Determining large hyperfine interactions of a model flavoprotein in the semiquinone state by pulse-EPR techniques&nbsp;

Martinez, Jesús Ignacio; Frago, Susana; Medina, Milagos; García-Rubio, Inés

doi:https://doi.org/10.5194/mr-2024-24

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https://doi.org/10.5194/mr-2024-24

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17 Jan 2025

| 17 Jan 2025

Status: a revised version of this preprint is currently under review for the journal MR.

Determining large hyperfine interactions of a model flavoprotein in the semiquinone state by pulse-EPR techniques

Jesús Ignacio Martinez, Susana Frago, Milagos Medina, and Inés García-Rubio

Abstract. Flavoproteins are a versatile class of proteins involved in numerous biological processes, including redox reactions, electron transfer, and signal transduction, often relying on their ability to stabilize different oxidation states of their flavin cofactor. A critical feature of flavin cofactors is their capacity to achieve, within particular protein environments, a semiquinone state that plays a pivotal role in mediating single-electron transfer events and is key to understanding flavoprotein reactivity.

Hyperfine interactions between the unpaired electron in the semiquinone state and magnetic nuclei in the isoalloxazine ring provide valuable insights into the electronic structure of this intermediate and its mechanistic roles. This study investigates the hyperfine interactions of isotopically labeled flavodoxin (Fld) with ¹³C and ¹⁵N at specific positions of the flavin mononucleotide (FMN) ring using advanced electron paramagnetic resonance (EPR) techniques. The combination of Continuous wave (CW) EPR at X-band and ELDOR-detected NMR and HYSCORE at Q-band revealed a strong and anisotropic hyperfine interaction with the nucleus ¹³C at 4a and yielded principal tensor values of 40, -13.5 and -9 MHz, the first of which is associated to the axis perpendicular to the flavin plane. On the other hand, as predicted, the hyperfine interaction with the ¹³C nucleus at position 2 was minimal. Additionally, HYSCORE experiments on ¹⁵N-FMN labeled Fld provided precisely axial hyperfine parameters, (74, 5.6, 5.6) MHz for ¹⁵N(5) and (38, 3.2, 3.2) MHz for ¹⁵N(10). These were used to refine quadrupole tensor values for ¹⁴N nuclei via isotope-dependent scaling. These results showcase the potential of combining CW-EPR, ELDOR-detected NMR, and HYSCORE with isotopic labelling to probe electronic and nuclear interactions in flavoproteins. The new data complete and refine the existing experimental map for the electronic structure of the flavin cofactor and expose systematic divergences between the calculated and experimental values of hyperfine couplings of the atoms most contributing to the SOMO. This could indicate a slight but significant shift of the unpaired electron density from position 4a towards the central nitrogens of the pyrazine ring as compared to the calculations. These results highlight the importance of integrating computational and experimental approaches to refine our understanding of flavin cofactor reactivity.

Received: 27 Dec 2024 – Discussion started: 17 Jan 2025

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Jesús Ignacio Martinez, Susana Frago, Milagos Medina, and Inés García-Rubio

Status: final response (author comments only)

RC1:
'Comment on mr-2024-24', Anonymous Referee #1, 30 Jan 2025
Summary:
The manuscript presents an experimental study on hyperfine couplings in flavin mononucleotide (FMN) within isotopically labeled flavodoxin using a combination of X-band CW EPR, Q-band HYSCORE, and EDNMR. The detection of hyperfine interactions for C4a and two nitrogen nuclei (N5 and N10) expands the experimental database of FMN hyperfine couplings and provides an interesting comparison to previously calculated values, particularly for C4a. These results are of relevance to the magnetic resonance (MR) community, making this manuscript a valuable contribution.
However, the manuscript requires revisions before it can be accepted for publication. The, experimental procedures and data analysis need improvement to enhance clarity and consistency. Additionally, a more structured discussion would strengthen the manuscript’s argumentation. Below are specific and technical comments that should be addressed.
Experimental parameters:
different experimental temperatures were used for the experiments according to the experimental section (50 K and 90 K, line 161 and 172) yet Fig. 5 (line 362) describes 80 K experiments.

