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Talk:The protonmotive force and respiratory control

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Revision as of 14:32, 8 September 2017 by Beno Marija (talk | contribs)

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I have attached the edited version of the manuscript v29. Two issues of style, one no italics and the for example and that is are , e.g., and ,i.e., no parenthesis either.
I think it is very important and well written article to summarize previous concepts and clarify terminology. The only main question I have (maybe you have strong concept beside that) is about the paragraphs in part 3. The 3.1 is bioenergetic states , then 3.2 and 3.3 is biophysics and then 3.4 and 3.5 are normalization and conversation in general. In my personal opinion - maybe it is better to sum up 3.2 and 3.3 ? And do we need Table 9 SI prefixes (IUPAC)?
EG: Sections restructured, normalization as new Section 4.
As already cited, I am very interested to possible/potential influences on so-called ROX. Specifically, the level of ROX may and may be influenced by various O2 consuming enzyme activities which in turn may be modified by the concentrations of oxygen in medium. For these considerations I began to highlight this possible reciprocal influences.
Thereby and briefly ( the text may be also further expanded), I would like to suggest to modify the paragraph on ROX on your manuscript as follows:
Pag 20. ROX:
 oxygen-consuming reactions in mitochondria not related to ETS, such as oxygen consumption in reactions catalyzed by monoamine oxidases.., (type A and B), monooygenases ( cytochrome P450 monooxygenases), dioxygenase (sulfur dioxygenase and trimethyllysine dioxygenase), several hydoxylases and so on. It is important to stress that the interplay between mitochondrial respiratory complexes, oxygen concentrations and the important enzymes activities has a significant reciprocal regulatory role, which should be clarified in all its physiological and pathophysiological implications, thereby also influencing oxygen consumption measurements.


Because of my brief career in the world of mitochondrial breathing, the article has served me to my training much more than my modest contributions can serve to you. .. I would like to thank you again for the opportunity that you have offered me in my personal formation in the exciting world of mitochondrial breathing that is fascinating me. At the moment we have done some modest works but I want to implement this line of investigation in the laboratory that I’m leading. I hope you can read some of our work in near future. For now, our work have been accepted for communication at an international congress (ECTRIMS 2017) and our plan is implement this work in order to perform an original article.
To make a clearer differentiation between the figure feet and the body text. Personally, sometimes it has cost me to differentiate it and consequently has made my reading less fluid. As a possible solution I suggest framing the figure feet or use a different font size than the rest of the text.
Personally the section that talks about the UCP I think is not very informative. I think that remaking it could be more informative, my proposal is to change that section by:
Uncoupling protein (UCPs): There are proton channels in the inner mitochondrial membrane that allow the passage of protons from the intermembrane space to the mitochondrial matrix. As consequence there is a loss of membrane potential resulting in heat production instead of ATP.
EG:
However, your comment is included in the following form: “Uncoupling mediated by uncoupling protein 1 (UCP1) is physiologically controlled, e.g. in brown adipose tissue. UCP1 is a proton channel of the inner mitochondrial membrane facilitating the conductance of protons across the inner mitochondrial membrane. As consequence of this effective short-circiut, the protonmotive force collapses, resulting in stimulation of electron transfer to oxygen and heat dissipation without phosphorylation of ADP.”


  • 2017-09-02 EG:
Changes in Table 4. Protonmotive force matrix
  1. ΔιH+ to ∆ι
  2. Fel,H+/e to Fel/e
  3. Fel,H+/n to Fel/n
  • Whereas ∆ΌH+ = Fd,H+/n is the partial protonmotive force specific for proton displacement (d,H+), ∆ι = Fel/e is the partial protonmotive force (el) acting on charged motive molecules (ions that are displaceable across the inner mitochondrial membrane) in general.
  • Rows: isomorphic formats, e and n
  • Columns: partial isomorphic forces, Fel and Fd,H+
In my very personal taste, I would restyle the draft focusing on very few, clear and essential take home messages coupled if possible with few experiment images, more than adding further things.
I always bear in mind that too many informations are not digested by readers, especially from general audience but even from specialists! Basically, in my very personal view, too many sophistications risk to increase confusion to the existing confusion.


