Peer review of: An evolutionary path to altered cofactor specificity in a metalloenzyme (2020)

Reviewers' comments: 

 

Reviewer #1 (Remarks to the Author): 

 

In this paper, the authors present a study on S. aureus Fe/Mn- and Mn-dependent SODs, showing 
that the different metal specificity of the two enzymes depends on subtle structural factors located 
to the second coordination sphere of the metal ion. The work is well designed and the results are a 
valuable contribution to research in the field of bioinorganic chemistry and beyond. I thus expect 
that this paper will obtain broad interest throughout the scientific community. Nevertheless, I think 
that the authors somehow overstated the novelty of their results: specifically, at the end of 
discussion they state that their study “represents the first experimental evidence of how a change in 
metal specificity has naturally evolved in a metalloenzyme”, and that they demonstrate “a critical 
and surprising role for secondary coordination sphere residues in mediating this specificity switch”. 
In fact, the evolution of metal specificity in metalloenzymes has already been addressed several 
times in the past; also, the importance of the second coordination sphere in modulating the physico-
chemical properties of metal-binding sites in proteins, including metal identity, is a steady concept in 
bioinorganic chemistry. Some examples can be found in, e.g., J Biol Inorg Chem. 2019 Jun;24(4):517-
531, J Inorg Biochem. 2018 Feb;179:40-53, J Biol Chem. 2015 Jul 31;290(31):18914-23, Acc Chem 
Res. 2014 Oct 21;47(10):3110-7, Protein Sci. 2010 Jan;19(1):75-84, Biochemistry. 2005 Jun 
14;44(23):8378-86 

 

 

Reviewer #2 (Remarks to the Author): 

 

I have read the manuscript entitled An evolutionary path to altered cofactor specificity in a 
metalloenzyme by Barwinska-Sendra et al and have a number of comments. 

The ability to examine the evolution of the two SODs and say something about metal use and metal 
stress is remarkable. To introduce mutations into an enzyme and alter the specificity of metal 
utilization is also impressive. With regards to the latter point, however, I do have some comments. 

There was a paper published in 2018 (Hunter et al, Chem. Eur. J 2018 24 5303) that reported on a 
single mutation that changed the metal specificity of the MnSOD in C. elegans such that it became 
cambialistic. The modification was not to a metal-binding ligand but was in the second sphere. It is 
not analogous to modification reported here as there were proton interactions but it does warrant 
mentioning as a single mutation has changed metal specificity. 

The FeSOD has been shown in most cases to carry out a robust Fenton-type reaction with the 
peroxide formed in the dismutase reaction. This either leads to enzyme deactivation or to formation 
of a powerful oxidant in solution. The MnSOD has not been shown to carry out any of this 

peroxidative chemistry. This has certainly led to some speculation as to why certain species use 
certain of these SOD in general and is also worth noting here even though it is more obscure in the S. 
aureus. 

In Table 1, in all cases the enzymes with high cambialistic ratios have overall lower activity than the 
enzymes that are specific for a metal. This is a truism for virtually all naturally occurring cambialistic 
enzymes; there is a loss in the overall activity associated with the ability to bind either metal and 
have activity. I don’t believe that this is necessarily associated with changes in auto-oxidation of 
cambialistic Mn/FeSODs. The autooxidation results and speculated connection with redox potential 
changes is, for me, particularly significant and worth discussing in a little more detail. 

Overall, I find this to be a compelling paper. 

 

 

 

Reviewer #3 (Remarks to the Author): 

 