HYSCORE was performed successfully with multiple tau values to determine the N coupling parameters (Figs. 5 and 6) yet only a single tau value was used for the C coupling detection. That is hard to understand, especially because the features the authors use to argue in Fig.4 c are clearly affected by tau blindspots.

What was the power of the EDNMR HTA pulse, and why were the parameters chosen?

The experimental section discusses FID-detected EPR spectra at Q band, presumably to set up the EDNMR experiments. Fig 3 discusses Echo detected spectra (line 284). What was done?

Orientation Selection and Simulations:
The origin of the orientation selection calculations in the figures is not described and, therefore, is hard to comprehend. Was a simulation of the EPR spectrum the basis for this? If so, why are they not shown?

Comments about the orientation selection in X band spectra do not necessarily hold true at Q band and should, therefore, not be made. (e.g. Fig 3)

The “complete nuclear system” (line 196) is not described.

Was orientation-selection considered in the HYSCORE simulations using saffron ? (Figure S1)

Experimental Hyperfine Spectra:
Highlight the features in EDNMR and HYSCORE, that are described in the text, in the figures.

Error of measurement parameters:
Line 245: The 0.3 mT error of /Delta{B}_out can be understood but the resulting error of 0.4 mT for the difference is unclear. The sum of the two errors should then be 0.6 mT.

The errors of the experimental hyperfine values are not explained at all, and the quadrupole coupling values are given without error. Do the errors result from performing multiple simulations? How were they judged?

Discussion
the discussion should be more concise, particularly regarding the agreement and discrepancies between experimental and calculated hyperfine parameters. This description extends over a full page and lacks a structured progression of points.

Figure 1:
The figure should appear way earlier to make the introduction argument more understandable.

Illustrating the spin density would improve the paper’s argumentation and the author’s choice of investigated nuclei.

Highlight the investigated nuclei in the structure

The orientation of the g-Tensor should be shown in the illustration.

Something in the rendering of the chemical structure has gone wrong, causing the misalignment of atoms and chemical bonds.

Spin Hamiltonian: Clarify the confusing indexing of SH parameters
Line 202: “n different nuclear spins (I_i)” – i should be used to avoid confusion with mu_n

Equation 1 then uses l and i as subscript. Only one is necessary to differentiate the nuclei.

g_n is also nucleus specific and should, therefore have the subscript i

the subscripts x,y,z ; 1,2,3 and parallel, perpendicular are used very interchangeably throughout the text, which causes considerable confusion

Further comments:
The authors make no comment about the data and code availability.

Line 36-40: Make it clear which reference belongs to which given example. Consider splitting the sentence to make it more readable.

Line 46: “the isoalloxazine ring”

Line 46-48: What is meant by this sentence, it is very confusing

Line 49-40: “ the ability to exchange …”

Line 69: How can protons be within the ring?

Line 73-75: Explain the quadrupole coupling aspect here, “is also helpful” is not an explanation

Line 75: I(15N)=1/2

Line 75: Which nitrogen nuclei? How do these studies relate to this new work?

Line 99: through?

Line 151: \mu M, not mM

Line 206 and 386: Authors cite Fuchs et al. 2002, the paper does not appear in the reference list.

Line 212: “use to be sensibly large” – what is meant by this?

Line 230-231: Calling the broad shoulders in the CW EPR spectra hyperfine splittings is a bit much.

Line 260-264: Rephrase the description of the EDNMR experiment. The HTA pulse is frequency-swept, not the detection sequence.

Line 270 and 280: Those are clearly not CW spectra but the echo detected spectra at Q band.

Figure 3. Subtraction, not Substraction

Line 273: “recorded at the center of the EPR spectrum”

Line 289: 2x \nu_L(13C) = 26 MHz, not 13. , “strong coupling regime”, In the strong coupling regime, signals in EDNMR are centered at A/2 and split by 2 \nu_L. The fact that the spectrum is asymmetric with regard to the central blindspot is also an important factor. The conclusion drawn here is right but the argument must be made better and with the correct values.

Line 320: the spectrum of 2C is in panel a) on the left, not the right.

Line 323 – 325: “ribbon shaped” and “streamer” are arbitrary words. Use indicators in the figure.

