Xanthine Oxidase—A Personal History
Round 1
Reviewer 1 Report
See attached file
Comments for author File: 
Comments.pdf
Author Response
All changes recommended by the reviewer have been made.Reviewer 2 Report
Type of manuscript: Perspective
Title: Xanthine Oxidase – A Personal History – Russ Hille
Journal: Molecules
https://susy.mdpi.com/user/review/review/35680590/ZO6ft5Th
The special issue “Molybdenum and Tungsten Enzymes – State of the Art in Research” is an impressive “creation” of Prof. Ralph Mendel and I think it is taking shape and under full development, as far as I know. The editor intends “to have a collection of “History reviews”, under a personal perspective, by pioneers/actors of the Mo-W field (in addition to possible other reviews and research papers that will probably be added in thttps://drive.google.com/file/d/1fDrN5FgzKx3X0licRBf_AVOHMyY61BM2/viewhis special issue)”. The articles, written in the first person, are supposed to give a personal account on the way the field developed, highlighting the author contributions (as a personal story) and revealing the interaction/collaboration with other key players.
“The History of the Molybdenum Cofactor—A Personal View” by Ralf R. Mendel, Molecules 2022, 27, 4934. https://doi.org/10.3390/ molecules27154934 was the starting point for the “stories”. In the first article of the series, we learn about the role of molybdenum (Mo) as an essential micronutrient in biology, and the way the metal is incorporated in the active center of Mo enzymes and how Mo is complexed by a pterin scaffold to form the molybdenum cofactor (Moco).
The article under review now, “Xanthine Oxidase – A Personal History” by Russ Hille, the second on the raw I presume, reports his personal perspective on Xanthine oxidase (XO), an enzyme that catalyzes the oxidation of xanthine to uric acid, involved in the degradation of purines, and activity associated with health issues and drug metabolism and other xenobiotics, and its inhibition is a target for the treatment of certain medical conditions.
I had an enormous pleasure in reading the article that provide, extensively and timely, the work developed in several laboratories on XO, including the author´s one, establishing the reaction mechanism and structure of xanthine oxidase. I congratulate Russ Hille for the fluid and catching text, where names such as Olson, Palmer, Massey, Bray, Ballou (within many others) are called for the completion of the picture, and how the author gain is own way in the field. The writing is appealing and topics such as: oxidative half reaction of XO, kinetics, intramolecular electron transfer, exploitation of EPR, XAS, EXAFS (isotopic labelling) and the renewed interested introduced by the resolution of the XO 3D structure (also with inhibitors) after the Desulfovibrio gigas Aldehyde oxidoreductase 3D structure previously solved are clearly explained. The details on the multiple Mo(V) faces shown by the enzyme, such as the very rapid EPR signals, are beautifully described. The orientation of the substrate interaction (different possibilities) with the Mo-site is a savory and complex scientific discussion, where Nishino and Leimkühler are remembered (excellent example for young readers to understand better how science is made).
I think the article should be accept as submitted and I renovate the pleasure I had in reading the manuscript.
Author Response
No changes suggested.Reviewer 3 Report
This article, by one the major players of this enzymes family (''The Veteran'', Science 2014), is very well written. It was a real pleasure to read it! This reviewer has no doubt to fully support its publication as it is.
Yet, this paper is so much more than just a beautifully written personal history. This is an excellent work for teaching! It explains how knowledge evolves, how different techniques and strategies must be chosen and articulated, to come together and originate the very detailed image that we have of XO today. As such, we should leverage it to allow the ''molybdoenzymes-outsiders'' to learn more about these enzymes and XO in particular. And for that, in this reviewer's opinion, this article could be further improved if it had a ''scholarly touch''. Some suggestions are:
(1) A chemical reaction is much needed. The hydroxylation of xanthine to urate with n O2 being reduced to superoxide anion radical and H2O2 is needed in the beginning; the structures of xanthine and urate must be present, to make it clear what the XO ''does'' (which C, which H, which future O). This will also help the reader to understand the EPR signals... the inhibition by alloxanthine...
(2) This reviewer understands that the ''final'' XO 3D structure, with the rearrangement of its cofactors (Mo, Fe/S, Fe/S, FAD), and of its molybdenum centre only came out later (pag 10-11, Figs. 4-5). However, it would be very useful to have a simple scheme of them in the beginning of the paper to help the readers following the e- transfer assays... modified flavins assays... up take of protons, FAD and Mo... the EPR spectra (the species that originate each signal type... H, O, C, S-labelled assays...), etc ... all that are being described by chronological order subsequently.
(3) More schemes (simple ones) of the hypothesis tested / strategies followed will be also a plus. Two examples: (a) One aspect often overlooked (inclusive by the Mo/W community) are the features of the reaction with O2. A scheme could be drawn to explain the number of superoxide and H2O2 molecules formed and the different kinetic phases of the reaction... (b) The "balls schemes" are very useful for illustrating how the e- are distributed among the 4 redox-active cofactors of XO... their relation with reduction potentials (the author has several of these schemes in his previous works/reviews).
Finally, even though the dehydrogenase form is not address in this paper, this reviewer suggests that some words about it should be included (to give the correct xanthine oxidoreductase picture to the readers).
Please, note that in pag 12, top, the sentence is repeated.
Author Response
The issue of “dehydrogenase” versus “oxidoreductase is now addressed in the Introduction, the suggested reaction mechanism and several new schemes have been added, and the duplicated sentence has been deleted.