Electron Transfer via Helical Oligopeptide to Laccase Including Chiral Schiff Base Copper Mediators
Round 1
Reviewer 1 Report
Working in the domain of bioelectrochemistry, I will judge mostly this part of the manuscript, leaving the complexes synthesis and characterization to more relevant reviewers.
This manuscript treats of the impact of chirality of the polypeptide linkers on the electron transfer between the electrode and laccase with docked mediator. Although the overall idea of the authors to use chiral polypeptides to inject electrons to laccase seems appealing for me, the manuscript doesn’t contain experimental evidences that the authors actually achieved any electron transfer between laccase and electrode. Systematically, blank experiments are missing that would allow to confirm the authors claims:
- Figure S1: The authors suggest here (and in the main text) that the observed peak starting at -0.01 V vs SCE is laccase T1 center. However, numerous electrochemical studies indicate that versicolor laccase has potential of Cu T1 at 780-800 mV vs NHE (530-550 mV vs SCE, see ref. 1, page 2405), i.e. more than 0.5 V higher! Therefore, I have strong reasons to believe that the observed peak at such low potential is the non-enzymatic oxygen reduction on bare electrode (carbon or gold, which is not clear from the figure legend). The blank experiment in the absence of laccase under O2, which would allow to confirm or refute it, is missing.
- Figure 4: Again, blank experiments of bare carbon paste electrodes in the absence of complexes are missing. Thus, it is unclear whether the observed peaks come from the complexes or the electrode itself. The legend is not sufficiently detailed – was it aerobic or anaerobic experiment?
- Figure 6: The values should be calculated for enzyme activity in U/mg or s-1 to give an idea of the real enzyme activity. The blank experiment in the absence of laccase is important and missing. It’s known that ABTS exhibits slow non-catalytic oxidation even in the absence of catalysts, so the background rate should be absolutely taken into account.
- Figure 7: Blank experiments in the absence of laccase are missing. The exact nature of the observed peaks is not clear. Why is there an increase of the anodic peak on Fig 7A in the presence of O2? If catalysis of oxygen reduction occurs, the decrease of the anodic + increase of the cathodic current should be observed. Moreover, even Cu2+ salts (e.g. CuSO4) can demonstrate catalysis of oxygen reduction at similar potential. Since free Cu2+ can be formed upon decomposition of the complexes, blank experiment without the complexes 1 and 2 but in the presence of CuSO4 should be made.
- Figure 9,10: It is unclear for me how these data demonstrate the influence of the chirality of polypeptides on the electron transfer.
These are just major points indicating that it contains serious flaws. I therefore suggest rejecting it in the present form and advice the authors to reconsider their electrochemical experiments.
Author Response
April 23, 2020
Dear The editor of Symmetry:
Please find an attached the revised manuscript of article symmetry-773223.
The answers for query and revised points of the text (which have been written in the revised manuscript as red letters) are as follows:
<Reviewer 1>
Figure S1: The authors suggest here (and in the main text) that the observed peak starting at -0.01 V vs SCE is laccase T1 center. However, numerous electrochemical studies indicate that versicolor laccase has potential of Cu T1 at 780-800 mV vs NHE (530-550 mV vs SCE, see ref. 1, page 2405), i.e. more than 0.5 V higher! Therefore, I have strong reasons to believe that the observed peak at such low potential is the non-enzymatic oxygen reduction on bare electrode (carbon or gold, which is not clear from the figure legend). The blank experiment in the absence of laccase under O2, which would allow to confirm or refute it, is missing.
@ The value of “-0.01 V” denoted starting point (not top) of a peak, and it removed it to avoid misunderstanding in the legend of Figure S1. Blank experiments were also added as Figures S2 and S3.
Figure 4: Again, blank experiments of bare carbon paste electrodes in the absence of complexes are missing. Thus, it is unclear whether the observed peaks come from the complexes or the electrode itself. The legend is not sufficiently detailed – was it aerobic or anaerobic experiment?
@ Figure 4 was CV data of Cu complexes obtained by conventional (anaerobic) CV measurement excluding electrodes.
