Metal Binding Proteins

Round 1

Reviewer 1 Report

The manuscript written by Dr. Permyakov describes the most relevant biochemical features of metalloproteins.

It is very well written and it is a sort of short textbook in bioinorganic chemistry. Pictures, on the contrary, are not of the highest quality. If possible, I encourage their improvement.

I have only three observations.

(i) Although this manuscript is focused on the biochemistry of metals, some information at the proteome level would be welcome. For example, about the fraction of proteins that are bound to metal cations etc.

(ii) Also, some additional information on metal cation cytotoxicity might enrich the quality of the manuscript.

(iii) An interesting nickel enzyme is missing: nickel superoxide dismutase (Proc Natl Acad Sci U S A. 2004 Jun 8;101(23):8569-74; Inorg Chem  2020 Apr 6;59(7):4772-4780).

Author Response

(i) Although this manuscript is focused on the biochemistry of metals, some information at the proteome level would be welcome. For example, about the fraction of proteins that are bound to metal cations etc.

I did not find such information. All I know is that more than a quarter of all the proteins contained in Protein Data Bank, possess bound metal ions. Most of all these proteins contain bound zinc ions, followed by iron, calcium and magnesium. I have added this information to the text (p. 1).

(ii) Also, some additional information on metal cation cytotoxicity might enrich the quality of the manuscript.

 I have added a chapter about metal cation toxicity.

(iii) An interesting nickel enzyme is missing: nickel superoxide dismutase (Proc Natl Acad Sci U S A. 2004 Jun 8;101(23):8569-74; Inorg Chem  2020 Apr 6;59(7):4772-4780).

Thank you, I have included this work in the text.

Reviewer 2 Report

The manuscript aims at a comprehensive overview of metal-binding proteins, which is by no means an easy task. The author made a substantial effort to provide useful information on a really diverse set of these proteins. I have a few recommendations, but not all of these are of equal importance, and there are many which I would find useful but I completely understand if the author would refrain from conforming to any of them.

My most important remark is that the oxydation states of the various metal ions should be clearly described and precisely used. For example, in lines 492-499 the author uses the notation Cu(I-IV), where the roman numbers refer to the identity of the copper ion, whereas in line 520 Fe(II) and Fe(III) refer to the oxydation state. I strongly suggest to use a unified nomenclature and/or clarification, if necessary. In addition., Cu(I) and Cu(II) are exisiting oxydation states of protein-bound copper, and Mn oxydation states in the oxygen-evolving complex of PSII should at least be mentioned.

On a related note, the manuscript could clearly benefit from providing a more detailed introduction on the following aspects:
- the metal ions with one oxydation state and those with two or more ones (I suggest maybe extending the periodic table or adding a novel summarizing table). I suggest to add the info on ion radiuses also here.
- the details of possible coordination modes (with schemes on different geometries, if possible)
- a short explanation on the role of metal-binding cofactors
- a brief explanation of "soft" and "hard" ligands 

The figures are generally of high quality but there are some with (at least for me) yellow background, I suggest to prepare uniform representations and to provide information about the molecular graphics software used. Some figures ocntain details that seem unnecessary and/or are not explained (e.g. Fig 9 shows a Tyr reside and also some atoms in wireframe).

I suggest to move Na/K-binding proteins up to Ca/Mg nbinding ones, and also adding the info about the in vivo concentrations of these ions..

The manuscript might also benefit of the inclusion of the CATH codes of the metal-binding domains, if the author also would think it useful, and/or some references to the RCSB PDB ligand browser on how to find protein structures with bound metal ions.

As the author mentions Cd(II) (Line 388), it might be interesting to add a short paragraph on cases where naturally bound ions are replaced in proteins by toxic ones (if the author thinks it fits the scope).