Just going through the new version of the MitoEAGLE manuscript. I like the figure 8 added, but I was not about the meaning of the abbreviation and the legend did not clarify it for me. Then I realized that you need Table 6 to fully understand. I was wondering if some of the definition in Table 6 that are fully needed to understand Fig. 8 should not be simply added in Fig. 8 (a little table included in a box with some symbols and expression used in the figure). I think it would make it way easier for the reader to understand the figure quickly (and to not have to look on a separate page to understand).
As discussed in a previous workshop together, I believe this is a great initiative since so much confusion is in the general science community, especially since the recent resurrection of the interest for mitochondria from different specialists not previously involved in mitochondrial research. My very personal point of view is that the manuscript should be a bit restyled in order to focus on LESS but VERY clear concepts that can be easily digested by non specialists, in order to have a larger audience and impact, otherwise the paper will mainly talk to specialist, many of which are at least partly familiar with these informations. Please have a look how effectively these papers addressed methodological issues on cell death and autophagy. They are very easy to read and had huge impact. I would personally try to simplify the key concepts, cut energically overclassifications and formulae, limit historical citations, and possibly add a couple of experiments to illustrate the different fluxes. ::::Limit abbreviations to a minimum. And more clearly explain what are OXPHOS and ETS, what it relies on, and what it means if 1 or both are decreased. Possibly with an illustrative trace. For the rest, with this first read, I have some other, more circumscribed comments:
  • -Page 6: I don’t understand where the content of page 6 goes. The IM separates the IMS from matrix. In some place of the manuscript I would more clearly states what the Respiratory chain includes (CI-CIV) compared to OXPHOS system (Respiratory chain + CV). Also not enough highlighted that OXPHOS rely also on Krebs. - (EG: see Fig. 1).
  • In page 6, other important mitochondrial components in addition to OXPHOS are mentioned, but many others have not: calcium metabolism, lipid, Coq10 and steroid biosynthesis, vitamin metabolism, etc
. ::::Maybe just simpler to mention that the mitochondrial proteome comprises more than 1200 proteins (Mitocharta), mostly encoded by nDNA, with a fantastic variety of functions, many of which still under investigation
 (EG: added).
  • -I personally do not like ETS, and prefer ETC=electron transfer capacity.
  • EG: ETS - system: this is the structural and functional unit; Capacity: this relates to flux. A distinction is required.
  • -page 8: I don’t like to define mitochondrial preparations tissue homogenates, permeabilized fibers/tissues, but rather would define as they are : tissue homogenates, permeabilized fibers/tissues. ::::Mitochondrial preparations are in my opinion mitochondrial enriched fraction or purified mitochondria (either by Percoll, sucrose or analogues). I don’t agree on the fact that these preparation largely maintain the mitochondrial structure, and very often not even the function. On the contrary most of these traetments introduce modifications in both, that can often be even quite drastic. This is one of the main limitation of the mitochondrial in vitro studies we have to accept until in vivo methods will be available, if ever. In fact one of the major plagues in mitochondrial papers, is that many groups basically measure O2 consumption on screwed out mitochondria, because of poorly prepared samples!!
  • -Page 9: found not clear
  • -Personally don’t like »P«
  • -ROX: not necessarily non mitochondrial respiration in absolute terms, but in the practice the ROX should be almost undectable if the instrument has been properly calibrated.
  • EG: There are non-ETS enzymes catalyzing O2-consuming reactions.
  • -it may be useful to mention how tricky it is to express fluxes by mass in weight g. The tissue composition in fact can change drastically between different diseases or individuals so that 1 g of dystrophic muscle will have half of its muscle mass replaced by fibrous tissue. Same is true for many other conditions, not to mention how drastically can differences in hydratation or even in the scale measurements (if very small pieces are used) of the tissue lead to errors.
  • -not really clear how to normalize for mitochondrial mass: (a) stress the importance to measure the normalizer on the SAME prep introduced into the chamber, which can be sometime difficult or impossible. (b) I don’t think TOM20 can be used to normalize respirometric data (with which technique?) (c) mtDNA quantification has been shown to be a poor marker of mitochondrial mass, and is frequently altered in diseases indedndently from mass. Better use CS or caldiolipin. (d) I personally don’t agree on recommending using FCR as internal marker, it clearly changes between individuals and even in the preparation phase
Of course I do not expect that everybody should agree, and open to discussion.
1- would it be useful to evoke/include the beta-oxidation cycle in the general framework of Fig1A ?
  • EG: We should do this in a follow-up part focussing on ETS pathways.
2- In Fig1, H+out/O2 and H+in/»P ratios are presented as fixed ratios. My understanding is that these are the ratios expected while one NADH (and not one succinate) is oxydised in theory, e.g. not taking into account proton slipping. If I am correct, this should be precised in the fig legend ?
  • EG edited the caption: H+out/O2 is the ratio of outward proton flux from the matrix space to catabolic O2 flux in the NADH-linked pathway. ::::H+in/»P is the ratio of inward proton flux from the inter-membrane space to the endergonic flux of phosphorylation of ADP to ATP. Due to proton leak and slip these are not fixed stoichiometries.
3- at the bottom of page 10, « In isolated mammalian mitochondria ATP production catalysed by adenylate kinase
. Â» => what is the point of this sentence? particularly regarding mitochondrial exploration ?
4- Page 19, paragraph 3.1 : « The protonmotive force is high in the OXPHOS state 
, elevated in the LEAK state
 Â» I would change elevated into very high or maximum to make it clearer. - DONE.
thanks again for letting me review this great manuscript.
After all the suggestions that have been added, I just checked in the MitoEAGLE the last version, at the moment I have no additional comments. I think it is a great contribution to the field, and it would be great if the community will start to following the recommendations.
I think it is time that the scientific community finally address this topic and tries to unify nomenclature and protocols so reproducible data can generated and compared across labs worldwide.
This is extremely well crated manuscript. Considering the expansion of the field of mitochondrial biology, standardization of nomenclature, terminology, and units will help the understanding of students and entering scientist to the field. I have attached a file with my suggestions hoping to contribute a little bit to your manuscript. My suggestions appear on pages 7 and 31.
With an emphasis on quality of research, data gathered can be useful far beyond the specific question of an experiment.
Considering Mte is referring to the quantity of mitochondria, why don't we use MtQ (with subscript Q). Q being the first letter of quantity, this might deliver the message to the point more easily.
EG:
It is not easy to introduce a symbol if it does not exist in the literature. Q is a difficult decision, being used with so many different meanings. Perhaps some other suggestions will improve on the present “mte”.
I would like to make two very brief comments:
1) On page 28, section “Sample concentration”, the sentence “Erich will add isolated mitochondria 
” is to be found.  This probably waits to be accordingly adjusted.
2) Based on my own microcalorimetric experience, I was wondering whether the “crowding effect”, i.e., the decrease in mass-specific metabolic rate with increasing sample size, should not have (at least briefly) been mentioned. To guarantee that the mass-specific O2 flux is independent of the size of a muscle specimen (as well as of the volume density of a cell suspension) in the measuring chamber (page26), the tissue slice must be small (thin) enough (or the cell density low enough, or the stirring rate high enough) to allow unrestricted O2 diffusion. Otherwise, the specific metabolic rate could – apparently – decrease with increasing sample size, probably due to an increasing proportion of anaerobic glycolysis in the depth of the tissue sample (or at the bottom of the cell suspension, respectively).
The problem has been extensively discussed by Richard Kemp in The Handbook of Thermal Analysis and Calorimetry [1], referring to a model calculation that I had published some years before [2].
[1] Kemp RB, Guan YH: Microcalorimetric studies of animal tissues and their isolated cells. In: Handbook of Thermal Analysis and Calorimetry, ed by Gallagher PK, vol 4, From Macromolecules to Man, ed by Kemp RB; Elsevier, Amsterdam 1999, chapt 11, pp 557-656
[2] Singer D, Schunck O, Bach F, Kuhn HJ: Size effects on metabolic rate in cell, tissue, and body calorimetry. Thermochim Acta 1995; 251: 227-240


