taken as quantitative. read the letter →
The pith
A machine-rendered reading of the paper's core claim, the machinery that carries it, and where it could break.
The reading
What carries the argument
The machinery is a Boltzmann/Monte Carlo sampling of the oxygen cavity: 451 oxygen atoms placed on a 1 Å grid inside the QM/MM-optimized protein model around the Mn4O5Ca cluster, each oxygen allowed to be water, hydroxide, or exit to solvent, together with proton conformers and sidechain rotamers of D1-E189. Energies come from solving the Poisson-Boltzmann equation for each conformer, and Metropolis Monte Carlo generates the grand-canonical Boltzmann distribution at pH 7. The discriminating variable is the S2 redox isomer: in the g=2 isomer Mn1 is oxidized and the cavity is closed to new ligands, while in the g=4.1 isomer Mn4 is oxidized, the Mn4-Ca2+ distance opens by 0.27 Å, and the O6 site between Mn4 and Ca2+ becomes occupiable. Density-functional optimization of the sampled structures then refines O6 into a 1.91 Å Mn4 ligand with a hydrogen bond to the mu-oxo O5, and the EXAFS comparison against experimental data places the computed structure as a pre-open intermediate.
What would settle it
A concrete test is to drive the S2-to-S3 transition under conditions where the g=2 isomer is the only populated state, for example at low temperature or with modified calcium, and measure with time-resolved Mn EXAFS whether any water insertion or S3 signature appears; if S3 forms without any detectable g=4.1 population, the gating claim fails. A second test is to trap the oxidized-intermediate state and look for the W3 proton: if W3 is still bound as water and no proton has been released to the lumen before manganese oxidation, the proposed deprotonation-before-oxidation sequence is wrong.
Extended reading notes
Core claim
The central claim is that S3-state formation cannot start from the S2 g=2 state: in that state the extra water/hydroxide is thermodynamically rejected, and the computed oxidation potential of Mn1 (1.4 V) is above the potential available from the photooxidized chlorophyll P680. Instead, the S2 g=2 state first converts to the S2 g=4.1 redox isomer, where the Mn4-Ca2+ distance is lengthened by 0.27 Å and a hydroxide (O6) bridging Mn4 and Ca2+ is occupied with about 25% probability, at the expense of the W3 water ligand of Ca2+. When the electron acceptor YZ is oxidized and D1-H190 becomes protonated, O6 is always present and W3 is never found, implying W3 deprotonates and releases a proton to the lumen before manganese oxidation. The resulting hydroxide completes the octahedral coordination of Mn4, which is then oxidized to Mn(IV) to form S3. Independently, sampling of more than 50 conformers of D1-E189 shows E189 moving away from Mn1 when a second hydroxide (O7) binds Mn1, an isoenergetic substitution in the S2 g=4.1 and S3 states; the computed EXAFS of the resulting S3 model is an intermediate between the closed and open S3 forms, with the final metastable S3 better described by the earlier open form.
Load-bearing premise
The load-bearing premise is that a classical continuum-electrostatics model with a fixed protein backbone, discrete sidechain rotamers, and high-spin manganese centers reliably predicts which water and hydroxide positions are thermodynamically favored; if protein dynamics open alternative channels or move residues outside the sampled conformations, the preference for O6/O7 and the order of proton release could change.
Editorial extensions
If this is right
- The S2 g=4.1 isomer is not a side population but a required gateway: conditions that destabilize it should block S3 formation, while conditions that populate it should accelerate the transition.
- Proton release precedes manganese oxidation in this step, with W3 as the deprotonated group; the proton goes to the lumen upon formation of the YZ-centered oxidized intermediate with protonated D1-H190.
- The two conflicting XFEL oxygen positions are compatible: one site (O6) completes Mn4 and the other (O7) replaces E189 at Mn1, so different crystals can resolve different parts of the same insertion process.
- The final S3 state is the open form; the structure produced by the S2-to-S3 insertion is an intermediate that relaxes afterward, which is why EXAFS of the computed model does not fully match the experimental S3 spectrum.
- E189's apparent indifference to mutation is explained by an isoenergetic alternative structure in which a hydroxide replaces it as the Mn1 ligand.