Line 338: The “proper simulations” of the intensities do not agree with the intensities detected experimentally. They are also not discussed further. What is the point then?

Line 432: ”Q band EDNMR and HYSCORE“

Line 440: Cryptochromes were not mentioned in the introduction of this paper. “… values should be …”

Line 444: “or if they vary”
Citation: https://doi.org/10.5194/mr-2024-24-RC1
- AC3:
  'Reply on RC1', Inés García-Rubio, 08 Mar 2025
  Section “Experimental parameters”:
  1.- Erratum in temperature (p. 362) has been corrected.
  2.- HYSCORE experiments were measured at several tau values for the 13C-labelled samples (tau = 96, 112, 124, 176 and 144 ns for the central position, for example). In all of the spectra, the spectral features due to 13C(4) are very, very weak, yet, the spectrum shown in figure 4 of the manuscript (tau=112 ns) a bit better quality than the others. 13C features, which are very near the noise level show much better in the spectrum for tau=112 ns because it has a slightly better s/n ratio, that is why we decided to display it alone in the manuscript. The following figure, is the sum of the spectra corresponding to 124, 176 and 144 ns. As one can see, the 13C features are clearly there, but the spectrum in the manuscript is able to show them better. Moreover, no evident feature present in any other spectra is missing in the spectrum for tau 112 ns.
  
  3.- To set and optimize the parameters, a single 1000 ns pulse was set and its power optimized to produce an FID. The FID integrated intensity was recorded as a function of the magnetic field. Then, the position of the magnetic field was fixed either at the center of the field-swept spectrum or at the high-end tail and an initial HTA ELDOR pulse was added. The ELDOR channel attenuation was initially set to 0 db, and several ELDOR-detected NMR spectra were taken varying the length of the ELDOR pulse from 1000 to 5000 ns. Then, the operation was repeated for several levels of ELDOR mw power. From the resulting spectra, the best s/n was found for HTA 1000 ns long and 0 db ELDOR power attenuation, so these parameters were adopted for longer accumulation of the spectra. The interpulse delay was chosen to be 1500 ns, long enough to let the potential FID of the first pulse decay. Information on the power of the HTA pulse and how the parameters were chosen has been added to the experimental section.
  4.- Both, FID- detected and Echo-detected spectra were recorded. The referee is perfectly right, the FID-detected field-sweep experiments were recorded as a part of the set-up procedure, but in the figure, echo-detected experiments are shown, just to indicate the field position where the ELDOR-detected NMR were taken.
  Section “Orientation Selección and Simulations:
  1.- Yes, the calculations of the orientation selection patterns is based on the simulation of the Q-band CW spectrum. An X-band simulation is now shown in Fig. 2 together with the experimental spectra.
  2.- Misleading comment on orientation selection in Fig. 3 caption and text has been changed. The orientation selection patterns calculated for Q-band frequencies, and shown in the figure show the orientations being excited for the spectra recorded at the maximum of the EPR absorption and the high-field tail. The patterns are, again, based on the simulation of the spectrum at Q-band frequencies, so, they should be reliable. This pattern, for the magnetic field set to the maximum of the spectrum shows that all orientations are excited. While the orientations in the plane of the flavin ring contribute more, there is still some contribution of the orientations close to the perpendicular to the plane.
  3.- The position of the nuclear features on the HYSCORE spectra were first calculated taking into account single nuclei one by one using endorfreq. Then, the simulation of the spectra was done with saffron using the complete set of nuclei which want to be simulated in order to obtain the feature’s intensities and combination lines. We have done some rephrasing in the manuscript in order to clarify this paragraph, the referee was right in pointing out some confusion.
  4.- In the HYSCORE simulations using saffron, orientation selection was indeed considered.
  
  Section “Experimental Hyperfina Spectra”:
  - Experimental features described in the text has been highlighted (Figs. 3 and 4).
  