As the text is long, there are some misspellings and/or sentences to be rephrased. E.g.:

Line 162: canonic -> canonical
Line 165-66: 11-residues, 13-residues -> 11 residues, 13 residues (btw, here a sequence logo could be useful)Line 289: worse -> weaker
Line 322: the sentence makes no sense, please rephrase
Line 333: zinc atom -> zinc ion
Line 620: Ferritins is -> ferritins are
Line 700: polynucleotide (I doubt this is the correct word here)
Line 810: Squire pyramid -> Square pyramid

Author Response

My most important remark is that the oxydation states of the various metal ions should be clearly described and precisely used. For example, in lines 492-499 the author uses the notation Cu(I-IV), where the roman numbers refer to the identity of the copper ion, whereas in line 520 Fe(II) and Fe(III) refer to the oxydation state. I strongly suggest to use a unified nomenclature and/or clarification, if necessary. In addition., Cu(I) and Cu(II) are exisiting oxydation states of protein-bound copper, and Mn oxydation states in the oxygen-evolving complex of PSII should at least be mentioned.

I have changed the notation of Cu atoms on the p. 15. I have added some information about oxidation state of Mn in the oxygen evolving complex of PSII on p. 24.

On a related note, the manuscript could clearly benefit from providing a more detailed introduction on the following aspects:
- the metal ions with one oxydation state and those with two or more ones (I suggest maybe extending the periodic table or adding a novel summarizing table). I suggest to add the info on ion radiuses also here.
- the details of possible coordination modes (with schemes on different geometries, if possible)
- a short explanation on the role of metal-binding cofactors
- a brief explanation of "soft" and "hard" ligands 

I have added a table of ionic radii for various oxidation states of metals (p. 2).

As for possible coordination modes, they are so diverse that it would be necessary to compile a whole atlas of these structures. I tried to show the main ones in this short overview and they are distributed throughout the text.

I have provided a short explanation of “soft” and “hard” ligands (p. 3 and 4)

I have added some information on metal cofactors (p. 5)

The figures are generally of high quality but there are some with (at least for me) yellow background, I suggest to prepare uniform representations and to provide information about the molecular graphics software used. Some figures ocntain details that seem unnecessary and/or are not explained (e.g. Fig 9 shows a Tyr reside and also some atoms in wireframe).

I have uniformed the background of all the figures and gave the information about the molecular graphics software. I have tried to remove unnecessary datails from the figures.

I suggest to move Na/K-binding proteins up to Ca/Mg nbinding ones, and also adding the info about the in vivo concentrations of these ions..

I do not want to move Na/K-binding proteins up to Ca/Mg-binding ones since first I want to provide information about proteins with high affinities to metal ions. I have added information about in vivo concentration of Na and K ions (p. 25).

The manuscript might also benefit of the inclusion of the CATH codes of the metal-binding domains, if the author also would think it useful, and/or some references to the RCSB PDB ligand browser on how to find protein structures with bound metal ions

In principle, it is possible to use http://www.cathdb.info site to look for some metal binding domains, but I think this service is still underdeveloped for metal binding proteins

As the author mentions Cd(II) (Line 388), it might be interesting to add a short paragraph on cases where naturally bound ions are replaced in proteins by toxic ones (if the author thinks it fits the scope).

I have added a chapter on toxic metals (p. 27).

As the text is long, there are some misspellings and/or sentences to be rephrased. E.g.:

Line 162: canonic -> canonical corrected
Line 165-66: 11-residues, 13-residues -> 11 residues, 13 residues (btw, here a sequence logo could be useful) corrected Line 289: worse -> weaker corrected
Line 322: the sentence makes no sense, please rephrase corrected
Line 333: zinc atom -> zinc ion corrected
Line 620: Ferritins is -> ferritins are corrected
Line 700: polynucleotide (I doubt this is the correct word here) corrected
Line 810: Squire pyramid -> Square pyramid corrected

Round 2

Reviewer 1 Report

In my opinion, no further modifications are necessary.

Reviewer 2 Report

I thank the author for considering and answering all my comments.