Reading between the lines
- If the g=2-to-g=4.1 interconversion is the gate, the rate of S3 formation should track the equilibrium population of the g=4.1 isomer; a quantitative EPR-visible correlation between isomer population and S3 yield would be a direct test the paper does not perform.
- The model's O6 and O7 sites are independent, so a transient S3 intermediate could carry two added hydroxides; a two-oxygen intermediate would appear as an extra Mn-Mn or Mn-Ca scattering feature in time-resolved EXAFS delayed relative to the first insertion.
- Because the computed structure sits between the closed and open S3 forms, the relaxation from intermediate to open form is a distinct physical step that could show up as a slow phase in time-resolved X-ray or EXAFS measurements after the fast electron-transfer phase.
- A testable extension would be to run the same Boltzmann sampling on a g=4.1 structure with Mn1 reduced rather than oxidized, predicting whether O7 binding is driven purely by electrostatics or requires the oxidized state.
Signed reviews
Editorial analysis
A structured set of objections, weighed in public.
Referee Report
Summary. This paper presents a computational model of the S2-to-S3 transition in the oxygen-evolving complex of photosystem II. The authors use QM/MM-optimized structures of the S2 g=2 and g=4.1 redox isomers, embed them in a Monte Carlo / continuum-electrostatics (MCCE/APBS) framework, and sample water/hydroxide positions on a 1 Å grid together with side-chain rotamers, protonation states, and Mn oxidation states. They report that water/hydroxide binding is thermodynamically unfavorable in the S2 g=2 state but favorable in the S2 g=4.1 state, leading to the proposal that the S2-to-S3 transition begins with a g=2-to-g=4.1 isomerization, followed by deprotonation of W3 and transfer of the resulting hydroxide to O6 at Mn4, and eventually binding of a second hydroxide (O7) at Mn1 with a conformational change of D1-E189. The resulting S3 structures are compared with experimental EXAFS and with XFEL structures from Suga et al. and Kern et al., and the paper argues that the computed S3 structure is an intermediate preceding the open S3 form.
Significance. The paper addresses a central open question in photosynthesis research: the molecular mechanism of the S2-to-S3 transition, and specifically the role of the redox isomer equilibrium and water/hydroxide insertion. The methodological approach, combining MCCE sampling of many oxygen positions, rotamers, and protonation states, is more comprehensive than standard single-structure QM/MM studies and provides a concrete, falsifiable proposal about the order of events (isomerization, deprotonation, insertion, E189 conformational change). The authors are transparent in providing optimized coordinates and spin densities in the Supporting Information, and they honestly report that their final S3 model fits EXAFS less well than an earlier open-form model. However, the central thermodynamic comparison lacks a well-defined bulk water/hydroxide reference state, the key free-energy differences are small (sub-kcal/mol to a few kcal/mol), and no sensitivity or error analysis is provided.
major comments (3)
- [Computational Methods, paragraph on Monte Carlo sampling] The central conclusion that water/hydroxide addition is unfavorable in S2 g=2 but favorable in S2 g=4.1 depends on the free energy of the "moved out of the protein into solvent" conformer that serves as the reference for binding. This free energy, and the standard-state concentration (or chemical potential) of the bulk water/hydroxide reservoir, is never defined in the manuscript. The occupancy numbers in the S2 g=4.1 state (O6 at 25% vs W3 at 75%) imply a free-energy difference of only about 0.65 kcal/mol, so a shift of roughly 1 kcal/mol in the bulk reference would reverse the predicted ordering. Please specify the exact energy expression for the solvent conformer, the standard-state convention, and whether the same reference is used for all four states; in addition, report a sensitivity analysis (e.g., varying the bulk reference energy, the protein dielectric constant, and the grid spacing) to demonstrate that the "unfavorable in g=2, favorable in g=4.1" result is not an artifact of the unspecified reference.
- [Figure 2 and the paragraph beginning "Figure 2 compares..."] The paper acknowledges that the EXAFS spectrum of the proposed S3 model matches the experimental data worse than the previously published open-form S3 model, and interprets this as evidence that the new model is an intermediate on the S2-to-S3 pathway. However, no free-energy or kinetic calculation is provided to show that this structure lies on the reaction path; a worse fit is equally consistent with the model being incorrect. The claim that this is "an intermediate structure during the S2-to-S3 state transition" is therefore a post hoc interpretation. Please state explicitly that this conclusion is not supported by the EXAFS comparison alone, and either add a genuine pathway calculation (e.g., minimum-energy path between S2 g=4.1 and the open S3 form) or soften the claim to a speculation.
