Charged Amino Acid Substitutions Affect Conformation of Neuroglobin and Cytochrome c Heme Groups

Round 1

Reviewer 1 Report

The authors have studied the heme conformational changes using RRS and SERS after mutations of Ngb and CytC. The results are interesting and encouraging and do support the conclusions.

However, it would be very insightful if the following data could be obtained:

1) measurement of electron flow in these mutants

2) expression of these mutants in a neuronal cell line and study how they affect the protection against apoptosis

Minor:

In the Introduction, second paragraph, Ngb implications in neuronal survival was mentioned (ref 10-20). Please elaborate more on this point. 

Author Response

We appreciate the time and effort the reviewer has dedicated to reviewing the manuscript. In this revised version, we have addressed the requests raised by the reviewer, and the changes have been marked in yellow for easy tracking.

The authors have studied the heme conformational changes using RRS and SERS after mutations of Ngb and CytC. The results are interesting and encouraging and do support the conclusions.

However, it would be very insightful if the following data could be obtained:

1) measurement of electron flow in these mutants

2) expression of these mutants in a neuronal cell line and study how they affect the protection against apoptosis

Answer. 1) We are currently carrying out a Raman spectroscopy study of the redox interaction of Ngb and Cyt c and their mutant forms in various combinations. However, due to the large volume of experimental material, as well as some technical difficulties in processing and analyzing the data obtained, we plan to write a separate manuscript based on these results.

2) To date, we have constructed a vector containing the Ngb WT gene for transfection of cell lines, and a model transient line based on HEK293 cells has been obtained. We chose this cell line because it lacks the Ngb gene, unlike neuronal lines. Preliminary experiments were carried out on the induction of apoptosis via the cytochrome c-dependent pathway, an apoptosis inducer was selected, and conditions for visualizing apoptotic cells were selected (see Figure in the attachment). We next plan to quantitatively characterize the neuroprotective effects of Ngb under conditions of oxidative stress, both for the wild type of the protein and for its mutant forms. However, due to the large volume of experimental material, after the completion of the study, we plan to present the results of this work as a separate publication.

Minor:

In the Introduction, second paragraph, Ngb implications in neuronal survival was mentioned (ref 10-20). Please elaborate more on this point. 

Answer. We added a fragment to the Introduction:

Significant amounts of evidence support a physiological role of Ngb in promotion of neuronal survival in conditions such as Alzheimer's and Huntington's diseases, oxidative stress, stroke, ischemia, hypoxia, retinal degeneration, etc. [10–20]. Ngb-mediated neuroprotection proposed to be due to Ngb involvement in various biochemical cascades of the cell [10,12,14,15,20]. Ngb has the ability to detoxify reactive oxygen and nitrogen species [11,13]. Furthermore, protein-protein interactions between Ngb and voltage-depended anion channels, α-subunit of heterotrimeric G-protein, and mitochondrial cytochrome c (Cyt c) can also play crucial role in Ngb-mediated promotion of neuronal survival [14,20–22].

Author Response File: Author Response.pdf

Reviewer 2 Report

I think that the manuscript entitled "Charged Amino Acid Substitutions Affect Conformation of Neuroglobin and Cytochrome c Heme Groups" is in principle suited for publication in CIMB, Section "Biochemistry, Molecular and Cellular Biology." The article explores the conformational changes induced by amino acid substitutions in Ngb and Cyt c mutants. Understanding the conformational changes in proteins like Ngb and Cyt c is crucial for elucidating their biological functions, particularly their roles in electron transfer processes. The interpretation of surface-enhanced Raman spectroscopy (SERS) and resonance Raman spectroscopy (RRS) data is well-supported by theoretical frameworks, ensuring the scientific soundness of the study. However, I have some remarks, especially regarding the figures presented in the manuscript.

 Comments:

Lines 57-60. "Ngb amino acid residues involved in interaction with Cyt c belong to E and F α-helices, while most of Cyt c interaction residues belong to either Cyt c universal binding site or non-ordered red Ω-loop (70–85) [29–33]." I believe this information pertains not to all cited references #29-33, but mainly to 32,33. Additionally, in the cited reference 33, it is not specified that Ω-loop (70–85) is "non-ordered red." Is this information from the authors or other researchers? Please check.

Lines 433-441. These lines need to be revised as they are duplicated in the work https://doi.org/10.1134/S1068162023060274.

Figure 1. I think the figure is cropped and not fully represented. Please check.

Figures 2,3. Why are the intensity peak value axes not included in the graphs?

Lines 278-279. "Dotted lines show the maximum positions of the most intensive peaks." Why are the peaks not marked on the graphs in the regions 1220-1260 cm-1 (Figure 3a) and 680-720 cm-1 (Figure 3b)?

Author Response

We appreciate the time and effort the reviewer has dedicated to reviewing the manuscript. In this revised version, we have addressed the requests raised by the reviewer, and the changes have been marked in yellow for easy tracking.