Figure 6: The values should be calculated for enzyme activity in U/mg or s-1 to give an idea of the real enzyme activity. The blank experiment in the absence of laccase is important and missing. It’s known that ABTS exhibits slow non-catalytic oxidation even in the absence of catalysts, so the background rate should be absolutely taken into account.
@ Unfortunately, quantitatively reliable values of activity could not be obtained because of addition of mediators and treatment of background throughout this study.
Figure 7: Blank experiments in the absence of laccase are missing. The exact nature of the observed peaks is not clear. Why is there an increase of the anodic peak on Fig 7A in the presence of O2? If catalysis of oxygen reduction occurs, the decrease of the anodic + increase of the cathodic current should be observed. Moreover, even Cu2+ salts (e.g. CuSO4) can demonstrate catalysis of oxygen reduction at similar potential. Since free Cu2+ can be formed upon decomposition of the complexes, blank experiment without the complexes 1 and 2 but in the presence of CuSO4 should be made.
@ Possible data corresponding to the blank tests may be Figure 4 and S1 in this case. No experimental proof of releasing free Cu(II) ion from chelate ligand could be observed in Figure 4 at least.
Figure 9,10: It is unclear for me how these data demonstrate the influence of the chirality of polypeptides on the electron transfer.
@ We judged the difference of current of CV, and difference of two samples compared for each case was only the chirality of polypeptides as bridges for electron conduction.
<Reviewer 2>
The present work is interesting and performed at a high experimental level, the well-known literature data are also well discussed, the background includes all relevant references. Therefore the article can be published in the journal Symmetry
@Thank you.
<Reviewer 3>
Pg. 4,142: Please rewrite or better specify the meaning of the following sentence “Unassigned electron density due to solvent molecules were remained”.
@According to your opinion, new sentence of “Low electron density due to solvent molecules could not be completely assigned as reasonable disorder nor smoothing.” was replaced.
Pg 6, 199: “suggesting that complexing the laccase with the Cu(II) complexes may result in the unfolding of the enzyme”. Unfolding of the enzyme would result in loss of the catalytic activity. Please rephrase the sentence.
@ According to your opinion, new sentence of “suggesting that complexing the laccase with the Cu(II) complexes may result in slightly structural changes of the enzyme.” was replaced.
Pg. 6, 203: “Both complexes dock at the lowest energy very close to the laccase “ Perhaps it is better to say: “The lowest energy docking poses for both complexes are very close to the laccase...”
@ Thank you. We employed your improved sentences.
In the conclusion part two sentences lack the full stop.
@ Thank you. We added two “.’s” in proper positions.
Please change in paragraph 2.4 the indefinite article to definite before the name of the programs used.
@ According to your comment, we added some words to indicated program package and each program using crystal structure analysis clearly.
Pg. 11, 325 Please correct the typo “oligoleptide”
@Thank you. We fixed this typo.
That’s all.
We would like to say thanks in advance for your prompt treatment of our manuscript.
Best regards,
Prof. Dr. Takashiro Akitsu
Department of Chemistry, Faculty of Science, Tokyo University of Science
1-3 Kagurazaka, Shinjuku-ku, Tokyo 162-8601, Japan
Tel. +81-3-5228-8271, Fax. +81-3-5261-4631
E-mail: [email protected]
Reviewer 2 Report
Account
The proposed article “Electron transfer via helical oligopeptide to laccase including chiral Schiff base copper mediators” by Kumpei Kashiwagi, Francesco Tassinari, Tomoyuki Haraguchi, Koyel Banerjee-Gosh, Takashiro Akitsu1, and Ron Naaman is devoted to the fact that the efficiency of a laccase-modified electrode, depends on the chirality of the oligopeptide linker used to bind the enzyme to the surface. It was earlier discovered that an enzymatic biofuel cell uses enzymes as catalysts on the anode for oxidizing fuel as an electron donor and enzymes on the cathode for oxygen reduction. Therefore the cell can convert chemical energy into electrical energy. Thus, a biofuel cell is a device providing electricity by converting chemical energy. In the present work, the authors studied a complex of Cu (II) with a Schiff-based dipeptide as an intermediary between the (cathode) electrode and oxygen-reducing laccase. ABTS analysis showed that laccase does not lose enzymatic activity when complexes are incorporated into laccase molecules. The surface of the gold substrate and laccase were associated with oligoleptide enantiomers acting as electron transfer paths. In these hybrid electrode materials, laccase showed enzymatic activity, and the complexes acted as a mediator. The authors showed for the first time that the electron transfer to laccase with the mediator via oligopeptide can occur as a spin filter without an external magnetic field.
