Author Response
The following is the authors’ response to the original reviews.
Reviewer #1 (Public Review):
“First, I agree with the authors of this manuscript that conformational changes in the XFEL structures with 2.8 A resolution are not reliable enough for demonstrating the subtle changes in the electron transfer events in this bacterial photosynthesis system. Actually, the data statistics in the paper by Dods et al. showed that the high-resolution range of some of the XFEL datasets may include pretty high noise (low CC1/2 and high Rsplit) so the comparison of the subtle conformational changes of the structures is problematic.
The manuscript by Gai Nishikawa investigated time-dependent changes in the energetics of the electron transfer pathway based on the structures by Dods et al. by calculating redox potential of the active and inactive branches in the structures and found no clear link between the time-dependent structural changes and the electron transfer events in the XFEL structures published by Dods, R.et al. (2021). This study provided validation for the interpretation of the structures of those electron-transferring proteins.
The paper was well prepared.”
Thank you very much for your positive and insightful comment. We greatly appreciate your suggestion regarding the high noise levels of the XFEL structures, as indicated by the low CC1/2 and high Rsplit values reported by Dods et al. Including this information in the Introduction section will draw readers’ attention to the concerns about the reliability of these XFEL structures. We have incorporated the following sentences into the Introduction section:
“Furthermore, the data statistics provided by Dods et al. indicate that the high-resolution range of some XFEL datasets exhibit high levels of noise, as evidenced by low CC1/2 and high Rsplit values. These observations raise concerns about the reliable comparison of subtle conformational changes among these structures. Hence, caution must be exercised when interpreting these XFEL structures in terms of their ability to accurately capture relevant conformational changes.”
The following sentences have also been added to the Conclusions section:
“Hence, it is crucial to exercise caution when interpreting time-dependent XFEL structures, especially in the absence of comprehensive evaluations of the energetics and accompanying structural changes. This cautionary note should serve as a counterargument in the future, highlighting the potential pitfalls associated with presenting time-dependent XFEL structures of insufficient quality and drawing conclusive interpretations of protein structural changes that may not be distinguishable from significant experimental errors.”
Recommendations for the authors
“Figure 1 needs clear labels or detailed notes in the figure legend for the labels such as M, L, Pm, Pl, etc.”
In Figure 1, we have increased the size of the labels to improve visibility. Additionally, we have expanded the figure legend to include detailed explanations of the abbreviations used, such as M, L, PM, PL, etc. We believe that these modifications have significantly improved the clarity and comprehensibility of Figure 1.
Reviewer #2 (Public Review):
“The manuscript by Nishikawa et al. addresses time-dependent changes in the electron transfer energetics in the photosynthetic reaction center from Blastochloris viridis, whose time-dependent structural changes upon light illumination were recently demonstrated by time-resolved serial femtosecond crystallography (SFX) using X-ray free-electron laser (XFEL) (Dods et al., Nature, 2021). Based on the redox potential Em values of bacteriopheophytin in the electron transfer active branch (BL) by solving the linear Poisson-Boltzmann equation, the authors found that Em(HL) values in the charge-separated 5-ps structure obtained by XFEL are not clearly changed, suggesting that the P+HL- state is not stabilized owing to protein reorganization. Furthermore, chlorin ring deformation upon HL- formation, which was expected from their QM/MM calculation, is not recognized in the 5-ps XFEL structure. Then the authors concluded that the structural changes in the XFEL structures are not related to the actual time course of charge separation. They argued that their calculated changes in Em and chlorin ring deformations using the XEFL structures may reflect the experimental errors rather than the real structural changes; they mentioned this problem is due to the fact that the XFEL structures were obtained at not high resolutions (mostly at 2.8 Å). I consider that their systematic calculations may suggest a useful theoretical interpretation of the XFEL study. However, the present manuscript insists as a whole negatively that the experimental errors may hamper to provide the actual structural changes relevant to the electron transfer events. My concerns are the following two points:
Is the premise of the authors for the electron transfer energetics obviously valid?
Could the authors find any positive aspect(s) in the XFEL study?
The authors' argument is certainly due to their premise "Em(HL) is expected to be exclusively higher in the 5-ps and 20-ps structures than in the other XFEL structures due to the stabilization of the [PLPM]•+HL•- state by protein reorganization" as noted in the Results and Discussion (p. 12, lines 180-182); however, it is unknown whether this premise can be applied to the ps-timescale electron transfer events. The above premise is surely based on the Marcus theory, as the authors also noted in the Introduction "The anionic state formation induces not only reorganization of the protein environment (ref. 5: Marcus and Sutin, 1985) but also out-of-plane distortion of the chlorin ring (ref. 6: two of the authors, Saito and Ishikita, co-authored, 2012)"; however, it is unknown whether protein reorganization can follow the ps-timescale electron transfer events. Indeed, Dods et al. mentioned in the Nature paper (2021) "The primary electron-transfer step from SP (special pair PLPM) to BPhL (HL) occurs in 2.8 {plus minus} 0.2 ps across a distance of 10 Å by means of a two-step hopping mechanism via the monomeric BChL molecule and is more rapid than conventional Marcus theory". It was also mentioned, "By contrast, the 9 Å electron-transfer step from BPhL to QA has a single exponential decay time of 230 {plus minus} 30 ps, which is consistent with conventional Marcus theory". As for the primary electron-transfer step from PLPM to HL, Wang et al. (2007, Science 316, 747; cited as ref. 8 in the Nature paper 2021) reported, by monitoring tryptophan absorbance changes in various reaction centers in which the driving forces (namely, the Em gaps between PLPM and HL) are different, that the protein relaxation kinetics is independent of the charge separation kinetics on the picosecond timescale. On the other hand, in the EPR study cited by the authors as ref. 7 (Muh et al. (1998) Biochemistry 37, 13066), although the authors described "two distinct conformations of HL- were reported in spectroscopic studies" (p. 3, lines 44-45), it should be noted that conformation of HL- was formed by 1 or 45 s illumination prior to freezing, and hence the second-order reorganized conformations may differ from picosecond-order conformations observed by the XFEL study (Nature, 2021) and/or the transient absorption spectroscopy (Science, 2007).