I agree with the almost final version of the text. I will follow the final position of all the partners but better BBA and Cell Metabolism if accepted. I find the text very pedagogic indeed.
Fig. 8A. Mitochondrial yield, Ymt, in preparation of isolated mitochondria.
Fig. 8B. Respirometry with tissue homogenate.
  • 2017-08-28 EG: Suggestions
  • From 'Mitochondrial respiratory control: MitoEAGLE recommendations'
To 'The protonmotive force and respiratory control: Building blocks of mitochondrial physiology'
  • Figure 8 and Table 6: mte vs. mt
  • 2017-08-27
Table 4 update by EG
  • Protonmotive force, symbol ∆pH+ changed from previous symbol ∆pmt, for consistency of force and flow, FH+/e and IH+/e
∆pH+ = ∆ιmt + ∆”H+ / F ; (Eq. 1)
  • 2017-08-27 Morten Scheibye-Knudsen
I think the mitochondrial marker section is a bit simplistic and I would suggest including something about appropriate markers (for example use markers of beta-oxidation if you are investigating beta oxidation etc.).
  • EG: Perhaps TOM20 is the better marker, if fatty acid oxidation (FAO) capacity is selectively stimulated relative to NADH- and succinate-linked pathway capacity, then enzymes involved in FAO are a good marker for FAO but a bad marker for mitochondrial content.
I think these are excellent points and I think the sentence you have added is adequate. Regarding a symbol for mitochondrial content based on markers, I do not think such a symbol exists and I think you can therefore use the "mte" abbreviation as you do now.
Regarding the journal, if you get a large consensus which includes a large contingency of authors I guess this could be of interest for a higher level journal because it could act as a reference point. ::::Perhaps even something like Cell Metabolism.