The present work is interesting and performed at a high experimental level, the well-known literature data are also well discussed, the background includes all relevant references. Therefore the article can be published in the journal Symmetry
reviewer
Comments for author File: 
Comments.docx
Author Response
April 23, 2020
Dear The editor of Symmetry:
Please find an attached the revised manuscript of article symmetry-773223.
The answers for query and revised points of the text (which have been written in the revised manuscript as red letters) are as follows:
<Reviewer 1>
Figure S1: The authors suggest here (and in the main text) that the observed peak starting at -0.01 V vs SCE is laccase T1 center. However, numerous electrochemical studies indicate that versicolor laccase has potential of Cu T1 at 780-800 mV vs NHE (530-550 mV vs SCE, see ref. 1, page 2405), i.e. more than 0.5 V higher! Therefore, I have strong reasons to believe that the observed peak at such low potential is the non-enzymatic oxygen reduction on bare electrode (carbon or gold, which is not clear from the figure legend). The blank experiment in the absence of laccase under O2, which would allow to confirm or refute it, is missing.
@ The value of “-0.01 V” denoted starting point (not top) of a peak, and it removed it to avoid misunderstanding in the legend of Figure S1. Blank experiments were also added as Figures S2 and S3.
Figure 4: Again, blank experiments of bare carbon paste electrodes in the absence of complexes are missing. Thus, it is unclear whether the observed peaks come from the complexes or the electrode itself. The legend is not sufficiently detailed – was it aerobic or anaerobic experiment?
@ Figure 4 was CV data of Cu complexes obtained by conventional (anaerobic) CV measurement excluding electrodes.
Figure 6: The values should be calculated for enzyme activity in U/mg or s-1 to give an idea of the real enzyme activity. The blank experiment in the absence of laccase is important and missing. It’s known that ABTS exhibits slow non-catalytic oxidation even in the absence of catalysts, so the background rate should be absolutely taken into account.
@ Unfortunately, quantitatively reliable values of activity could not be obtained because of addition of mediators and treatment of background throughout this study.
Figure 7: Blank experiments in the absence of laccase are missing. The exact nature of the observed peaks is not clear. Why is there an increase of the anodic peak on Fig 7A in the presence of O2? If catalysis of oxygen reduction occurs, the decrease of the anodic + increase of the cathodic current should be observed. Moreover, even Cu2+ salts (e.g. CuSO4) can demonstrate catalysis of oxygen reduction at similar potential. Since free Cu2+ can be formed upon decomposition of the complexes, blank experiment without the complexes 1 and 2 but in the presence of CuSO4 should be made.
@ Possible data corresponding to the blank tests may be Figure 4 and S1 in this case. No experimental proof of releasing free Cu(II) ion from chelate ligand could be observed in Figure 4 at least.
Figure 9,10: It is unclear for me how these data demonstrate the influence of the chirality of polypeptides on the electron transfer.
@ We judged the difference of current of CV, and difference of two samples compared for each case was only the chirality of polypeptides as bridges for electron conduction.
<Reviewer 2>
The present work is interesting and performed at a high experimental level, the well-known literature data are also well discussed, the background includes all relevant references. Therefore the article can be published in the journal Symmetry
@Thank you.
<Reviewer 3>
Pg. 4,142: Please rewrite or better specify the meaning of the following sentence “Unassigned electron density due to solvent molecules were remained”.
@According to your opinion, new sentence of “Low electron density due to solvent molecules could not be completely assigned as reasonable disorder nor smoothing.” was replaced.
