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  1. Author Response:

    Reviewer #1 (Public Review):

    The authors have investigated the structure of photosystem I (PSI) of the cyanobacterium Gloeobacter that markedly differs in its optical properties from that other cyanobacteria. Interestingly, the PSI of Gloeobacter does not possess the so-called red chlorophylls (Chls) that are responsible for long-wavelength absorption and emission. So far, there were only suggestions for the identity of these red Chls in the literature. These suggestions were based on the structure of PSI of other cyanobacteria that exhibit Chl dimers and trimers with small interpigment distances. According to our general knowledge, the small distances give rise to electron exchange between the pigments, which leads to a quantum mechanic mixing of excited states and charge transfer states, that can lead to low-energy states. In their high-resolution structural analysis of Gloeobacter with cryo-electron microscopy the authors unambiguously unravel the molecular identity of the red Chl states in cyanobacteria by noting that two Chls involved in dimers and trimers in other cyanobacteria are simply absent in Gloeobacter. This is a very clear and simple identification that has a great impact on our understanding of light-harvesting in PSI. Moreover, as the authors also note, Gloeobacter is much more susceptible to photodamage occuring at high light intensities than other cyanobacteria. The authors suggest that the dimer of red Chls, identified as described above, is responsible for photoprotection in the other cyanobateria. This is a very interesting suggestion that will stimulate further experimental and theoretical work.

    We want to thank the reviewer for their highly positive and encouraging evaluation of our manuscript. Based on the comments of the other reveiwers, we have removed the discussions regarding photoprotection from the revised manuscript.

    Reviewer #2 (Public Review):

    Weaknesses: Although the authors extensively compared the structural and spectral characteristics of Gloeobacter PSI with Synechocystis and T. vulcanus PSIs, the function of Low1 and Low2 in photoprotection are mainly claimed from the structural but not the functional differences. Due to lack of a genetic operation system of Gloeobacter, it is difficult to test the structural observations and function of Low1 and Low2 from physiological aspects. Therefore, the function of Low1 and Low2 in the photoprotection of oxyphototrophs still needs further functional investigations in the future.

    First of all, we thank the reviewer for their positive evaluation and important comments and suggestions to improve our manuscript. In view of the comments and suggestions of this reviewer as well as the other reviewers, we completely removed the discussions regarding the roles of low-energy Chls in the photoprotection from the revised manuscript.

    Reviewer #3 (Public Review):

    Comment 1: The structural data were obtained at high resolution and represent the strength of this work. The comparison between the structures is interesting and can indeed provide suggestions on the location of the red forms. However, it is essential to make clear to the reader that those suggestions need to be validated by experiments and/or calculations and that it is not possible to assign the energy of pigments only by looking at the structure.

    First of all, we thank the reviewer for their positive evaluation and important comments and suggestions to improve our manuscript. We agree with the reviewer’s comment, and added the sentences “However, it should be noted that the energy levels of Chls cannot be assigned only by the structural analysis of PSI. Further mutagenesis studies and theoretical calculations will be required for understanding the correlation of Low1 and Low2 with the fluorescence bands at around 723 and 730 nm.” to the section of “Correlation of Low1 and Low2 with characteristic fluorescence bands” in the revised manuscript.

    Comment 2: The authors interpret their results in the framework of photoprotection. However, they do not provide evidence that the PSI without red forms is more photosensitive. It should be emphasized that the role of the red form is not yet known. Several proposals were made, including photoprotection, but no conclusive results are available. The three references used here to support the role of red forms in photoprotection (Shubin et al. 1995; Shibata et al. 2010 and Schlodder et al. 2011) do not appear to be appropriate. They are studies of excitation energy transfer and do not discuss photoprotection. In the three cited papers, the fluorescence quenching of the red forms is due to energy transfer to P700/P700+. Actually, the authors of these works use the evidence for energy transfer at low temperature to the reaction center to suggest that the red forms are close to P700. These results are relevant for the present work but need to be discussed in a different context. The same is true for Gobets et al. 2001.

    We agree with the reviewer’s comment that it is not clear that PSI without low-energy forms is more photosensitive, although Gloeobacter is generally much more photosensitive than other cyanobacteria. Based on the commnets of this and also other reviewers, we removed all sentences regarding photoprotection from the revised manuscript, in order to avoid misleading.

    Comment 3: Another point of attention is that the red forms have a different energy in different cyanobacteria. This also means that the organization and/or location of the chlorophylls responsible for the red forms might vary in the different species. The authors of this manuscript assume that are only two red forms emitting at 723 and 730 nm, with one of them present in all PSI and the other in some of them. The situation is much more complex, as it is well described in the literature.

