Origin of Low-Lying Red States in the Lhca4 Light-Harvesting Complex of Photosystem I

The antenna complexes of Photosystem I present low-lying states visible as red-shifted and broadened absorption and fluorescence bands. Among these, Lhca4 has the most evident features of these “red” states, with a fluorescence band shifted by more than 25 nm from typical LHC emission. This signal arises from a mixing of exciton and charge-transfer (CT) states within the excitonically coupled a603–a609 chlorophyll (Chl) dimer. Here we combine molecular dynamics, multiscale quantum chemical calculations, and spectral simulations to uncover the molecular mechanism for the formation and tuning of exciton-CT interactions in Lhca4. We show that the coupling between exciton and CT states is extremely sensitive to tiny variations in the Chl dimer arrangement, explaining both the red-shifted bands and the switch between conformations with blue and red emission observed in single-molecule spectroscopy. Finally, we show that mutating the axial ligand of a603 diminishes the exciton–CT coupling, removing any red-state fingerprint.

The manuscript is very well-written, the methodology is properly and clearly presented and the conclusions are adequately supported by the data.In addition, the manuscript presents significant new physical insights which will be appealing for a broad physical chemistry audience, not only for specialists working in photosynthesis research.
Therefore, I strongly recommend the manuscript for publication in The Journal of Physical Chemistry Letters after minor revision (my comments are indicated below).

Specific comments:
1-Based on bond distances from MDs, it is suggested that Chl is bound weaklier to Asn with respect to His.I would suggest some care when discussing bonding from molecular mechanics simulations.Can the authors comment on the force field parameters that describe the Chl -Asn and Chl -His bonds?2-A hydrogen bond between the Asn98 amide group and the carbonyl group of Chl a609 is observed in the WT.In the N98H mutant, this interaction is not present, "as the His N-H group does not have the correct orientation to make the H-bond".However, the structure for the mutant was obtained by manually swapping the sidechain.Is it possible that, if another His rotamer was chosen, this H-bond could be established?

Comments on the figures:
Fig1: The legend should indicate the meaning of the dashed lines as indicated in FigS1 "Blue dashed lines represent hydrogen bonding".In addition, it will be good to indicate the ligation to a609 (in panel c) and to a603 and a609 (in panel d).
FigS5: The colors used are too similar and difficult to distinguish.I suggest to use colors with more contrast among each other and to remove the solid color under the curves, as well as to indicate the meaning of the dashed lines.
FigS7: The legend is missing the reference number.This figure seems to have a wrong numbering, in the mean text it is mentioned after FigS10.The sentence "where Chl a603 water-ligated" should not be "with Chl a603 water-ligated" or "where Chl a603 is water-ligated"?FigS8: The last sentence in the legend seems unfinished.The colors used are too similar and difficult to distinguish.I suggest to use colors with more contrast among each other.

Minor grammar corrections and typos:
Page3 (line 29-32): The order of this sentence seems wrong "These states exhibit broad bandwidths, even at 4K, and exceptionally red-shifted emission extending the absorption of the complex to the far red."Perhaps it should read: "These states exhibit broad bandwidths, even at 4K, extending the absorption of the complex to the far red, and yielding exceptionally red-shifted emission."Page10 (line 45-46): It reads "Notably, the HOMO-LUMO energy gap does to show large variations (...)" It probably means: "Notably, the HOMO-LUMO energy gap does not show large variations (...)" Page S3: The sentence that starts with "the structure of the dimer was optimized at (...)" is missing a capital T.
FigS3: "repolicas" should be "replicas" Author's Response to Peer Review Comments: Reviewer: 1 The authors present an elegant and comprehensive combination of molecular dynamics, multiscale quantum chemical calculations, and spectral simulations with the aim to understand the molecular mechanism for formation and fine-tuning of exciton and charge-transfer (CT) interactions in Lhca4.Interestingly, the authors show how extremely sensitive is the coupling between exciton and CT states to very small changes in the relative orientation between the two excitonically coupled a603-a609 chlorophyll pair.By unravelling the precise mechanism of exciton-CT mixing and its consequences on the energy landscape and spectral signatures, the authors provide unvaluable information on how to control the energy transfer dynamics and quenching processes in lightharvesting complexes, which in turn, has far-reach implications in the development of new materials for solar-energy conversion (for instance for photovoltaic applications).
The manuscript is very well-written, the methodology is properly and clearly presented and the conclusions are adequately supported by the data.In addition, the manuscript presents significant new physical insights which will be appealing for a broad physical chemistry audience, not only for specialists working in photosynthesis research.Therefore, I strongly recommend the manuscript for publication in The Journal of Physical Chemistry Letters after minor revision (my comments are indicated below).
Authors' Reply: we sincerely thank the Reviewer for their positive assessment of our work.