Pg 6, 199: “suggesting that complexing the laccase with the Cu(II) complexes may result in the unfolding of the enzyme”. Unfolding of the enzyme would result in loss of the catalytic activity. Please rephrase the sentence.
@ According to your opinion, new sentence of “suggesting that complexing the laccase with the Cu(II) complexes may result in slightly structural changes of the enzyme.” was replaced.
Pg. 6, 203: “Both complexes dock at the lowest energy very close to the laccase “ Perhaps it is better to say: “The lowest energy docking poses for both complexes are very close to the laccase...”
@ Thank you. We employed your improved sentences.
In the conclusion part two sentences lack the full stop.
@ Thank you. We added two “.’s” in proper positions.
Please change in paragraph 2.4 the indefinite article to definite before the name of the programs used.
@ According to your comment, we added some words to indicated program package and each program using crystal structure analysis clearly.
Pg. 11, 325 Please correct the typo “oligoleptide”
@Thank you. We fixed this typo.
That’s all.
We would like to say thanks in advance for your prompt treatment of our manuscript.
Best regards,
Prof. Dr. Takashiro Akitsu
Department of Chemistry, Faculty of Science, Tokyo University of Science
1-3 Kagurazaka, Shinjuku-ku, Tokyo 162-8601, Japan
Tel. +81-3-5228-8271, Fax. +81-3-5261-4631
E-mail: [email protected]
Reviewer 3 Report
In the present article Kashiwagi et al. describe the preparation of an electrode in which chiral oligopeptide linkers are used to bind a laccase/mediator complex to the electrode. The mediators are two Cu-complexes, based on Schiff base ligands with and without a photochromic azobenzene moiety. The reduction efficiency of the electrode was found to depend on the chirality of the oligopeptide linker that the authors explained with the Chiral-Induced Spin Selectivity effect.
In my opinion, the article is worth to be published in the Journal. There are few points that should be addressed in a revised manuscript prior to publication:
Pg. 4,142: Please rewrite or better specify the meaning of the following sentence “Unassigned electron density due to solvent molecules were remained”.
Pg 6, 199: “suggesting that complexing the laccase with the Cu(II) complexes may result in the unfolding of the enzyme”. Unfolding of the enzyme would result in loss of the catalytic activity. Please rephrase the sentence.
Pg. 6, 203: “Both complexes dock at the lowest energy very close to the laccase “ Perhaps it is better to say: “The lowest energy docking poses for both complexes are very close to the laccase...”
In the conclusion part two sentences lack the full stop.
Please change in paragraph 2.4 the indefinite article to definite before the name of the programs used.
Pg. 11, 325 Please correct the typo “oligoleptide”
Author Response
April 23, 2020
Dear The editor of Symmetry:
Please find an attached the revised manuscript of article symmetry-773223.
The answers for query and revised points of the text (which have been written in the revised manuscript as red letters) are as follows:
<Reviewer 1>
Figure S1: The authors suggest here (and in the main text) that the observed peak starting at -0.01 V vs SCE is laccase T1 center. However, numerous electrochemical studies indicate that versicolor laccase has potential of Cu T1 at 780-800 mV vs NHE (530-550 mV vs SCE, see ref. 1, page 2405), i.e. more than 0.5 V higher! Therefore, I have strong reasons to believe that the observed peak at such low potential is the non-enzymatic oxygen reduction on bare electrode (carbon or gold, which is not clear from the figure legend). The blank experiment in the absence of laccase under O2, which would allow to confirm or refute it, is missing.
@ The value of “-0.01 V” denoted starting point (not top) of a peak, and it removed it to avoid misunderstanding in the legend of Figure S1. Blank experiments were also added as Figures S2 and S3.
Figure 4: Again, blank experiments of bare carbon paste electrodes in the absence of complexes are missing. Thus, it is unclear whether the observed peaks come from the complexes or the electrode itself. The legend is not sufficiently detailed – was it aerobic or anaerobic experiment?
@ Figure 4 was CV data of Cu complexes obtained by conventional (anaerobic) CV measurement excluding electrodes.