    We agree with the reviewer’s comment that a wide variety of low-energy Chls are observed by spectroscopic techniques using different cyanobacteria. We do not assume that Low1 and Low2 are the only two types of low-energy Chls. Absorption spectroscopy showed various bands and shoulder around/over 700 nm, reflecting a complexity and difficulty of the identification of low-energy Chls in PSI. In contrast, fluorescence spectroscopy is a convenient method to observe low-energy Chls in PSI. It is known that under the liquid-nitrogen condition, two types of prominent fluorescence peaks from lowenergy Chls were mainly found at around 723 and 730 nm. The 723 and/or 730-nm fluroescence bands are conserved in most cyanobacteria, although their band widths and peaks may vary to some extents under different experimental conditions. To explain these contents, we modified the third paragraph of the Introduction section in the revised manuscript (pages 3-4).

    Comment 4: The authors assign the red forms in the different species looking at the low temperature emission spectra, assuming that the width of the spectra should be the same for all red forms. However, the width of the spectra of the red forms can vary depending on several factors, as shown in many papers and it is not correct to assume that it is constant. The presence of different red chlorophyll pools can be detected experimentally with different methods (see literature).

    We agree with the reviewer’s comment. As described in Author reply 3, characteristic bands at about 723 and 730 nm appear in fluorescence spectroscopy measured at 77 K. We mentioned the complexity of these fluorescence bands that may have different band widths and slight peak shifts. To explain these contents, we added the sentences to the third paragraph of Introduction section in the revised manuscript (pages 3-4).

    Comment 5: The authors attribute the sensitivity of Gleobacter to light to the absence of red forms in PSI. No data supporting this conclusion are presented. Many other factors can be responsible for this sensitivity (e.g. PSII). Moreover, the authors do not show that PSI of Gleobacter is more sensitive to light than other PSI.

    We fully agree with the reviewer’s comment, and have removed all sentences regarding photoprotection from the revised manuscript to avoid misleading.

    Comment 6: In the result paragraph about the functional significance of low1, the authors suggest that b-carotene in PSI is responsible for energy quenching, but no data supporting this statement are shown and I could not find them in the literature. The cited papers focus on non-photochemical quenching in the light-harvesting complexes of plants. I could not follow the reasoning in the last paragraph of the results. No data are shown and it is unclear how the authors reached their conclusions.

    We agree with the reviewer’s comment, and have remove all sentences regarding photoprotection and quenching (including the role of β-carotene commented by the reviewer) from the revised manuscript, in order to avoid misleading.

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  2. Evaluation Summary:

    This work reports the structure of the photosystem I of Gloeobacter, a cyanobacterium that does not contain low energy absorbing chlorophylls, the so-called red forms. By comparing this structure to those of other cyanobacteria that contain red forms, the authors aim to identify the chlorophylls responsible for low-energy absorption in PSI. Their second aim is to understand the role of the red forms. The topic is interesting, the structural data are very good, but the conclusions regarding the role of the red forms are not supported by data.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)

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  3. Reviewer #1 (Public Review):

    The authors have investigated the structure of photosystem I (PSI) of the cyanobacterium Gloeobacter that markedly differs in its optical properties from that other cyanobacteria. Interestingly, the PSI of Gloeobacter does not possess the so-called red chlorophylls (Chls) that are responsible for long-wavelength absorption and emission. So far, there were only suggestions for the identity of these red Chls in the literature. These suggestions were based on the structure of PSI of other cyanobacteria that exhibit Chl dimers and trimers with small interpigment distances. According to our general knowledge, the small distances give rise to electron exchange between the pigments, which leads to a quantum mechanic mixing of excited states and charge transfer states, that can lead to low-energy states. In their high-resolution structural analysis of Gloeobacter with cryo-electron microscopy the authors unambiguously unravel the molecular identity of the red Chl states in cyanobacteria by noting that two Chls involved in dimers and trimers in other cyanobacteria are simply absent in Gloeobacter. This is a very clear and simple identification that has a great impact on our understanding of light-harvesting in PSI. Moreover, as the authors also note, Gloeobacter is much more susceptible to photodamage occuring at high light intensities than other cyanobacteria. The authors suggest that the dimer of red Chls, identified as described above, is responsible for photoprotection in the other cyanobacteria. This is a very interesting suggestion that will stimulate further experimental and theoretical work.

    Was this evaluation helpful?
  4. Reviewer #2 (Public Review):

    Low-energy chlorophylls (Chls) in photosystem I (PSI) are essential for regulating energy balance for energy transfer and energy quenching, one of the photoprotection mechanisms that converts excitation energy to harmless heat. However, the location of the low-energy Chls is under debate both experimentally and theoretically.

