Nitrogenase structural evolution across Earth’s history

Reviewer #1 (Public review):

This was a clearly written manuscript that did an excellent job summarizing complex data. In this manuscript, Cuevas-Zuviría et al. use protein modeling to generate over 5,000 predicted structures of nitrogenase components, encompassing both extant and ancestral forms across different clades. The study highlights that key insertions define the various Nif groups. The authors also examined the structures of three ancestral nitrogenase variants that had been previously identified and experimentally tested. These ancestral forms were shown in earlier studies to exhibit reduced activity in Azotobacter vinelandii, a model diazotroph.

This work provides a useful resource for studying nitrogenase evolution. However, its impact is somewhat limited due to a lack of evidence linking the observed structural differences to functional changes. For example, in the ancestral nitrogenase structures, only a small set of residues (lines 421-431) were identified as potentially affecting interactions between nitrogenase components. Why didn't the authors test whether reverting these residues to their extant counterparts could improve nitrogenase activity of the ancestral variants?

Additionally, the paper feels somewhat disconnected. The predicted nitrogenase structures discussed in the first half of the manuscript were not well integrated with the findings from the ancestral structures. For instance, do the ancestral nitrogenase structures align with the predicted models? This comparison was never explicitly made and could have strengthened the study's conclusions.

Reviewer #2 (Public review):

This work aims to study the evolution of nitrogenanses, understanding how their structure and function adapted to changes in the environment, including oxygen levels and changes in metal availability.

The study predicts > 5000 structures of nitrogenases, corresponding to extant, ancestral, and alternative ancestral sequences. It is observed that structural variations in the nitrogenases correlate with phylogenetic relationships. The amount of data generated in this study represents a massive undertaking that is certain to be a resource for the community. The study also provides strong insight into how structural evolution correlates with environmental and biological phenotypes.

The challenge with this study is that all (or nearly all) of the quantitative analyses presented are based on RMSD calculations, many of which are under 2 angstroms. For all intents and purposes, two structures with RMSD < 2 angstroms could be considered 'structurally identical'. A lot of insight generated is based on minuscule differences in RMSD, for which it is not clear that they are significantly different. The suggestion would be to find a way to evaluate the RMSD metric and determine whether these values, as obtained for structures being compared, are reliable. Some options are provided in earlier studies: PMID: 11514933, PMID: 17218333, PMID: 11420449, PMID: 8289285 (and others).

It could also be valuable to focus more on site-specific RMSDs rather than Global RMSDs. The high conservation in the nitrogenases likely ensures that the global RMSDs will remain low across the family. Focusing on specific regions might reveal interesting differences between clades that are more informative regarding the evolution of structure in tandem with environment/time.

Author response:

Public Reviews:

Reviewer #1 (Public review):

This was a clearly written manuscript that did an excellent job summarizing complex data.

In this manuscript, Cuevas-Zuviría et al. use protein modeling to generate over 5,000 predicted structures of nitrogenase components, encompassing both extant and ancestral forms across different clades. The study highlights that key insertions define the various Nif groups. The authors also examined the structures of three ancestral nitrogenase variants that had been previously identified and experimentally tested. These ancestral forms were shown in earlier studies to exhibit reduced activity in Azotobacter vinelandii, a model diazotroph. This work provides a useful resource for studying nitrogenase evolution.

However, its impact is somewhat limited due to a lack of evidence linking the observed structural differences to functional changes. For example, in the ancestral nitrogenase structures, only a small set of residues (lines 421-431) were identified as potentially affecting interactions between nitrogenase components. Why didn't the authors test whether reverting these residues to their extant counterparts could improve nitrogenase activity of the ancestral variants?

We thank the reviewer for their thoughtful comments. We acknowledge that our current study is primarily focused on a computational exploration of the structural differences in both extant and ancestral nitrogenase variants, which allowed us to generate a comprehensive structural dataset. Although we did not carry out experimental reversion tests in this study, we agree that directly assessing the functional consequences of reverting the specific residues (lines 420 to 429) to their extant counterparts is an important next step to elucidate their functional role. Indeed, these findings provide a valuable foundation for our future work, which is designed to include experimental characterization of these variants and further elucidate the role of critical residues in nitrogenase activity and evolution. We believe that these experiments will offer the direct functional validation that the reviewer has rightly pointed out, and we look forward to reporting on these results in a future study.

Additionally, the paper feels somewhat disconnected. The predicted nitrogenase structures discussed in the first half of the manuscript were not well integrated with the findings from the ancestral structures. For instance, do the ancestral nitrogenase structures align with the predicted models? This comparison was never explicitly made and could have strengthened the study's conclusions.

We thank the reviewer for this suggestion. Our original analysis (previously shown in Figure S9, now Figure S10) included insights into structural align comparisons. In response, we have reorganized the results section (lines 351-355) to explicitly address this comparison.

Reviewer #2 (Public review):

This work aims to study the evolution of nitrogenases, understanding how their structure and function adapted to changes in the environment, including oxygen levels and changes in metal availability. The study predicts > 5000 structures of nitrogenases, corresponding to extant, ancestral, and alternative ancestral sequences. It is observed that structural variations in the nitrogenases correlate with phylogenetic relationships. The amount of data generated in this study represents a massive undertaking that is certain to be a resource for the community. The study also provides strong insight into how structural evolution correlates with environmental and biological phenotypes.

The challenge with this study is that all (or nearly all) of the quantitative analyses presented are based on RMSD calculations, many of which are under 2 angstroms. For all intents and purposes, two structures with RMSD < 2 angstroms could be considered 'structurally identical'. A lot of insight generated is based on minuscule differences in RMSD, for which it is not clear that they are significantly different. The suggestion would be to find a way to evaluate the RMSD metric and determine whether these values, as obtained for structures being compared, are reliable. Some options are provided in earlier studies: PMID: 11514933, PMID: 17218333, PMID: 11420449, PMID: 8289285 (and others). It could also be valuable to focus more on site-specific RMSDs rather than Global RMSDs. The high conservation in the nitrogenases likely ensures that the global RMSDs will remain low across the family. Focusing on specific regions might reveal interesting differences between clades that are more informative regarding the evolution of structure in tandem with environment/time.

We thank the reviewer for their suggestions. We agree that while global RMSD values below 2Å typically indicate high structural similarity, relying solely on these measures can mask subtle yet potentially functionally meaningful differences. Our aim was not to test for overall structural identity but rather to quantify fine-scale variations between highly conserved nitrogenase structures, including extant and ancestral variants. Nevertheless, in light of the reviewer’s suggestions, we have implemented an additional metric ( rmsd₁₀₀) for a more nuanced comparison. The results of our additional analyses (Figure S3) align closely with our original results (Figure 2), supporting our decision to retain the un-normalized results in the main text. As an additional measure, we also computed site-specific RMSDs for the active site’s environments (Figure S6) to further delineate subtle structural variations.