Figure 6: The values should be calculated for enzyme activity in U/mg or s-1 to give an idea of the real enzyme activity. The blank experiment in the absence of laccase is important and missing. It’s known that ABTS exhibits slow non-catalytic oxidation even in the absence of catalysts, so the background rate should be absolutely taken into account.
@ Unfortunately, quantitatively reliable values of activity could not be obtained because of addition of mediators and treatment of background throughout this study.
Figure 7: Blank experiments in the absence of laccase are missing. The exact nature of the observed peaks is not clear. Why is there an increase of the anodic peak on Fig 7A in the presence of O2? If catalysis of oxygen reduction occurs, the decrease of the anodic + increase of the cathodic current should be observed. Moreover, even Cu2+ salts (e.g. CuSO4) can demonstrate catalysis of oxygen reduction at similar potential. Since free Cu2+ can be formed upon decomposition of the complexes, blank experiment without the complexes 1 and 2 but in the presence of CuSO4 should be made.
@ Possible data corresponding to the blank tests may be Figure 4 and S1 in this case. No experimental proof of releasing free Cu(II) ion from chelate ligand could be observed in Figure 4 at least.
Figure 9,10: It is unclear for me how these data demonstrate the influence of the chirality of polypeptides on the electron transfer.
@ We judged the difference of current of CV, and difference of two samples compared for each case was only the chirality of polypeptides as bridges for electron conduction.
<Reviewer 2>
The present work is interesting and performed at a high experimental level, the well-known literature data are also well discussed, the background includes all relevant references. Therefore the article can be published in the journal Symmetry
@Thank you.
<Reviewer 3>
Pg. 4,142: Please rewrite or better specify the meaning of the following sentence “Unassigned electron density due to solvent molecules were remained”.
@According to your opinion, new sentence of “Low electron density due to solvent molecules could not be completely assigned as reasonable disorder nor smoothing.” was replaced.
Pg 6, 199: “suggesting that complexing the laccase with the Cu(II) complexes may result in the unfolding of the enzyme”. Unfolding of the enzyme would result in loss of the catalytic activity. Please rephrase the sentence.
@ According to your opinion, new sentence of “suggesting that complexing the laccase with the Cu(II) complexes may result in slightly structural changes of the enzyme.” was replaced.
Pg. 6, 203: “Both complexes dock at the lowest energy very close to the laccase “ Perhaps it is better to say: “The lowest energy docking poses for both complexes are very close to the laccase...”
@ Thank you. We employed your improved sentences.
In the conclusion part two sentences lack the full stop.
@ Thank you. We added two “.’s” in proper positions.
Please change in paragraph 2.4 the indefinite article to definite before the name of the programs used.
@ According to your comment, we added some words to indicated program package and each program using crystal structure analysis clearly.
Pg. 11, 325 Please correct the typo “oligoleptide”
@Thank you. We fixed this typo.
That’s all.
We would like to say thanks in advance for your prompt treatment of our manuscript.
Best regards,
Prof. Dr. Takashiro Akitsu
Department of Chemistry, Faculty of Science, Tokyo University of Science
1-3 Kagurazaka, Shinjuku-ku, Tokyo 162-8601, Japan
Tel. +81-3-5228-8271, Fax. +81-3-5261-4631
E-mail: [email protected]
Round 2
Reviewer 1 Report
In the revised version of the manuscript "Electron transfer via helical oligopeptide to laccase including chiral Schiff base copper mediators" the authors have made minor corrections and added a couple of figures to the SI. However, this didn’t resolve the major issue of the manuscript: the absence of electrochemical signature of an active laccase on any of the figures.
Laccase is an oxygen-reducing enzyme, therefore, in the presence of oxygen and electronic communication with the electrode, a cathodic catalytic current must be observed. I don’t see any catalytic (i.e. without anodic counterpart) current increase that wouldn’t be higher than on blank electrode and that might be attributed to the laccase activity. As an example, on the new Fig S2 provided by authors, the reduction current in the absence laccase is more than 10 higher than the same experiment in the presence of laccase. Logically, it should be opposite.
Thus, my final decision remains unchanged