  • 2017-08-24
Journal: Int J Biochem Cell Biol, discussed at MITOEAGLE Barcelona 2017 and MiPschool Obergurgl 2017; page limit is surpassed.
  • 2017-08-24 David Fell:
I've read through your draft and I think there is an issue that needs to be clarified, which is the distinction between 'responds to' and 'controlled by'. In MCA terms, this is equivalent to the difference between (flux) control coefficients and response coefficients. I could prepare a short document for you and your co-authors to explain my thinking on this issue. If you accept my points, the second step would be to work out how to alter the text without increasing its length. A question I have for you as well is: when you say 'respiratory control' are you exclusively concerned with control of oxygen consumption flux, or do you mean more generally it and other variables of the system, such as phosphorylation flux, or even PMF? If the latter, then there needs to be some clarification about that, in my view.
  • EG: You raised two good and important points. I would welcome clarification between ‘responds to’ and ‘controlled by’, and editing critically the present version of the ms. will be a big improvement. Yes, ‘respiratory control’ should be clarified in a wider sense not restricted to oxygen flux, since we explicitely talk about states and rates. Again, clarification will help.
The terminology here is a bit loose, and come close to invoking the idea of the "high energy" phosphate bond. See discussion in Nicholls's book on this term. Furthermore, all chemical processes in the cell occur in the "eneretic downhill direction". I am not sure if these two symbols are needed. But if needed, then I suggest the definition is that >>P refers to ATP synthesis from ADP and Pi, and >>P refers to ATP hydrolysis.
EG:
is this better?: phosphorylation in the context of OXPHOS is clearly defined as phosphorylation of ADP to ATP. On the other hand, the term phosphorylation is used in the general literature in many different contexts, e.g. protein phosphorylation. This justifies consideration of a symbol more discriminative than P as used in the P/O ratio (phosphate to atomic oxygen ratio), where P indicates phosphorylation of ADP to ATP or GDP to GTP. We propose the symbol »P for the endergonic direction of phosphorylation coupled to catabolic reactions, and likewise the symbol «P for the corresponding exergonic hydrolysis (upwards and downwards arrows, respectively, in Fig. 2).
Dan Beard
Which ATPase activity are we talking about here? I think it is a contaminating ATP hydrolysis activity, not the ATP synthase activity. Need to clarify. YES-CLARFIED.
In this situation, ATP synthesis is greater than 0 because ATP hydrolysis is greater than zero. Thus, the inequality here is correct, but could lead to confusion as an incomplete statement.
EG:
Oxygen consumption in State 4 is an overestimation of LEAK respiration if the contaminating ATP hydrolysis activity recycles some ATP to ADP, J«P , which stimulates respiration coupled to phosphorylation, J»P>0.
Dan Beard
By this definition, would calcium current be interpreted as a proton leak? Ca current is balanced by Na/Ca exchange, which is balanced by Na/H exchange or K/H exchange. – No, this is cation cycling, not a property of the membrane.
EG:
Cation cycling: Calcium current is balanced by Na/Ca exchange, which is balanced by Na/H exchange or K/H exchange. This is another effective uncoupling mechanism different from proton leak and slip.
Should this reference be added?
Dan Beard
Vinnakota KC, Singhal A, Van den Bergh F, Bagher-Oskouei M, Wiseman RW, Beard DA (2016) Open-loop control of oxidative phosphorylation in skeletal and cardiac muscle mitochondria by Ca2+. Biophys J 110:954-61.
I think that this would be a better reference for the contribution of calcium cycling to leak current: Stucki and Ineichen. Eur. J. Biochem 48:365-375 (1974). I wonder if we should still call calcium cycling a “leak”. It is a “leak current” in the language of electrophysiology. I wonder if it would be worth make this distinction clear: Proton leak is a leak current of protons. There can be other cation contributors to leak current including calcium and probably magnesium (but I don’t know the magnitude.) Under physiological conditions the proton leak is the dominant contributor to the overall leak current.
  • Figure 1A: It might be simpler to show just one single arrow rather than multiple arrows between CI/CII and Q. I like the introduction of the term ">>P”, but when I first looked at Figure 1 I didn't understand what the ">>" represented because it hadn’t yet been defined, so consider defining it in the figure legend.
  • EG: Yes, the definitions are added to the figure legend, where the multiple convergent electron transfer pathways are also explained.
  • Figure 1B: The use of "-“ to reflect stoichiometry could be confusing to some readers, and it might be worth explaining it in the legend.
  • Page 9: I find the term "evolutionary background" confusing. In this context, "evolutionary or acquired differences in the genetic basis of mitochondrial function (or dysfunction) between subjects" would be clearer, at least from my perspective.
  • Figure 3: Should ATPase activity and the J<<P arrow be shown here? Could it be removed or somehow shown as lower than the rate of phosphorylation? Showing it as it is currently is relevant to intact cells, but less relevant to isolated mitochondria with low ATPase activity.
  • EG: – But highly relevant for permeabilized cells and fibres.
  • Page 16: I think a statement, either here or earlier, that explicitly distinguishes the terms uncoupled, noncoupled, and dyscoupled would be valuable. As written, the distinction is only made implicitly, but I think it would be valuable to new researchers to have the meanings spelled out clearly.
  • Page 18, last two sentences: I think the point of these two sentences could be made more clearly.
  • Page 35, “300 mitochondria per cell”: I think it would be better to express this idea by instead using the volume density of mitochondria per cell.
  • 2017-08-23 Version 22' (see Table 4):
  • Is there a general symbol available for amount of mitochondria, as estimated by a mitochondrial marker?
  • Amount of mt-elements, mte (= quantity of mt-marker)
  • Mitochondrial concentration in the instrumental chamber with volume V, Cmte=mte∙V-1
  • Specific mitochondrial density in tissue X with tissue mass mX, Dmte=mte∙mX-1
  • Mitochondrial content per cell, mtecell=mte∙Ncell-1 (where Ncell is the number of cells)
I also have some comments and corrections (see my comments and suggestions in attached file):
1.Figure 1 - since fatty acids are mentioned previously, I would include also fatty acid oxidation pathway in Figure
2.p. 8 - the "protein phosphorylation" is more common term, that also includes phosphorylation of enzymes. Suggestion: change "phosphorylation of enzymes" to "protein phosphorylation"
3.p 12 State 4 definition. What about state 4 (or state 4o) as state after inhibition of phosphorylation system by Omy or Catr? suggestion is to mention this conditions also at state 4 definition.
4.p 30. Add sentence of example - "For example, endurance exercise training increases citrate synthase activity."
5.p 34 description of substrate-level phosphorylation - I believe there is a mistake. There should be "glucose" instead of "glycogen"
6.p 35 include also respiratory reference state as mitochondrial marker
7.The thing I missing is scheme of connection of classical Chance terminology to the recommended one. If you don't mind I'll think a little bit how to make this visible, and provide version of scheme or modification of Figure 6 later.


In collaboration with my supervisor, Professor Pedro F. Oliveira, we have read the latest version of the manuscript and made some suggestions mentioned as annotations in the PDF file (annex). We want to congratulate you and all the contributing co-authors for the excellent work.
EG response: Many thanks for your helpful suggestions. I have incorporated almost all of your changes in the upcoming version 22, contributing together with previous updates to an improvement of our ms.
Particularly important is the part of thermodynamics, because of many researchers tend to forget this component of bioenergetics.
EG response: I would like to share Tuuli’s point of view, representative of several comments received during the past few days.
Nevertheless, I hope that we can move more ‘heavy’ text into tables and notes to tables, to make the final version more easily readable. I invite our co-author’s comments on the suggestions for ‘new’ symbols (see Tab. 4), which we have to introduce if IUPAC guidelines are insufficient (I am not aware of consistent symbols in the literature). I am particularly fond of Table 3, which gives so much more ‘straight’ insight into the protonmotive force, when ‘seen’ together with flux in the two ‘isomorphic’ formats


