Probing the Bioinorganic Chemistry of Cu(I) with 111Ag Perturbed Angular Correlation (PAC) Spectroscopy
Round 1
Reviewer 1 Report
The manuscript of V. Karner et al. reviews the uses of perturbed angular correlation of gamma-spectroscopy of 111Ag/111mCd pair for probing metal-binding sites of copper-containing proteins. The manuscript is interesting and has significant impact because of the rising interest in Cu metabolism and the lack of sensitive analytical methods to study Cu-containing proteins. There are some recommendations below that could enhance the relevance of the review. Still, the manuscript may be published as is, without revision.
1. Some notes on absolute and relative method sensitivity (the amount of protein/isotope needed for NQI estimation, its comparison to competing methods) are strongly desirable. Will it is possible to use it for in vivo studies?
2. It would be good to provide some basic information about the 111Ag isotope and its benefits (sources and availability, handling, presence/absence of Auger emission)
3. It is worth mentioning in section 3.1 that H2C[M] (type I copper site) of azurin/plastocyanin is a very important class of Cu-binding sites. One or several type I copper sites are present in many Cu- proteins, most notably in blue multicopper oxidases (laccases, ascorbate oxidase, ceruloplasmin, haephestin…).
4. Concerning section 3.5.1. We wish the authors luck, but even non-specific replacement of redox-active Cu by Ag(I) has been very challenging for most Cu-enzymes. To our knowledge, only type I copper sites (H2C[M]) are readily loaded with Ag(I) in vivo. Silver-SOD1 or properly folded silver-containing multicopper oxidases fail to form either in vivo or in vitro. It may be because of the coupling between ion oxidation and protein folding (it is almost surely so for ceruloplasmin, see PMID 20965708) or because ionic radius of Ag(I) is distinctly larger the radius of Cu(I) (the sites in many oxidoreductases are very tight). If Cu ions are removed from Cu-oxidoreductases by brute force (e.g. by cyanide), the proteins denaturate irreversibly and no longer bind either Cu or Ag (such studies were done for ceruloplasmin and laccase after their discovery).
5. In section 3.5, it may be also worth mentioning Met-only Cu(I)/Ag(I)-binding sites. It is an emerging group of copper-binding sites in Cu-enzymes and Cu-transporting proteins (e.g. CTR1 and CTR2 channels, see also a peculiar silver/laccase record 5AFA in PDB). Apparently, such sites bind the metal ions transiently, and the described method with nanosecond response time may be of great interest.
Author Response
Dear reviewer,
We thank you for your feedback on our manuscript. Please see the attachment below for a point-by-point response to your comments.
Sincerely,
Victoria L. Karner
Author Response File: 
Author Response.pdf
Reviewer 2 Report
The present minireview is related to the application of 111Ag PAC spectroscopy for investigation of Cu(I) bioinorganic chemistry. The review is well written and can be accepted after consideration of the following issues:
1) Authors should include more keywords.
2) Introductions, lines 34 - 37: the appropriate references should be included.
3) The abbreviations should be defined at their first appearance.
Additional comments:
4) The main question addressed by the present minireview is the use of 111Ag PAC spectroscopy in identification of Cu(I) binding sites in biological systems.
5) The minireview on this topic could be relevant in the field of bioinorganic chemistry considering the significance of copper(I/II) in biological systems.
6) The article provides an overview of the results achieved in the application of 111Ag PAC spectroscopy for analysis of copper proteins, as well as possible future application in the field.
7) The cited references are appropriate. In some parts of the text references are missing, e.g. Introduction section, lines 34 - 37: the appropriate references should be included.
8) The Figures are appropriate. They can be of higher resolution.
Author Response
Dear reviewer,
We thank you for your feedback on our manuscript. Please see the attachment below for a point-by-point response to your comments.
Sincerely,
Victoria L. Karner
Author Response File: Author Response.pdf