- [Supporting Information, Table S1] The DFT (B97D) energy difference between the Mn1-OH7 and Mn1-E189 forms of the S3 state is reported as 2.4 kcal/mol, and the two states are called "isoenergetic." Given that B97D with a modest basis set (LanL2DZ for metals, 6-31G** for light atoms) is generally accurate to only a few kcal/mol for transition-metal redox and isomerization energetics, this 2.4 kcal/mol difference cannot be used to support a robust preference for O7 binding. The claim that O7 replaces E189 as a ligand of Mn1 should be supported by tests with other functionals, larger basis sets, or a clear statement that the two structures are degenerate within the accuracy of the method and that the proposal rests on the broader electrostatic sampling rather than this single DFT energy difference.
minor comments (5)
- [Abstract] The last sentence contains a grammatical error: "In the S3 state in the population of protonated D1-E189 increases" should be "In the S3 state, the population of protonated D1-E189 increases."
- [Computational Methods] The manuscript states that "the parameters for the OEC and ligands used here as reported previously in Amin et al." but does not specify which parameters are transferred and whether any were re-optimized or re-fitted for the S2/S3 states. A short table or list of transferred parameters and their references would improve reproducibility.
- [Computational Methods, Monte Carlo sampling] The manuscript does not state the number of Monte Carlo steps, convergence criteria, or whether the reported occupancies are averages over multiple independent runs. Reporting these details would allow readers to assess the statistical uncertainty in the key 25%/75% occupancies and the 0% occupancy in S2 g=2.
- [Computational Methods, DFT settings] The sentence "All the Mn(IV) ions were defined in the high spin state" should be justified, because different spin states of the Mn ions can alter the electrostatics and ligand-binding energetics. Please provide a reference or a test for the spin-state assumption.
- [Figure 2 caption and text] The phrase "reduced distance space" in the caption is unusual; the text refers to the "Fourier Transformed spectrum." Please use consistent terminology and define χ(R).
Circularity Check
No significant circularity: the water/hydroxide binding step is an MC output, and the EXAFS comparison is an independent adverse check.
full rationale
No circular step meets the evidentiary bar. The load-bearing conclusion that water/hydroxide binding is unfavorable in S2 g=2 but favorable in S2 g=4.1 is an output of grand-canonical Monte Carlo sampling over 451 oxygen grid positions and 7569 proton conformers, with O6 and O7 occupancies reported as 25% and 0% in the two S2 isomers; these occupancies are not fitted to the S3 XFEL densities that the paper later compares against. The model does import OEC/ligand parameters and pKa methodology from Amin et al. 2013 and 2015, which are self-citations by overlapping authors, but those parameters were developed and tested on model compounds and the Kok cycle, and the current conclusion is not simply a restatement of those papers. The EXAFS comparison is an external, independent check and is adverse to the proposed structure, as the paper states that the new model 'does not match the experimental EXAFS data as well as the QM/MM-optimized open-form of the S3 state'; this is the opposite of circular confirmation. The skeptic's concern that the bulk water/hydroxide reference chemical potential is unspecified and that 25% O6 occupancy implies only a marginal preference is a real well-posedness and robustness risk, but it is a correctness issue, not a reduction of the prediction to its inputs; no equation in the paper defines the result through a fitted parameter or self-citation chain. Therefore the circularity score is low.
Assumptions & free parameters
free parameters (5)
- Protein dielectric constant =
4
- Grid spacing for oxygen placement =
1.0 Å
- Mn(III/IV) solution redox potential Em,sol =
from Amin et al. 2013
- EXAFS Debye-Waller factor =
0.003 Å
- EXAFS Fermi energy E0 =
6540.0 eV
assumptions (5)
- domain assumption The QM/MM optimized S2 g=2 and g=4.1 structures are accurate starting points for sampling.
- domain assumption Continuum electrostatics with dielectric 4 and Monte Carlo sampling reproduces the thermodynamics of water/hydroxide binding and protonation at pH 7.
- domain assumption The oxidation states and high-spin coupling of Mn centers are correctly assigned in both S2 isomers and S3.
