In this report Camponeschi et al. present NMR assignments of the dimeric membrane-anchored human CDGSH protein (“mitoNEET”). Each subunit contains a Fe₂S₂ cluster and the current study aims at investigating its electronic properties in both the oxidized and the reduced states. Due to the paramagnetism of the cluster the resonance assignment requires separate sets of experiments for residues located outside a ~ 10 Å sphere and residues near the cluster. The former involves standard 3D backbone triple-resonance and side-chain experiments. In order to reduce the “blind sphere” around the cluster and observe very fast relaxing resonances 2D ¹⁵N-IR-HSQC-AP, developed a couple of years ago by one of the authors, and protonless ¹³C-detected CON experiments were carried out. Finally, a number of protons from Fe-coordinating residues were assigned using 1D NOE experiments in conjunction with X-ray structure derived distances. Conclusions about the electron distribution within the Fe₂S₂ cluster were drawn from the envelope of the hyperfine-shifted spectral region, which has some functional implications.

Although I am not an expert for iron-sulfur proteins it appears to me that the system studied here is of high biological relevance (nice literature overview in the Introduction, by the way). Overall, the manuscript is very well-written, except for paragraph 3.2.2 (see below). It describes sound experimental work and comprehensible interpretation of the results. However, it mostly represents an application of established techniques to a well-studied protein. Considering its editorial policy the current paper falls outside the scope of Magnetic Resonance.

Paragraph 3.2.2 (“Paramagnetic experiments on [Fe₂S₂]-mitoNEET reduced and oxidized”) is to a large extent phrased in a lab-style language and should be rewritten.

Some examples:

Heading: are the experiments paramagnetic or the samples ?

Lines 190, 194, 200: “Spectra were recorded on a Bruker Av600 MHz” (spectrometer ?)

Line 198: “Each experiment consisted from ~ 300k up to ~900k scans.”

Line 202: “…using 16.5 ms and 13.7 ms as acquisition and a t_1max delay....”

Line 209: “…2048 scans each fid were collected…”

Line 211: “…the IPAP approach was used for homodecoupling…” Does that mean virtual decoupling of ¹J_C’Ca ?

Further minor issues:

According to the Material and Methods section ¹⁵N relaxation experiments (R₁, R₂, hetNOE) were performed for the diamagnetic part of the protein. The purpose and result of these experiments is not reported at any point in the manuscript.

Proton 1D spectra were recorded with a spectral width of 320 ppm, much wider than required for spectral range observed here. Were any specialized wide-band pulses employed that would be able to excite a ~ 200-kHz region ?

It is mentioned that the CON experiment was optimized for paramagnetic systems (section 4.2.3). Which modifications were applied ? Simply shorter magnetization transfer periods ? An INEPT delay of 8 ms is specified in the experimental section, which is shorter than 1/(2*¹J_C’Ca). Is that sufficient to incorporate the IPAP module ?

In this study, Camponeschi et al use NMR to characterize mitoNEET, a mitochondrial Fe2S2 protein. By using 1D NOE experiments, 13C direct-detected experiments, and the optimization of NMR experiments for paramagnetic systems, the authors show significantly reduction of the “blind” sphere of the protein around the paramagnetic cluster, thus allowing the detection of residues possibly involved in the biological function of mitoNEET. The study has significant implications in the fields of paramagnetic NMR and FeS proteins. Some revisions are recommended. 

In details: 

1. I have some general questions about the mitoNEET protein I hope the authors can help answer. 

a) If  mitoNEET can repair Fe-S proteins by donating its own Fe2S2 cluster, how does it reacquire the Fe2S2 cluster? Can the authors comment on the source of its Fe2S2 cluster? 

b) The redox states of mitoNEET are crucial for its function and stability. How are the redox states of mitoNEET regulated in cells? 

2. Some experimental details are needed. 

a) For M9 media growth, how much (15NH4)2SO4 and 13C-glucose were supplemented? 

b) What kind of anaerobic environment was used? 

c) Does the phosphate buffer contain any NaCl? 

d) I assume there were additional steps to remove the extra K4Fe(CN)6 or sodium dithionite? 

2) What's the Fe2S2: protein ratio 'as purified'? It would be helpful to include UV data to show the load of Fe2S2 on the protein in both redox states. 

3) The authors purified the protein in an anaerobic environment, I assume it's because the Fe2S2 is susceptible to oxidative damage. Would addition of 10mM K4Fe(CN)6 to the protein solution damage the Fe2S2 cluster? 

4) Is the purified mitoNEET protein a homodimer as shown in Fig. 1A? 

5) In Fig. 1A, can the authors highlight the residues that are affected by different redox states? 

6) Fig 1B, how were the chemical shift differences between two redox states calculated? 

7) It's intriguing to me that the redox state change would mainly affect the regions involved in inter-subunit contacts. Do the authors have any hypothesis why? 

8) There is no mention of Fig. 1C in the text. The author might add some. 

9) Can the authors provide some explanations why no hyperfine shifted signals were observed for the reduced [Fe2S2]+-bound form of mitoNEET? 

10) The authors should provide the data showing the broadening of signal B collected in D2O. 

11) The authors might want to highlight the additional residues assigned by 15N-IR-HSQC-AP in the structure of mitoNEET. 

12) The labels in Fig.3 are too small to read, the authors might want to improve that.