The Amino Acids Motif -32GSSYN36- in the Catalytic Domain of E. coli Flavorubredoxin NO Reductase Is Essential for Its Activity
, Susana F. Fernandes, Bruno A. Salgueiro, Jéssica C. Soares, Célia V. Romão, Cláudio M. Soares, Diana Lousa, Filipe Folgosa * and Miguel Teixeira *Round 1
Reviewer 1 Report
The paper submitted by Martins et al. present a study on flavodiiron proteins (FDPs) activity to understand the role of amino acids motif -G[S,T]SYN- in the catalytic domain by experimental data and molecular modelling. While the results seem interesting and prove the authors conclusions, I have major concern of the novelty and the importance of this work. In this regard, prior resubmission, I suggest the authors to focus on these points:
- Improve language style of the whole manuscript
- Rephrase the abstract
- Improve the aim of the work at the end of the introduction i.e. what is the practical scope of the paper?
- Could the authors comment on how did measure ~250 μM of O2 for amperometric study?
- Greatly improve the number of reference adding more substantial work on the introduction and discussion of results
Author Response
The paper submitted by Martins et al. present a study on flavodiiron proteins (FDPs) activity to understand the role of amino acids motif -G[S,T]SYN- in the catalytic domain by experimental data and molecular modelling. While the results seem interesting and prove the authors conclusions, I have major concern of the novelty and the importance of this work. In this regard, prior resubmission, I suggest the authors to focus on these points:
- Improve language style of the whole manuscript
The whole manuscript was carefully edited to improve the language style.
- Rephrase the abstract
The abstract was rephrased.
- Improve the aim of the work at the end of the introduction i.e. what is the practical scope of the paper?
As stated in the Introduction, our aim was to assess the role of a highly conserved motif in flavodiiron proteins, particularly taking into account that the determinants of their substrate specificity are still unknown, in spite the large number of 3D structures already available.
- Could the authors comment on how did measure ~250 μM of O2 for amperometric study?
As described in the “Methods” section of the manuscript, the amperometric studies were performed in a Clark-type electrodes selective for O2 (Oxygraph-2K, Oroboros Instruments, Innsbruck, Austria). In each experiment, the buffer was allowed to equilibrate at air while oxygen concentration was monitored by the electrode (considering its internal measurements of the atmospheric pressure). At the end of each assay, a sodium dithionite solution was added to the assay to establish the “zero” oxygen concentration. Therefore, the oxygen concentration of each assay was determined to be between 250 and 260 mM. These values are in accordance with the previously determined by Benson and Krause in 1980 (Limnology and Oceanography, 25, 1980, 662-671), i.e. ~8.3 mg/L (~259 mM) at 25oC and 1atm.
- Greatly improve the number of reference adding more substantial work on the introduction and discussion of results
Both the introduction, Results and Conclusions sections were modified, and more relevant references were included.
Reviewer 2 Report
The manuscript described the essential role of a strictly conserved amino acids motif -G[S,T]SYN- close to the catalytic diiron center in E. coli FIRd, especially for NO reduction. Through the construction of site-directed mutants, the general characteristics of wild type enzyme and mutants were compared, including cofactor content, thermalstability, integrity of the diiron center and their reductase activity towards NO and O2. Molecular modelling and structural analysis revealed the mutants affect the hydration of internal cavities, product release and/or proton uptake.
The manuscripts appears carefully prepared and the results are well-documented. I only have few minor comments to improve the paper:
Page 1, Line 15: FlRd’s activity; Does FIRd specifically represent the enzyme from E. coli?
Page 2, Line 53: Abbreviation of ¨FMN¨ should be defined when it first showed up.
Page 2, Line 78-79: The author should give an explanation why these mutants were constructed in this study.
Page 2, Line 84: “G-[S,T,I]-[T,S]-Y-N-[A,S]-Y-]F,L,A]-[I,M,V]” should be “G-[S,T,I]-[T,S]-Y-N-[A,S]-Y-[F,L,A]-[I,M,V]”.