I think that the manuscript entitled "Charged Amino Acid Substitutions Affect Conformation of Neuroglobin and Cytochrome c Heme Groups" is in principle suited for publication in CIMB, Section "Biochemistry, Molecular and Cellular Biology." The article explores the conformational changes induced by amino acid substitutions in Ngb and Cyt c mutants. Understanding the conformational changes in proteins like Ngb and Cyt c is crucial for elucidating their biological functions, particularly their roles in electron transfer processes. The interpretation of surface-enhanced Raman spectroscopy (SERS) and resonance Raman spectroscopy (RRS) data is well-supported by theoretical frameworks, ensuring the scientific soundness of the study. However, I have some remarks, especially regarding the figures presented in the manuscript.

 Comments:

1. Lines 57-60. "Ngb amino acid residues involved in interaction with Cyt c belong to E and F α-helices, while most of Cyt c interaction residues belong to either Cyt c universal binding site or non-ordered red Ω-loop (70–85) [29–33]." I believe this information pertains not to all cited references #29-33, but mainly to 32,33. Additionally, in the cited reference 33, it is not specified that Ω-loop (70–85) is "non-ordered red." Is this information from the authors or other researchers? Please check.

Answer 1. We checked the references and made corrections to the text:

Ngb amino acid residues involved in interaction with Cyt c belong to E and F α-helices, while most of Cyt c interaction residues belong to either Cyt c universal binding site or Ω-loop (70–85) [32,33]. Glu60 and Glu87 (Ngb) presumably interact with Cyt c residues Lys72 and Lys25 [29–31].

2. Lines 433-441. These lines need to be revised as they are duplicated in the work https://doi.org/10.1134/S1068162023060274.

Answer 2. We have shortened and rewritten this fragment:

The Soret bands in the UV-vis absorption spectra of Ngb E60K and K95E mutants are characterized by small shifts (1-3 nm) (Fig. S2, a and b). In addition, spectra of reduced forms of Ngb E60K and E60K/E87K characterized by a well-defined Q-peak (582 nm). Aforementioned spectral features may be due to a change in the spin state of heme iron and a change in the electrostatic field near the heme [47,48].

3. Figure 1. I think the figure is cropped and not fully represented. Please check.

Answer 3. We checked the Figure 1and made changes to it (c) and (d) so that it does not appear cropped.

4. Figures 2,3. Why are the intensity peak value axes not included in the graphs?

Answer 4.1. We do not use absolute intensities of Raman or SERS peaks, but we employ ratios of peak intensities. So, for better representation each Raman or SERS spectrum was normalized to the sum of intensities in the whole spectral range and all spectra were shifted vertically one over another. We added vertical scale bars for the intensity peak and corrected the figure captions.

Lines 278-279. "Dotted lines show the maximum positions of the most intensive peaks." Why are the peaks not marked on the graphs in the regions 1220-1260 cm-1 (Figure 3a) and 680-720 cm-1 (Figure 3b)?

Answer 4.2. The peak with maximum position at 690 cm^-1 corresponds to the symmetrical vibrations of pirrol rings in cytochrome C. We did not focus on this peak, since it was more convenient to use the more intense peak at 1374 cm^-1, which is also associated with symmetrical vibrations of the pyrrole rings.

The peak at 1233 cm^-1 originate from the protein bonds vibrations (-CN-, -NH-, so-called amide III Raman peak). So, it is not a heme peak and therefore we did not use it to analyse heme properties. Hence, we did not show them in Picture 3 in order not to overload the image.

In addition, we have made a number of corrections to the Materials & Methods and Conclusions sections (see manuscript), and adjusted the statement of the aim of this study.

Round 2

Reviewer 1 Report

Adding new data, as suggested, would improve the manuscript.

OK

Author Response

We appreciate the time and effort the reviewer has dedicated to reviewing the manuscript. In this revised version, we have addressed the requests raised by the reviewer, and the changes have been marked in yellow for easy tracking.

Adding new data, as suggested, would improve the manuscript.

Answer: We agree that additional data can improve almost any manuscript, including the one under review. However, we believe that within the framework of the set aim and the proposed research methods, we have obtained detailed information about the conformational changes in the hemes of cytochrome c and neuroglobin when replacing charged amino acid residues involved in the formation of the protein interaction interface. Such results may be of interest as a separate publication for a wide range of CIMB readers.

We are currently carrying out studies of the redox interaction of cytochrome c and neuroglobin, as well as their mutant variants in various combinations using Raman spectroscopy. Resonance Raman spectroscopy is a promising method to study the electron transfer between ferrous neuroglobin and ferric cytochrome c. This approach is relevant because both proteins in question are heme proteins, and the redox state of heme changes during the electron transfer. Moreover, neuroglobin and cytochrome c hemes belong to different types (hemes b and c), therefore, their Raman spectra have characteristic peaks that make it possible to distinguish the signal from the heme of each protein when recording their joint spectrum. Due to the high rate of autoxidation of human neuroglobin, which causes technical difficulties in detecting the redox reaction occurring between reduced neuroglobin and oxidized cytochrome c, we are conducting a large-scale study, within the framework of which we have developed an original protocol for detecting this interaction using difference Raman spectra. At the moment, the data obtained for various combinations of mutant proteins are being processed and verified; we plan to prepare a manuscript based on these materials later.

As for the studies on the model cell line described in response 1 of the first round of review, we have so far obtained preliminary results in this area, which require systematization, verification and continued research on mutant variants of neuroglobin.

In connection with the above, it is not possible for us to supplement this article with any additional materials.