1. I can see that the section on normalisation has greatly expanded. I think this is a really valuable section and gives a very thoughtful consideration to the different options for normalisation. I particularly appreciated the section on the use of mitochondrial markers, and the warnings about the potential pitfalls of using such markers. It is not absolutely necessary but under the problem of accuracy of measurement, I would be tempted to highlight the potential issues of measuring a marker in the contents of the oxygraph chamber at the end of a respirometry protocol, when recovery of e.g. enzyme activity may be less than 100%. Overall, I think this section makes a strong case for the use of FCR without being forceful.
2. It would be my own personal preference, but I would switch the order of the two sections "Mitochondrial concentration, Cmt, and mitochondrial markers" and "Mass-specific flux, JmX,O2" (Page 27). This way, the discussion of the use of mitochondrial markers follows on from the introduction of possible markers.
3. I agree with the focus of the manuscript being on mitochondrial preparations (with reference to intact cells where appropriate), but what might be missing for the non-expert reader might be the purpose of using such preparations, as opposed to intact cells. I would suggest a sentence or two following the definition of mt-preparations (pages 5 and 6), which can lead into the next section. Feel free to edit, but perhaps something along the lines of:
"The (non-permeabilized) plasma membrane prevents the free passage of many water-soluble mitochondrial substrates and inhibitors, limiting the scope of investigations into mitochondrial respiratory function, whilst the cycling of ATP/ADP in intact cells prohibits an analysis of the control exerted by ADP on respiration."
4. A minor, pedantic point on page 14. Proton slip can also happen at the ATP-synthase, in which case the proton will not slip back to the original compartment (as stated in the definition), but slip downhill to the matrix without contributing to ATP synthesis. Would you class this as proton leak or proton slip? I have seen both terms used in the literature.
5. Page 20, last line... change spelling of "Mitchel's" to "Mitchell's".
I have suggested a small addition to the normalization chapter with basically just a short paragraph trying to tie the chapter together and also add a few examples to clarify. In addition to this I have also suggested a few minor changes that I think increases the readability of a few sentences. As I said, these are complex questions and will probably benefit from being clear and easy to read.
One thing that I realized as I read the manuscript is that brackets [] are used quite freely and somewhat irresponsibly. In several cases the brackets should be exchanged for parenthesis () as to not be confused with concentrations. It is important to remember the target audience, and even if a lot of the information goes closer to physics, most of the readers are likely to be biologists, chemists and such. Therefore I think we should try and look over where the brackets are actually necessary and exchange them for parentheses where they aren't.


A few “minor” comments. The manuscript is looking pretty clear and comprehensive.
EG: Thanks – it’s in version 21‘ on the page. ‘Conservation of energy’ – of course, it should be ‘exergy’ (not in the sense of First-Law energy conservation), but this will be later in the ms. To avoid unnecessary confusion, I just deleted the ‘exergy conservation’ part of the sentence, it is not necessary to explain ‘uncontrolled’
Been through the previous version a time or two and mostly understand and agree. The primary part that is giving me pause is the discussion on correcting flux values for external mitochondrial markers.  ::::Working on formulating my thoughts coherently.
EG response: Here an important distinction:
‱Correction: for instrumental or chemical background, to eliminate methodological artefacts.
‱Normalization: for external or internal markers – this is different from ‘correction’, it is putting the data into specified contexts.
Michael:My biggest concern is the approach to normalization. As articulated in the comments within the manuscript, I do not believe that we currently have an external marker that meets the requirements of representing mitochondrial abundance, either from a qualitative or quantitative point of view. No external marker can be assured to have a fixed relationship with mitochondrial quality or quantity in order to serve as a surrogate or indicator for those properties, so to suggest that normalization in this manner is necessary and valid is not correct (in my opinion). This does not mean that expressing flux as a function of these proposed markers is incorrect – depending on the question being asked, it may be quite valid and enlightening to express flux as a function of enzyme X, Y, or Z. But I don’t believe that such approaches should be used at this time to extrapolate to mitochondria as a complex functional organelle.
  • 2017-08-21 Garry Buettner:
On manuscript pages 11 and 12, you use the term “saturating levels of oxygen”. My first reaction was that the atmosphere was 100% O2. I thought you should rather have air-saturated.
But I soon realized this was meant to refer to Km. Perhaps use an appositive such as , >>Km, in the text and figure caption. The term saturating with gases can be confusing.
In a redox biology work, I take great care on the use of “reduce” -- lower values? or gain electron(s)?
EG
I agree. Since ‚saturating‘ may be insufficiently discriminated from ‘saturated’ levels of oxygen, I added now throughout the text ‘kinetically saturating’ ..
Chuck Hoppel (whom I regard as one of our top experts) suggested to replace ETS state by oxidative state (OX). I guess this would not be welcome by redox biologists?