  Section “Error of measured parameters”:
  1.- Error in the expression (4) should be propagated from the individual errors to the subtraction. This is obtained from the squared root of the sum of the squared individual errors. The value ±0.4 mT is properly calculated.
  2.- We have added on page 18 a brief explanation on how the errors in the parameters of the Spin Hamiltonian were estimated. The errors for the estimated quadrupole couplings have been added as well.
  Section “Discussion”:
  - Following the reviewer’s suggestion, discussion has been shortened and restructured in order to make it clearer.
  Section “Figure 1”:
  - Suggested changes in the position and information collected in Fig. 1 have been implemented.
  Section “Spin Hamiltonian”:
  - SH equations and subindexes for tensor principal values have been corrected, simplified and explained.
  Section “Further Comments”:
  Upon acceptance, raw data and simulation code will be made available in a open-access repository.
  
  Typos have been corrected.
  
  The paragraph describing results and analysis of EDNMR experiments has been rewritten in order to make it more clear (see also “ELDOR-detected NMR “suggestions of reviewer 2).
  
  The description of the EDNMR experiment (former 260-264 lines) was rephrased.
  
  It is true that the intensities of the 13C(4) simulations do not reproduce well the experimental spectra. However the position of the HYSCORE features already allows estimating the magnitude of the hyperfine coupling of the nucleus with high precision.
  
  Citation: https://doi.org/10.5194/mr-2024-24-AC3
RC2:
'Comment on mr-2024-24', Anonymous Referee #2, 15 Feb 2025

Thanks to a combination of advanced EPR techniques, isotope labelling and a multifrequency approach the authors perform a detailed characterisation of the electron spin delocalisation over the isoalloxazine ring of a flavin semiquinone radical, with a special focus on positions characterised by the highest spin density, for which only limited data is currently available.
The Introduction paragraph provides the required background to understand the specific research question addressed by this work. Experimental data and the related analysis are presented in a clear way; in the Discussions section, these are interpreted in light of previous experimental and computational studies on similar systems.

Specific comments as well as technical corrections are highlighted in the attached document.

Citation: https://doi.org/10.5194/mr-2024-24-RC2
- AC2: 'Reply on RC2', Inés García-Rubio, 08 Mar 2025
  
  Reviewer 2
  Specific comments
  Materials & Methods:
  Point 1.- The applied phase cycle has been referred in the text.
  Point 2.- Information on the power of the ELDOR pulse and the process of sequence optimization has now been included in the text.
  Point 3.- The reference was added to the text. Fuchs et al. 2002.
  
  ELDOR-detected NMR
  Point1.- The text was changed in the manuscript to correct the mistake.
  Point2.- The corrections have been implemented in the text. We thank the reviewer for spotting these errors.
  Point3.- Although the study was performed at W-band, Davies ENDOR was used in Schleicher et al 2021 to obtain the couplings of 13C-labeled flavins but the signal of 13C(4) nucleus was not detected. The success of EDNMR as compared to ENDOR might have to do with the transitions being partially forbidden. This clarification has been added to the text.
  Point 4.- The detection frequency was placed off center in the resonator dip. The measurements of both samples were tuned as similarly as possible in order to minimize problems with comparison of the spectra. We do not think the signal at 11 MHz is (at least entirely) an artifact of the subtraction spectrum due to different acquisition conditions, which were kept as close as possible, since in the parameters of 13C(4) obtained from HYSCORE spectra also predict one of the nuclear frequencies to be about this magnitude for orientations close to the parallel orientation.
  The referee is right about the labeling of the x-axis in the EDNMR spectra, we have changed it in the new version of the manuscript. We also have softened the statement about not having orientation selection at the center of the spectrum and we have detailed the spin Hamiltonian used for calculation of the orientation selection patterns. The patterns have been corrected since there was a mistake with the original excitation bandwidth, which is now explicitly mentioned.
  Points 5, 6, 7, 8 and 10.- Description of the ¹³C(4a) features in the EDNMR spectrum and analysis has been revised and rewritten following the reviewer’s suggestions in order to make it more accurate and more clear.
  Point 9.- Several HTA pulse lengths and powers were tried, as indicated now I the Materials & Methods section. However these trial spectra were performed in order to spot the best s/n ratios in order to optimize the HTA pulse. Unfortunately, no detailed comparison was performed on the signal widths.
  13C HYSCORE
  Point 1.- Here is one full spectrum for [13C(2,4a)-FMN]-FLd
  