In this study, Anna Barwinska-Sendra and co-workers report on the characterization of point-
mutants in superoxide dismutases (SODs) that alter their relative metal reactivity. The authors chose 
two SODs from S. aureus, the former corresponding to a Mn-specific SOD and the latter to a 
cambialistic one (i.e. able to react either using Fe or Mn as metal cofactor). They combined amino 
acid sequence and X-ray structure comparisons to identify three sites likely responsible for 
explaining such changes in metal reactivity. They subsequently swapped amino acids from one to 
another SOD and observed indeed changes in metal reactivity that they attributed to changes in 
redox potentials due to changes in the second sphere of coordination environment. Determining the 
factors that can alter metal cofactor reactivity are important for both medicine and biotechnological 
applications. Overall the work is robust and well conducted. The authors have carefully checked, 
using X-ray crystallography at high-resolution, that the mutations do not affect either the whole 
structure or the metal environment. They provide an extensive bioinformatic analysis to complete 
their work showing that most likely, the cambialistic SODs derive from Mn-specific ones, which 
would have further evolved after a gene duplication event. However, the study lacks metal binding 
affinity constant measurements to better characterize the effect of these mutations. A more 
systematic approach to identify which amino acid would be the best ones at these positions to either 
select Mn- or Fe-dependent activity. The “cambialistic” ratio is indeed informative, but for some of 
the mutants, the drop in the overall activity seems more important than the change in metal-
reactivity. Indeed, while the Fe-dependent activity increases (from a low level) the Mn-dependent 
one seems to collapse, leading to a better “cambialistic” ratio that may be artificially overestimated. 
The authors have clearly identified a very interesting factor but the reviewer remains a bit 
disappointed because one would have expected some explanations about why/how changing these 
residues alter the metal reactivity. For example new redox potential determinations would have 
been appreciated. What is really modified when changing the second coordination sphere? This 
information would have been valuable for future applications in developing synthetic 

metalloenzymes. In the reviewer’s opinion, the manuscript does not fit the broader readership of 
Nature Communication and would be more suitable to a specialized journal. 

 

Minor comments: 

The authors refer to Mn(II) oxidation state in line 197, but it sounds more to electronic structure or 
redox potential because as it is Mn(II) the oxidation state cannot be modified unless it is not Mn(II) 
any more. Please clarify. 

The authors often refer to metal specificity. The reviewer would suggest metal reactivity because 
metal specificity would better correspond to either Mn or Fe preferentially bound to the active site. 
Please clarify this point. 

 

 

 

Reviewer #4 (Remarks to the Author): 

 

This paper aims to identify key structural and physical properties changes that allow SOD enzymes 
and mutants thereof from a bacterium to utilize two different ions, Mn and Fe. HF EPR is used to 
determine the zero-field interaction of the various mutants which is very sensitive to the electronic 
structure and coordination symmetry. The spectral differences are shown in Figure 3a which are 
small but certainly significant. The authors then site a paper where in model compounds these 
differences in zero-field interaction (D and E) are correlated with redox potential. As this is an 
enzyme and the paper aims to characterise structural and physical differences, the actually redox 
potentials should be measured to confirm this is actually true in this case. This data could be 
obtained by an EPR redox titration, clearly the Authors demonstrate that the EPR signal is quite 
strong and so this type of EPR experiment should be feasible (at X-band or W-band) even if a bit 
tedious. The Authors state in the conclusions and elsewhere that the alter redox potential is a main 
cause of the change in reactivity, but it was never measured. The uncertainly in this model complex 
correlation to this enzyme is highlighted by Supplementary Figure 5 were D/E ratios do not fit the 
model compounds. The redox potentials needs to be measured for the paper to be impactful, as the 
Authors write in their conclusion on page 13, line 319: ‘enabling future studies to determine how the 
architecture of a metalloenzyme imposes cofactor specificity’. Meaningful redox potentials would be 
very important for this future aim. 

 

Minor points 

On page 7, line `63, the Authors state that the positions of the mutated residues are no altered. This 
is at first a confusing statement because they must mean the positon of the backbone as the side-

chains are clearly different. This sentence and the next ones (like page 7, line 176 & page 8, line 194) 
should be rewritten. In this regard, the accuracy of the X-ray structure should be discussed, in 
particular the bond lengths and angles around the Mn ion (i.e. describe briefly Supplementary Table 
4 and the errors). 

 

Page 8, line 186. ‘zero field interaction’, zero-field is one word and needs the hyphen 

 

Figure 3A and tables Supplementary 5: The HFEPR spectra were analyzed using second-order 
expressions, which should be accurate enough to extract the D and E values (Supplementary Table 
5). However, it would be beneficial to the reader to include a simulation of each spectrum using a 
full matrix diagonalization approach. This is a very easy task today and numerous free software 
packages are available to achieve this. These spectra should appear in the Supporting Material.