Ich finde die Tabellen generell sehr hilfreich und Informativ. Ich muss aber sagen, das das Teil „Normalisation: flows and fluxes“ ein bisschen vielleicht zu viel ist. Ist natĂŒrlich korrekt und ausfĂŒhrlich, aber fĂŒr genau so ein Teil wĂ€re meiner Meinung nach auch eine sehr klare, sehr direkte Zusammenfassung fĂŒr LeserInnen nötig, wer schnell Referenz und klare Richtlinien suchen.
1. I think, the noncoupling, uncoupling and discoupling should be defined. We discussed it with the Andras, Luiz and Zuzana on Thursday that a brief description would be helpful for the reader to clarify everything. It might be written in the 2.1 Section (definitions). The term controlled and uncontrolled might be confusing. We might distinguish what is the difference between coupled and controlled versus noncoupled and non-controlled.
2. The definition of coupling, efficiency and power should be moved before the coupling versus bound processes. Firstly, the definition should be explained and after that a difference between coupling and bounding. Moreover, I found this sentence in the coupling versus bounded processes redundant (“Coupling occurs in an energy transformation between processes, if a coupling mechanism allows work to be performed on the endergonic or uphill output process (work per unit time is power; dW/dt [J/s] = Pout [W]; with a positive partial Gibbs energy change) driven by the exergonic or downhill input process (with a negative partial Gibbs energy change.)”) with the following in the next section: “In energetics (ergodynamics) coupling is defined as an exergy transformation fuelled by an exergonic (downhill) input process driving the advancement of an endergonic (uphill) output process.”
3. From my point of view, Table 3 is excellent and helpful according to our Thursday session, which I found interesting and now it is much more understandable.
4. I think that the paragraph Molar quantities should be moved to the 3.4 section, because you are speaking in this paragraph about intensive and extensive quantities which is defined only later in section 3.4. It might be moved after the Extensive quantity paragraph.
5. At page 28: “This problem is avoided when O2 fluxes measured in substrate-uncoupler-inhibitor titration protocols are normalized for flux in a defined respiratory reference state, which is used as an internal marker and yields flux control ratios, FCR (Fig. 7).”
Does it refer only to the SUIT protocols? I think, we should write there for example in SUIT protocols.
6. From my point of view, these new tables are quite helpful, the understanding is much more easier and the text is more traceable.


File:Protonmotive force matrix.jpg
  • 2017-08-21
New Table 3 in Version 21
Bernard Tandler and I went through the manuscript and edited for English style. Additionally, suggest that this is two manuscripts. One the terminology and the second a biophysical discussion of the subject with representative equations as required for explanations. I suggest to separate so we make a clear and declarative statement about terminology. Then the important biophysical component stands on its own.
Also in Figure 2 I find the use of ETS under transport &..., Membrane-ETS, and to describe the overall process confusing. From substrates moving through the outer mitochondrial membrane, transported through the inner mitochondrial membrane, and metabolism by dehydrogenases producing NADH or FADH to enter the electron transport system, this seems to me to be the oxidative system.


I added very few changes in the manuscript. Also, I think that could be useful to include a definition for control of metabolism and regulation. In one of my commentaries, I wrote a couple of sentences from David Fell's book that could be interesting. I know that is adding more text but is just a few words.
  • 2017-08-19 Garry Buettner:
  • Ad MITOEAGLE: Have all CAPS is a bit gaudy. It makes text look strange and uninviting. Because „ito“ are not „first“ letters, I suggest lower case.
EG: I agree. MITOEAGLE was originally used in our successful COST Action grant application, accoring to the COST rules to use capital letters for the project acronym. Since then I have seen several COST projects using small and capital letters for their acronym. MitoEAGLE looks better. Probably, we will need a Management Committee E-vote. Let's try MitoEAGLE for the manuscript, and decide on the general strategy.
You hit exactly the target of MitoEAGLE. With an emphasis on quality of research (and life), “data gathered can be useful far beyond the specific question of an experiment.”
Added in the intro:
The mission of the MitoEAGLE network aims at developing harmonized experimental protocols and implementing a quality control and data management system to interrelate results obtained in different studies and to generate a rigorously monitored database on mitochondrial respiratory function under varying experimental conditions. With an emphasis on quality of research, data gathered can be useful far beyond the specific question of an experiment.
Fig. 7. Different meanings of rate may lead to confusion, if the normalization is not sufficiently specified.
  • I would use L and g rather than mL and mg. Although l for liter is still in use, L is better and is the future. For example, the American Chem Soc Journals go with L as do many others. Elsevier is lagging, but becoming flexible.
EG: This makes sense, and we should get used to it. I added a conversion table, to facilitate harmonization.
Attached are my specific comments. I have added them to Garry's version, in the hope that this may make it easier for you.
Some thoughts:
1.Concerning normalization, i.e., the "mg protein" versus "cell" issue Garry raised, "cell" is limited to cellular experiments, whereas "mitochondrial preparations" (introduced earlier) refer to tissue and cells. As there are many applications using (permeabilized) tissue, fibers etc (we have even used zebrafish hearts), I suggest to either use a single normalisation that serves all purposes or then give specific recommendations for different applications. Also, there are arguably additional parameters that one may wish to use to standardise respiration, e.g., mitochondrial mass. The choice of normalisation may well depend on the biological question the user wishes to address, and I suggest to make this point clear.
2.Figure 2 refers to "ROS", though this aspect is not discussed further in the article. If you wish to retain reference to electron leak to oxygen in the Figure, I recommend that you refer to the univalent reduction of oxygen to superoxide anion radical instead, as this is the known product, whereas "ROS" is not well defined, and we probably don't want to recommend its use.
3.It may be worth considering using the term dioxygen instead of oxygen; in any case, I suggest to use one term only if possible, and the abbreviation (O2) consistently after its introduction.