  Far from being devoid of signals, the (-+) quadrant is dominated by 14N nitrogen signals but there is no evident 13C signals observed in the (-+) quadrant. A careful comparison between spectra of [13C(2,4a)-FMN]-FLd and [13C(2)-FMN]-FLd does not bring any signals that could be attributed to 13C(4a) in the (-+) quadrant. Therefore, when we wanted to focus of the 13C signals we have only shown only the (++) quadrant.
  Point 3.- We agree with reviewer that representation in squared frequency axes would give a first estimation of the hyperfine parameters. We considered that we could skip this step of the analysis in the manuscript in order to make it lighter, as we already obtained a first estimation of the parameters form our X-band CW-EPR and Q-band EDNMR results that are enough for the simulation refinement.
  Point 4.- Typo in line 334 has been corrected.
  Point 5.- We have added on page 18 a brief explanation on how the uncertainties in the parameters of the Spin Hamiltonian were estimated.
  Point 6.- Specific references to figures in the supplementary material have been added at all points were the Supplementary Material was referred to.
  Points 7-8.- Reviewer’s suggestions on Figs. S1 and S2 have been followed. Note that although the approximate field position with respect to the EPR spectrum is the same in Figs 3a & S2, the exact field position is different due to differences in the microwave frequencies. EDNMR and HYSCORE were performed at different spectrometers at different times.
  
  ¹⁵N and ¹⁴N HSYCORE
  Points 1 & 2.-Figures 5 and 6. Due to the reviewer’s comment, all field positions at which the experiments were performed were checked and, indeed, some inconsistencies together with flat-out errors were found. For example, the magnetic field values of the EDNMR experiments were swap, so we realized the tail experiments were actually performed at the high-field tail and not the low-field tail of the EPR spectrum. After careful verification, we believe the field positions and corresponding diagrams are now correct in all figures. About the reason to choose the high-field end: Since the dominating anisotropy in the EPR spectrum is due to the axial hyperfine couplings of N(5), N(10), which are much larger in the direction perpendicular to the isoaloxacine plane, positioning the magnetic field at any of those ends would select the “parallel” orientation. The high-field end was chosen because there is a small g-anisotropy, which at Qband gives a slightly better orientation selection for the high-field end.
  Point 3.- In order to have the best possible orientation selection, we tried to do the experiments at the highest possible magnetic field that gives a good signal. For 14N signals this was possible at a quite high field. On the other hand, in the ¹⁵N labeled sample such a field position did not provide usable modulations and we had to move to a lower field. This is most probably related to the quadrupole coupling providing a way to mix the nuclear levels and yielding forbidden transitions that give good modulation of the echo. For ¹⁵N, the quadrupole interaction is missing and for an orientation very close to the perpendicular to the plane, which is an eigenaxis of the hyperfine coupling, the transitions are allowed and no modulation is observed.
  The difference in the field setting between the spectra of the two samples is due to the difference in the microwave frequency of the two experiments.
  Point 4.- The sum of the spectra was performed after Fourier transformation. Since the echo is strongly modulated, its intensity varies very much along the time trace. However, no significant difference in the envelope intensity is worth mentioning between tau = 96 ns and tau = 168 ns.
  
  Point 5.- For the sake of clarity, in Figure 5 we have removed all antidiagonal lines except the one of ¹⁵N.
  Point 6.- The sample is not deuterated but since some unidentified signals appear to lie on this diagonal, the antidiagonal line was drawn in the analysis phase. We have removed this antidiagonal line in order not to generate confusion.
  Point 7.- After the display of the hyperfine parameters in the text, we have introduced a small paragraph explaining how the uncertainties were estimated. Within the mentioned uncertainty (+- 0.3 MHz) the shape of the correlation ridges is not compatible with rhombicity above the mentioned uncertainty. On the (++) quadrant, two separate and well defined peaks were found assigned to the parallel features of each of the two nitrogen nuclei. If there was any moderate rhombicity these features would be smeared out into a ridge.
  We thank the reviewer’s thorough comments, which allowed to identify and correct the above mentioned mistakes and increase the quality of the article.
  Technical corrections.- All typos spotted by the reviewer were corrected.
  