Specific comments:
1-Based on bond distances from MDs, it is suggested that Chl is bound weaklier to Asn with respect to His.I would suggest some care when discussing bonding from molecular mechanics simulations.Can the authors comment on the force field parameters that describe the Chl -Asn and Chl -His bonds?
Authors' Reply: We agree with the Reviewer that the Chl-ligand interactions, including Mg coordination, are force-field dependent.The Chl-ligand interactions are all described with the nonbonded terms of the Amber force field, that is, point-charge electrostatics and van der Waals (vdW) nonelectrostatic terms.Electrostatic and vdW parameters of Asn and His were taken from the well tested Amber ff14SB protein force field.For the Chl, we have used QM-derived parameters by Ceccarelli et al. with modifications by Zhang et al. as described in the SI.This ff was successfully applied to a number of natural light harvesting systems (Balevičius et al., Sci Rep. 2017, 7, 13956;Prandi et al. Commun Biol. (2022), 5, 145; Bourne-Worster et al., PNAS 2023, 120, e2210811120 and others), however, the distances between Chl and a binding residue were never specifically investigated.
The only direct difference between His-binding and Asn-binding is represented by the different parameters of His and Asn in the protein force field.Although we agree with the Reviewer that some care is needed in interpreting the Mg coordination from MD, the difference observed in our simulations between the two binding residues should be at least qualitatively correct.
2-A hydrogen bond between the Asn98 amide group and the carbonyl group of Chl a609 is observed in the WT.In the N98H mutant, this interaction is not present, "as the His N-H group does not have the correct orientation to make the H-bond".However, the structure for the mutant was obtained by manually swapping the sidechain.Is it possible that, if another His rotamer was chosen, this H-bond could be established?

Authors' Reply:
We thank the reviewer for the suggestion.We included the absolute values of center-to-center distances for the CT pair into the main text.

Authors' Reply:
We have added a reference to Figure 1c when mentioning the hydrogen bond to the carbonyl.

Comments on the figures:
Fig1: The legend should indicate the meaning of the dashed lines as indicated in FigS1 "Blue dashed lines represent hydrogen bonding".In addition, it will be good to indicate the ligation to a609 (in panel c) and to a603 and a609 (in panel d).
Authors' Reply: We thank the Reviewer for the suggestion.The image was changed to include the ligation.The dashed lines are now explained in the figure caption.
FigS5: The colors used are too similar and difficult to distinguish.I suggest to use colors with more contrast among each other and to remove the solid color under the curves, as well as to indicate the meaning of the dashed lines.

Authors' Reply:
We agree with the Reviewer that the colored area below the curve leads to a too cluttered figure due to the overlapping curves.We removed the solid color under the curves and made the lines thicker to make it easier to read.FigS7: The legend is missing the reference number.This figure seems to have a wrong numbering, in the mean text it is mentioned after FigS10.The sentence "where Chl a603 water-ligated" should not be "with Chl a603 water-ligated" or "where Chl a603 is water-ligated"?Authors' Reply: We thank the reviewer for pointing out the typo in the reference and the caption.The experimental reference and the figure caption was corrected.
We also reordered the SI figures so that former Figure S7  FigS8: The last sentence in the legend seems unfinished.The colors used are too similar and difficult to distinguish.I suggest to use colors with more contrast among each other.

Authors' Reply:
We thank the reviewer for the suggestion and for noticing the errors in the caption.In the original version of this Figure, we used the same coloring scheme as in the main text.However, we agree that changing the colors improves the readability, therefore we changed the color scheme to a more contrasted one.We also fixed the last sentence in the caption, which now reads: "We note that for WT the lowest CT state has 603 + 609 -character while for the N98H mutant it has 603 -609 + character." Minor grammar corrections and typos: Page3 (line 29-32): The order of this sentence seems wrong "These states exhibit broad bandwidths, even at 4K, and exceptionally red-shifted emission extending the absorption of the complex to the far red."Perhaps it should read: "These states exhibit broad bandwidths, even at 4K, extending the absorption of the complex to the far red, and yielding exceptionally red-shifted emission." Authors' Reply: We agree with the reviewer that the proposed sentence is more clear and understandable.We corrected the sentence in the main text.Page10 (line 45-46): It reads "Notably, the HOMO-LUMO energy gap does to show large variations (...)" It probably means: "Notably, the HOMO-LUMO energy gap does not show large variations (…)" Authors' Reply: We thank the reviewer for pointing out this typo.The sentence was corrected in the main text.
Page S3: The sentence that starts with "the structure of the dimer was optimized at (...)" is missing a capital T.

Authors' Reply:
The typo was corrected in the main text.
FigS3: "repolicas" should be "replicas" Authors' Reply: We thank the reviewer for noticing the typo.The figure caption was corrected.
3-FigureS11contains important information about the mechanism of exciton-CT coupling.I suggest to include a more detailed discussion about this figure in the main text (at some point in page 16).4-In page 16 (line 29-31), the authors mention "Revealing the molecular mechanism of formation of the low-lying exciton states" should not be "low-lying exciton-CT states"?Similarly, in FigS11 the text on the figure "Lowest exciton state" should not be "Lowest exciton-CT state"?Minor comments on the text: Page6 (line 9-10): indicate the a603-a609 distance values.Page6 (line 49): indicate Fig1c.
is now Figure S11, and former Figures S8-S11 are now Figures S7-S10.Now the supporting figures are mentioned in order in the main text.