  • 2017-08-18 Garry Buettner:
I think in the section on whole cells a somewhat different approach to normalization would be better. I offer my suggestions and some edits with that, but the edits are not complete as your reaction to the suggestion is needed.
1.In our work with whole cells, be it bioenergetics or other goals, we normalize everything to cell number to produce mol cell-1 or mol cell-1 s-1 . With oxygen consumption by cells attomoles is most convenient, so we use amol cell-1 s-1 . This allows scaling to be simple. We use cell density asl cell L-1 . Results mesh directly with M. Scaling and manipulations are easy with many fewer mistakes by beginners. See Wagner BA, Venkataraman S, Buettner GR. (2011) The rate of oxygen utilization by cells. Free Radic Biol Med. 51:700-712. PMID: 21664270 http://dx.doi.org/10.1016/j.freeradbiomed.2011.05.024 PMCID: PMC3147247
2.In cell culture, mol cell-1 should be the coin of the realm See Doskey CM, van ‘t Erve TJ, Wagner BA, Buettner GR. (2015) Moles of a substance per cell is a highly informative dosing metric in cell culture. PLoS ONE 10(7): e0132572. PMID: 26172833 http://dx.doi.org/doi:10.1371/journal.pone.0132572 Open Access PMCID: PMC4501792
3.An application of this is in: Doskey CM, Buranasudja V, Wagner BA, Wilkes JG, Du J, Cullen JJ, Buettner GR. (2016) Tumor cells have decreased ability to metabolize H2O2: Implications for pharmacological ascorbate in cancer therapy. Redox Biology. 10:274-284. PMID: 27833040 http://dx.doi.org/10.1016/j.redox.2016.10.010 Open Access PMCID: PMC5106370
On manuscript pages 11 and 12, you use the term “saturating levels of oxygen”. My first reaction was that the atmosphere was 100% O2. I thought you should rather have air-saturated.
But I soon realized this was meant to refer to Km. Perhaps use an appositive such as , >>Km, in the text and figure caption. The term saturating with gases can be confusing.
In a redox biology work, I take great care on the use of “reduce” -- lower values? or gain electron(s)?


I have completed the review and English editing with my colleague Bernard Tandler, Ph.D.. I am working to add these in edit mode. I worked on version 9 and when the version came from Javier I switched to using that version. I realize version 10 has now come. I will complete the editing on version 9 and send. Then I will compare to version 10.
The manuscript appears to be two different stories. One on physiological terminology and the other more biophysical. I will recommend making these two different manuscripts.