- domain assumption Proton release to the lumen can be inferred from computed total charge change in the 15 Å model sphere.
- domain assumption Standard QM/MM and DFT functionals (B97D/B3LYP) with LanL2DZ/6-31G** basis sets are adequate for this system.
Cite this review
Pith. "Pith review of Thermodynamics of the S2-to-S3 State Transition of the Oxygen-Evolving Complex of Photosystem II." pith.science (2026). https://pith.science/paper/ZXTDPYC3
@misc{pith2026190805056,
author = {Pith},
title = {Pith review of: Thermodynamics of the S2-to-S3 State Transition of the Oxygen-Evolving Complex of Photosystem II},
year = {2026},
howpublished = {\url{https://pith.science/paper/ZXTDPYC3}},
note = {Machine review of arXiv:1908.05056}
}
read the original abstract
The room temperature pump-probe X-ray free electron laser (XFEL) measurements used for serial femtosecond crystallography provide remarkable information about the structures of the catalytic (S-state) intermediates of the oxygen-evolution reaction of photosystem II. However, mixed populations of these intermediates and moderate resolution limit the interpretation of the data from current experiments. The S3 XFEL structures show extra density near the OEC that may correspond to a water/hydroxide molecule. However, in the latest structure, this additional oxygen is 2.08 {\AA} from the Oe2 of D1-E189, which is closer than the sum of the van der Waals radii of the two oxygens. Here, we use Boltzmann statistics and Monte Carlo sampling to provide a model for the S2-to-S3 state transition, allowing structural changes and the insertion of an additional water/hydroxide. Based on our model, water/hydroxide addition to the oxygen-evolving complex (OEC) is not thermodynamically favorable in the S2 g = 2 state, but it is in the S2 g = 4.1 redox isomer. Thus, formation of the S3 state starts by a transition from the S2 g = 2 to the S2 g = 4.1 structure. Then, electrostatic interactions support protonation of D1-H190 and deprotonation of the Ca2+-ligated water (W3) with proton loss to the lumen. The W3 hydroxide moves toward Mn4, completing the coordination shell of Mn4 and moving with its oxidation to Mn(IV) in the S3 state. In addition, binding additional hydroxide to Mn1 leads to a conformational change of D1-E189 in the S2 g = 4.1 and S3 structures. In the S3 state in the population of protonated D1-E189 increases.
Reference graph
Works this paper leans on
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[2]
and ends at D (S3). The B*, C* and D* are possible isomers of the B, C and D states, where O7 replaces D1-E189 as a ligand for Mn1. Conclusions Simulations based on classical Monte Carlo sampling of many possible oxygen positions to generate the Boltzmann distribution suggest the transition from the S2 to the S3 state starts by a transition from the S2 g ...
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[3]
Strong hydrogen bonds (shown in dashed lines) are formed between D1-E189, OH7 and µ-O1
The ligand environment of Mn1 with O7 (hydroxyl anion) and protonated D1-E189. Strong hydrogen bonds (shown in dashed lines) are formed between D1-E189, OH7 and µ-O1. The OH7 (i.e., the hydroxyl anion form of O7) has a stronger dipole moment than the carboxylate group of the amino acid and replaces the E189 ligand in the S2 g = 4.1 and in the S3 state due...
work page 2017
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(38) Becke, A. D. Density-Functional Exchange-Energy Approximation with Correct Asymptotic Behavior. Phys. Rev. A 1988, 38 (6), 3098–3100. https://doi.org/10.1103/PhysRevA.38.3098. (39) Becke, A. D. Density-functional Thermochemistry. III. The Role of Exact Exchange. J. Chem. Phys. 1993, 98 (7), 5648–5652. https://doi.org/10.1063/1.464913. (40) Grimme, S....
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https://doi.org/10.1007/s11120-019-00637-6. (61) Blomberg, M. R. A.; Siegbahn, P. E. M. A Quantum Chemical Study of Tyrosyl Reduction and O—O Bond Formation in Photosystem II. Mol. Phys. 2003, 101 (1–2), 323–333. https://doi.org/10.1080/00268970210162781. (62) Limburg, J.; Vrettos, J. S.; Liable-Sands, L. M.; Rheingold, A. L.; Crabtree, R. H.; Brudvig, G....
Reviewed August 14, 2026 · model on record in the stance chip above.
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