Therefore, I consider there is a possibility that the authors' findings may reflect not experimental errors but the actual ps-timescale phenomena presented by the first-time XFEL study on the timescale of the primary charge-separation reactions of photosynthesis. Thus I would like to suggest that the authors reconsider the premise for the electron transfer energetics on the picosecond timescale.
In any case, to discuss the experimental errors in the XFEL study, it is better to calculate the Em(QA) changes in the 300-ps and 8-us XFEL structures, which showed distinctive structural changes even at the 2.8 Å resolution as discussed by Dods et al. Then, if the Em(QA) values are changed as expected from theoretical calculations, such calculated results may suggest a useful theoretical interpretation of the XFEL study as a positive aspect. If the Em(QA) values are not higher in the 300-ps and 8-us structures than in the other structures, it may be argued that the experimental errors would be so large that the XFEL structures are irrelevant to the electron transfer events expected from theoretical calculations.”
We appreciate the reviewer's constructive suggestions, which significantly contributed to the improvement of our manuscript. We have performed additional calculations to address the reviewer's suggestion. We calculated the changes in Em(QA) in the XFEL structures. The Em(QA) values in the 300-ps and 8-μs structures were not significantly higher than those in the other structures (Figure 8).
These findings align with the scenario proposed by the reviewer, suggesting that the experimental errors are substantial, rendering the XFEL structures irrelevant to the electron transfer events. The results further reinforce our argument that the observed structural changes in the XFEL structures are not directly linked to the expected changes in electron transfer events.
We have incorporated these important points into the revised version as follows:
“One might argue that the loss of the link between the formation of the charge-separated state and the Em(HL) change (Figure 5) is not due to experimental errors but rather represents the actual ps-timescale phenomena during the primary charge-separation reactions (e.g., Dods et al. noted that “the primary electron-transfer step to HL is more rapid than conventional Marcus theory” 8). However, even if this were the case, this hypothesis regarding the relevance of the XFEL structures to the electron-transfer events can be further explored by examining the changes in Em(QA) among the XFEL structures, considering the relatively slow electron-transfer step to QA that allows sufficient protein relaxation to occur (e.g., Dods et al. stated that “the electron-transfer step to QA has a single exponential decay time of 230 ± 30 ps, consistent with conventional Marcus theory” 8). That is, if the Em(QA) values are not higher in the 300-ps and 8-μs structures than in the other structures, it suggests that significant experimental errors exist, rendering the XFEL structures irrelevant to the electron transfer events. Consistent with this perspective, the present results demonstrate that the Em(QA) values in the 300-ps and 8-μs structures are not significantly higher than those in the other structures, including the dark state structure (Figure 8). Consequently, the lack of a clear relationship between the charge separated state and the changes in Em(QA) at 300 ps and 8-μs further strengthens the argument that the XFEL structures are irrelevant to the electron transfer events.”
Recommendations for the authors
“In addition to my main concerns, the following points should also be taken into consideration:
The authors presented from QM/MM calculations out-plane distortion of HL (and HM) induced upon the reduction using the dark structure for dataset a (Table 5). However, to compare with the XFEL structures corresponding to the charge-separated state [PLPM]+HL-, positive charge should be located at the special pair (or, either PL or PM). In the present work, it is noted that counter ions were added to neutralize the entire system (in Methods: p. 6, lines104-105), but the location(s) of the positive charge is unclear.”
We appreciate the valuable suggestion provided by the reviewer. To address this concern, we have calculated out-of-plane distortion of HL•– in the presence of PL•+. The results have been included in Table 5. Note that the results obtained in the presence of PL•+ are substantially the same as those obtained in PL0 (Table 5).
For clarity, we have rephrased the sentence referring to counter ions as follows:
“To neutralize the entire system, counter ions were added randomly around the protein using the Autoionize plugin in VMD 22.”
“In relation to the calculations, the authors showed the induced out-plane distortion of HM for dataset a; however, the results for HM seem not to be mentioned anywhere. Instead, the calculations for HL of the dark structure for dataset b should be useful, especially for comparing with the time-dependent changes in the dataset b XFEL structures as shown in Figure 7.”
We have made Table 6 to present the results for dataset b. The results are consistent with those for dataset a (Table 5).