  Citation: https://doi.org/10.5194/mr-2024-24-AC2
- AC4: 'Reply on RC2', Inés García-Rubio, 08 Mar 2025
  
  Here is the pdf of the reply because the figures did not come out properly
  
  Citation: https://doi.org/10.5194/mr-2024-24-AC4
RC3:
'Comment on mr-2024-24', Anonymous Referee #3, 23 Feb 2025

Inés Garcia-Rubio and coworkers present EPR measurements to investigate the electronic structure of the flavin mononucleotide (FMN) cofactor in Anabaena flavodoxin. Specifically, cw-EPR as well as ELDOR-detected NMR and HYSCORE are applied to obtain isotropic as well as anisotropic contributions of hyperfine interactions (and also ¹⁴N quadrupole couplings) of those flavin positions that carry the highest electron spin densities: ¹³C(4a), N(5) and N(10). The present study serves to complement earlier investigations by the Zaragoza group as well as by others, thereby addressing certain gaps in the existing literature.
The presented experiments (partly conducted in the EPR labs of Jeschke at the ETH Zurich) and their analyses were carefully carried out and deserve to be published in “Magnetic Resonance”, provided that a few points are taken into account.
One rather strong statement is that there are needs “to also improve other aspects of electronic structure prediction” (see lines 487 to 488). This statement is based on the significant overestimation of the (isotropic and anisotropic) hyperfine coupling of ¹³C(4a) by (quantum chemical) calculations and the underestimation of the respective couplings from N(5) and N(10). However, some of the referenced hyperfine predictions are quite old (2002: Garcia, JPCA 106 (2002) 4729) or refer to another protein (photolyase: Schleicher, Sci. Rep. 11 (2021) 18234). The above assertion is therefore quite bold and would have to be substantiated by new calculations according to the current state of the art. Have the authors tried to obtain a better agreement between the newly measured hyperfine couplings and their predictions by systematically adjusting input structures and/or theoretical methods? Clearly, hyperfine couplings of flavins seem not to vary a lot among different flavoprotein surroundings – the authors mention this is lines 102 to 105 – but a renewed effort would nevertheless be worthwhile.
With regard to the above-mentioned statement (see lines 102 to 105) that hyperfine couplings of flavins in different proteins do not differ greatly, it would be useful to compare such couplings with those of flavin in isotropic solution. Corresponding data for the ¹³C nuclei in the isoalloxazine ring were recently published, see J. Phys. Chem. Lett. 13 (2022) 5160. This paper could also be listed at the end of page 2, where “the characterization of hyperfine structure using different techniques …” is mentioned.
The authors state (in lines 44 to 45) that “the semiquinone state is usually not detectable in redox processes involving free flavins, …”. Work published in JACS 140 (2018) 16521 shows that stabilization of the flavin radical in aqueous solution is well possible by trapping in agarose matrices.
The authors make use of the widening of the two shoulders (named “O1” and “O2”) in CW-EPR spectra (see Fig. 2) for the determination of A_z(13C4a). Spectral simulations would be quite helpful to substantiate such a simple analysis.
Figures 3 to 6: The respective insets (with information on orientation selection) are far too small! The small print cannot be recognized (due to low resolution) even on zooming in.
Referencing should be improved in various places:
Line 45: “… but in some flavoproteins it is highly stabilized by the protein environment.” At least some examples should be given.
Lines 82 to 83: “… because the N(5)-C(4a) locus of the flavin concentrates most of its chemical prowess.” Some references should be given.
Lines 119 to 120: “… that can have a relevant effect on the mechanisms where flavin is involved.” Some examples should be given.
Lines 465: “… the previously mentioned underestimation of the hyperfine splitting …”

The respective references should be listed here (again).
Lines 468 to 470: “It is worth noting that reported calculations …” The respective references should be given here (again).
Lines 490 to 491: “… which may have important consequences on the understanding of the electron transfer mechanism that specifically involve these positions.” Some references should be given.
Lines 495 to 496: “For example, more electron density in position C(4a) would favor reoxidation whereas more electron density in N(5) would promote hydride transfer.” This statement needs some references.
The authors should correct various typos:
Line 53: “electron transfer pathways” instead of “electron tranfer pathways”.
Line 60: “magnetoreception” instead of “magnetorecepcion”.
Line 99: “through” instead of “though”
Lines 218 and 418: “isoalloxazine” instead of “isoaloxacine”