Page 3, Line 89-90: “the only variation being the first serine residue which, in some cases, is conservatively substituted by a threonine” is not rigorous, the second serine can be substituted by a threonine as well, and the tyrosine can be substituted by alanine or serine.
Page 4, Line 152-154: “with the exceptions of S34V and Y35F mutants which present melting temperatures ca 10 ºC lower when compared with the other mutants and with the wt enzyme.” Could the author explain or suspect the reasons of melting temperature decrease of the two mutants S34V and Y35F? While includes S34V in the double mutant (S33V_S34V), no decrease anymore.
Figure S1: No wild type was found in the legend.
Figure 8 is missing in the main text.
Page 11, Line 369-370: If the author can discuss the catalytic mechanism difference between NO reduction and O2 reduction, will better explain the results observed for NO and O2 activity.
Comments for author File: 
Comments.pdf
Author Response
The manuscript described the essential role of a strictly conserved amino acids motif -G[S,T]SYN- close to the catalytic diiron center in E. coli FIRd, especially for NO reduction. Through the construction of site-directed mutants, the general characteristics of wild type enzyme and mutants were compared, including cofactor content, thermal stability, integrity of the diiron center and their reductase activity towards NO and O2. Molecular modelling and structural analysis revealed the mutants affect the hydration of internal cavities, product release and/or proton uptake.
The manuscripts appears carefully prepared and the results are well-documented. I only have few minor comments to improve the paper:
- Page 1, Line 15: FlRd’s activity; Does FIRd specifically represent the enzyme from E. coli?
Yes, in line 15, “FlRd” refers to the FDP (or flavorubredoxin) from E. coli. It was clarified in the manuscript.
- Page 2, Line 53: Abbreviation of ¨FMN¨ should be defined when it first showed up.
Corrected in the manuscript.
- Page 2, Line 78-79: The author should give an explanation why these mutants were constructed in this study.
A brief explanation about the intention of the mutants under study was given in the final part of the introduction section.
- Page 2, Line 84: “G-[S,T,I]-[T,S]-Y-N-[A,S]-Y-]F,L,A]-[I,M,V]” should be “G-[S,T,I]-[T,S]-Y-N-[A,S]-Y-[F,L,A]-[I,M,V]”.
Corrected in the manuscript.
Page 3, Line 89-90: “the only variation being the first serine residue which, in some cases, is conservatively substituted by a threonine” is not rigorous, the second serine can be substituted by a threonine as well, and the tyrosine can be substituted by alanine or serine.
The sentence mentioned by the reviewer refers only to class B FDPs (flavorubredoxins), i.e. from all the FDPs from class B sequences available, the only variation observed is in the first serine, which is substituted by a threonine.
Page 4, Line 152-154: “with the exceptions of S34V and Y35F mutants which present melting temperatures ca 10 ºC lower when compared with the other mutants and with the wt enzyme.” Could the author explain or suspect the reasons of melting temperature decrease of the two mutants S34V and Y35F? While includes S34V in the double mutant (S33V_S34V), no decrease anymore.
At the present moment, we have no explanation for the decrease of the melting temperature of these mutants, as the homology models built to not show any significant changes in the mutants compared to the wt protein. We have to have 3D structures of those mutants to clarify the observed differences.
Figure S1: No wild type was found in the legend.
Corrected in the figure.
Figure 8 is missing in the main text.
Corrected in the manuscript.
Page 11, Line 369-370: If the author can discuss the catalytic mechanism difference between NO reduction and O2 reduction, will better explain the results observed for NO and O2 activity.
The “Conclusion” section of the manuscript was modified to include a short discussion of the so far proposed NO reduction mechanism, and a corresponding figure was included 8now Figure 9). It is also important to note that there is no information in the literature regarding the oxygen reduction mechanism by FDPs.
Round 2
Reviewer 1 Report
I have no further comment