    In this work, the authors answered this question through a 2.04 Å resolution cryo-EM structure of the PSI trimer from a primitive cyanobacterium Gloeobacter violaceus PCC 7421, which only grows under extremely low light conditions. The structure showed absence of one dimeric (Chl1A/Chl2A, Low1) and one trimeric Chls (Chl1B/Chl2B/Chl3B, Low2), as well as some subunits commonly found in other cyanobacteria. Structural and spectral comparisons of Gloeobacter PSI with PSIs from other two cyanobacteria revealed the location and interactions of Low1 and Low2 within but not in the interface among the PSI monomers. Then the authors also demonstrated the function of Low 1 as a main photoprotection site for most oxyphototrophs even under normal light conditions, whereas Low2 is involved in either energy transfer or energy quenching in some of the oxyphototrophs.

    Strengths:
    This work reported the highest resolution structure of PSI trimers ever determined by X-ray crystallography and single particle cryo-EM, which provides solid structural information for clear presentations of the cofactors and side chain interactions of PSI. The structural analyses not only revealed the location of low energy Chls in cyanobactrial PSI, but also demonstrated the evolutionary changes of the low-energy Chls in the photoprotection machinery from photosynthetic prokaryotes to eukaryotes. This work will contribute to broaden the theory and diversity of the photoprotection mechanisms, and the structure with the highest resolution will provide an excellent model for further functional studies of the photoprotection of photosynthetic organisms.

    Weaknesses:
    Although the authors extensively compared the structural and spectral characteristics of Gloeobacter PSI with Synechocystis and T. vulcanus PSIs, the function of Low1 and Low2 in photoprotection are mainly claimed from the structural but not the functional differences. Due to lack of a genetic operation system of Gloeobacter, it is difficult to test the structural observations and function of Low1 and Low2 from physiological aspects. Therefore, the function of Low1 and Low2 in the photoprotection of oxyphototrophs still needs further functional investigations in the future.

    Was this evaluation helpful?
  5. Reviewer #3 (Public Review):

    The structural data were obtained at high resolution and represent the strength of this work. The comparison between the structures is interesting and can indeed provide suggestions on the location of the red forms. However, it is essential to make clear to the reader that those suggestions need to be validated by experiments and/or calculations and that it is not possible to assign the energy of pigments only by looking at the structure.

    The authors interpret their results in the framework of photoprotection. However, they do not provide evidence that the PSI without red forms is more photosensitive. It should be emphasized that the role of the red form is not yet known. Several proposals were made, including photoprotection, but no conclusive results are available. The three references used here to support the role of red forms in photoprotection (Shubin et al. 1995; Shibata et al. 2010 and Schlodder et al. 2011) do not appear to be appropriate. They are studies of excitation energy transfer and do not discuss photoprotection. In the three cited papers, the fluorescence quenching of the red forms is due to energy transfer to P700/P700+. Actually, the authors of these works use the evidence for energy transfer at low temperature to the reaction center to suggest that the red forms are close to P700. These results are relevant for the present work but need to be discussed in a different context. The same is true for Gobets et al. 2001.

    Another point of attention is that the red forms have a different energy in different cyanobacteria. This also means that the organization and/or location of the chlorophylls responsible for the red forms might vary in the different species. The authors of this manuscript assume that are only two red forms emitting at 723 and 730 nm, with one of them present in all PSI and the other in some of them. The situation is much more complex, as it is well described in the literature.

    The authors assign the red forms in the different species looking at the low temperature emission spectra, assuming that the width of the spectra should be the same for all red forms. However, the width of the spectra of the red forms can vary depending on several factors, as shown in many papers and it is not correct to assume that it is constant. The presence of different red chlorophyll pools can be detected experimentally with different methods (see literature).

    The authors attribute the sensitivity of Gleobacter to light to the absence of red forms in PSI. No data supporting this conclusion are presented. Many other factors can be responsible for this sensitivity (e.g. PSII). Moreover, the authors do not show that PSI of Gleobacter is more sensitive to light than other PSI.

    In the result paragraph about the functional significance of low1, the authors suggest that b-carotene in PSI is responsible for energy quenching, but no data supporting this statement are shown and I could not find them in the literature. The cited papers focus on non-photochemical quenching in the light-harvesting complexes of plants. I could not follow the reasoning in the last paragraph of the results. No data are shown and it is unclear how the authors reached their conclusions.

    Was this evaluation helpful?