Other (minor) points:
Section 2.2.1 is “CW-EPR” but contains also a description of pulse EPR experiments.
Section 2.3 (line 190): Simulations of CW-EPR are mentioned but none of them shown in the manuscript (and also not in the supporting information).
Line 240: Equation 3 includes terms A_z(N5) and A_z(N10), but in Equation 2 these are named A_parallel. This is not wrong, but somewhat inconsistent.
Line 320: “The spectrum of …, on the right, shows …” should be “on the left”.
Line 424: Please replace “… was never obtained for this protein …” with “… was never obtained for Fld ….”.
Line 440: Please correct “… so the reported experimental values of should be very useful …”
Line 466: Please correct “… the ability of the of the …”

Citation: https://doi.org/10.5194/mr-2024-24-RC3
- AC5: 'Reply on RC3', Inés García-Rubio, 10 Mar 2025
  
  We thank reviewer 3 for the appreciation of our paper and the constructive comments. Here are the replies to the specific points: 3^rd paragraph (on additional hyperfine calculations).- We appreciate the reviewer's comments and agree with him that further effort should be made to improve the available calculations. On the other hand, we believe that such work is beyond the scope of the present manuscript. In this paper, we mainly show the experimental approach to obtain the hyperfine parameters for some specific nuclei in the flavoprotein. These show relevant differences with those predicted by the calculations, and this point is an important problem in two aspects: first, this probably indicates that the calculations do not adequately predict some details of the flavin structure within the protein; second, in some cases the calculated hyperfine parameters are used to understand specific properties, for example in the development of models for the magnetochemistry involved in the chemical compasses of birds, and these values could be inaccurate. Developing better calculations to overcome these problems is a very interesting open topic for future research, but we do not consider it for the present paper. 4th and 5^th paragraphs (Statements and references on flavins in solution and semiquinone state in free flavins): Reference suggested by the reviewer were included and commented in the text.
  
  6^th paragraph (use of X-band CW-EPR spectra): We agree with the reviewer that the use of the CW-EPR spectrum to determine A_z hyperfine parameter for ¹³C4a nucleus should require the support of simulations. However, in this case we only make use of this evidence to infer a first approximation of the parameter, wich is then refined from EDNMR and Q-band HYSCORE experiments. In the latter case, we do make use of the corresponding simulation to accurately determine the hyperfine parameters. On the other hand, most of the signal broadening of the X-band CW-EPR spectra is due to unresolved g anisotropy and dozens of weak hyperfine splittings that cannot be properly introduced into the simulation. Therefore, we consider that such simulations are not necessary in our case.
  7^th paragraph (Figs. 3 to 6): inserts related with orientation selection were changed to increase readibility.
  On references: New references were added in all the places suggested by the reviewer.
  Also typos and minor points were corrected.
  
  Citation: https://doi.org/10.5194/mr-2024-24-AC5
AC1: 'Comment on mr-2024-24', Inés García-Rubio, 04 Mar 2025

We will submit the revised manuscript and answers to reviewers shortly

Citation: https://doi.org/10.5194/mr-2024-24-AC1
EC1: 'Upload revised manuscript', Nino Wili, 12 Mar 2025

Dear Authors,
thank you for your detailed answers. Please submit the revised manuscript to the MS records.

Citation: https://doi.org/10.5194/mr-2024-24-EC1

Jesús Ignacio Martinez, Susana Frago, Milagos Medina, and Inés García-Rubio

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https://doi.org/10.5194/mr-2024-24-supplement

Jesús Ignacio Martinez, Susana Frago, Milagos Medina, and Inés García-Rubio

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Short summary

Flavoproteins are essential biomolecules involved in many vital processes. Understanding their reactivity requires studying their intermediate semiquinone form. Using advanced spectroscopy and isotopic labeling, we measured key interactions within this state, uncovering subtle changes in the distribution of electron density. These findings refine our understanding of flavoprotein function and highlight the value of combining experiments and computations for studying complex biological systems.


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