We have read the most updated version of the file (version 18), and we do not want to add further comments.
Therefore, the article has our approval.
To help with this, I suggest including a "Table 2" that summarizes essential aspects of the 4 primary coupling states defined in section 2, which would compliment the already included classic Chance and Williams states in Table 1. I've attached working version that would be useful from my perspective that includes a succinct definition of LEAK, OXPHOS, ETS and ROX, with information on corresponding MMP, resp. rate, limiting and induction factors).
It also seems appropriate, given the topic of this review, to include a careful definition of the general coupling control factor (1-L/P) here a surrogate for RCR still widely used in the literature as an index of "coupling control" ... I have also included a couple of minor comments/questions on the attached pdf for your consideration.
  • 2017-08-18 Erich edited some equations in Version 18:
Protonmotive force, ∆pmt: The protonmotive force, ∆pmt,
∆pmt = ∆ιmt + ∆”H+ / F ; (Eq. 1)
is composed of an electric part, ∆ιmt, which is the difference of charge (electrical potential difference) across the inner mitochondrial membrane, and a chemical part, ∆”H+/F, which stems from the difference of pH (chemical potential difference) across the inner mitochondrial membrane and incorporates the Faraday constant, F. In other words, the protonmotive force is expressed as the sum of two terms, with somewhat complicated symbols in Eq. 1, which can be more easily explained as isomorphic partial protonmotive forces.
Electrical, el: Fe,el = ∆ιmt is the electrical pat of the protonmotive force expressed in units joules per coulomb, i.e., volt [V=J/C], and defined as partial Gibbs energy change per motive elementary charge of protons, e [C]. Fn,el = ∆ιmt∙F is the electrical force expressed in units joules per mole [V=J/mol], and defined as partial Gibbs energy change per motive amount of charge, n [mol].
Chemical, diffusion or dislocation, d: Fn,d = ∆”H+ is the corresponding chemical part of the protonmotive force expressed in units joules per mole [J/mol], and defined as partial Gibbs energy change per motive amount of protons, n [mol]. Fe,d = ∆”H+/F is the chemical force expressed in units joules per coulomb, i.e., volt [V=J/C], and defined as partial Gibbs energy change per motive amount of protons expressed in units of electric charge, e [C].
Faraday constant, F: The Faraday constant is the product of the elementary charge and the Avogadro (or Loschmidt) constant, F = e∙NA [C/mol], and yields the conversion between protonmotive force, Fe = ∆pmt [J/C], expressed per motive charge, e [C], and protonmotive force or chemiosmotic potential difference, Fn = ∆pmt∙F [J/mol], expressed per motive amount of substance, n [mol],
Fn = Fe ∙ F ; (Eq. 2.1)
Fe = Fe,el + Fe,d = ∆ιmt + ∆”H+/F ; e-isomorph [J/C=V] (Eq. 2.2)
Fn = Fn,el + Fn,d = ∆ιmt∙F + ∆”H+ ; n-isomorph [J/mol] (Eq. 2.3)
Protonmotive means that protons are moved across the mitochondrial membrane at constant force, and the direction of the translocation is defined in Fig. 2 as H+in → H+out,
Fn,d = ∆”H+ = -ln(10)∙RT∙∆pHmt ; (Eq. 3)
where RT is the gas constant times absolute temperature. ln(10)∙RT = 5.708 and 5.938 kJ∙mol­-1 at 25 and 37 °C, respectively. ln(10)∙RT/F = 59.16 and 61.54 mV at 25 and 37 °C, respectively. For a ∆pH of 1 unit, the chemical force (Eq. 3) changes by 6 kJ∙mol­-1 and the protonmotive force (Eq. 2.2) changes by 0.06 V.
Since F equals 96.5 (kJ∙mol­-1)/V, a membrane potential difference of -0.2 V (Eq. 2.2) equals a chemiosmotic potential difference, Fn, of 19 kJ∙mol-1 H+outSubscript text (Eq. 2.3). Considering a driving force of -470 kJ∙mol-1 O2 for oxidation, the thermodynamic limit of the H+out/O2 ratio is reached at a value of 470/19 = 24, compared to a mechanistic stoichiometry of 20 (H+out/O=10).
v"Alternatively, coupling of electron flow and phosphorylation is disengaged by uncouplers which induces a burst of oxygen consumption without performance of biochemical work (Fig. 1)."
What I intended to say is that "performance of biochemical work" could be an ambiguous term “Energy production“? – I do not like it (1st law of thermodynamics?). i agree with you if we follow first law of thermodynamics we probably should link uncoupling to HEAT and coupling to WORK or BIOCHEMICAL WORK.
Great job!
The amendment to the abstract is excellent. I also agree with the force/energy argument - I was being a bit "physicist" about it.
Leak:“when oxygen flux is maintained mainly to facilitate to compensate for the proton leak at a high chemiosmotic potential, when ATP synthase is not active.”
EG response: NOT facilitate the proton leak, but to drive protons back, i.e. compensate for the proton leak.
Brown RA answer: Trying to be helpful; I see facilitate is not a good word but is there not a better way of describing what is in yellow – the addition of to drive protons back made it clearer to me than the word compensate – many thanks
Brown: I will have a look at the rest.
“when oxygen flux is maintained mainly to try to compensate for loss of PMF due to proton leak at a high chemiosmotic potential, when ATP synthase is not active.” (comment - if compensation does not work mito fails; no? This phrasing allows for return to steady state, failure and inbetween)
EG: Many thanks for your input. I have integrated your suggestions into the new version which is now on the website.
Dorit: Attached is the conclusion part in which I suggest to introduce 2 sentences marked in yellow.
  • 2017-08-17
Perspective added in Version 17 (EG)
To provide an overall perspective of mitochondrial physiology we may link cellular bioenergetics to systemic human respiratory activity, without yet addressing cell- and tissue-specific mitochondrial function. A routine O2 flow of 234 ”mol∙s-1 per individual or flux of 3.3 nmol∙s-1∙g-1 body mass corresponds to -110 W catabolic energy flow at a body mass of 70 kg and -470 kJ/mol O2. Considering a cell count of 514∙106 cells per g tissue mass and an estimate of 300 mitochondria per cell (Ahluwalia 2017), the average oxygen flow per million cells at Jm,O2peak of 45 nmol·s-1·g-1 (60 ml O2·min-1·kg-1) is 88 pmol∙s­-1∙10­-6 cells, which compares well with OXPHOS capacity of human fibroblasts (not ETS but the lower OXPHOS capacity is used as a reference; Gnaiger 2014). We can describe our body as the sum of 36∙1012 cells (36 trillion cells). ::::Mitochondrial fitness of our 11∙1015 mitochondria (11 quadrillion mt) is indicated if O2 flow of 0.02 pmol∙s­-1∙10-6 mt­ at rest can be activated to 0.3 pmol∙s-1∙10-6 mt at high ergometric performance.
Fig. 6. Four-compartmental model of oxidative phosphorylation with respiratory states (ETS, OXPHOS, LEAK) and corresponding rates (E, P, L). Modified from Gnaiger (2014).
  • 2018-08-16 EG - added Fig. 6 and text in Version 16:
Fig. 6 summarizes the three coupling states, ETS, LEAK and OXPHOS, and puts them into a schematic context with the corresponding respiratory rates, abbreviated as E, L and P, respectively. This clarifies that E may exceed or be equal to P, but E cannot theoretically be lower than P. E<P must be discounted as an artefact, which may be caused experimentally by (i) using high and inhibitory uncoupler concentrations (Gnaiger 2008), (ii) non-saturating [ADP] or [Pi] (State 3), (iii) high oligomycin concentrations applied for measurement of L before titrations of uncoupler, when oligomycin exerts an inhibitory effect on E, or (iv) loss of oxidative capacity during the time course of the respirometric assay with E measured subsequently to P (Gnaiger 2014). On the other hand, E>P is observed in many types of mitochondria and depends on (i) the excess ETS capacity pushing the phosphorylation system (Fig. 1B) to the limit of its capacity of utilizing ∆pmt, (ii) the pathway control state with single or multiple electron input into the Q-junction and involvement of three or less coupling sites determining the H+out/O2 coupling stoichiometry (Fig. 2A), and (iii) the biochemical coupling efficiency expressed as (E-L)/E, since any increase of L causes an increase of P upwards to the limit of E. The excess E-P capacity, ExP=E-P, therefore, provides a sensitive diagnostic indicator of specific injuries of the phosphorylation system, when E remains constant but P declines relative to controls (Fig. 6). ::::Substrate cocktails supporting simultaneous convergent electron transfer to the Q-junction for reconstitution of TCA cycle function stimulate ETS capacity, and consequently increase the sensitivity of the ExP assay.
... In general, it is inappropriate to use the term ATP production for the difference of oxygen consumption measured in states P and L. The difference P-L is the upper limit of the part of OXPHOS capacity which is free (corrected for LEAK respiration) and is fully coupled to phosphorylation with a maximum mechanistic stoichoimetry, ≈P = P-L (Fig. 6).
I think that it is integrating well the 'State 1-5 - nomenclature' with the 'new terminology'. In 2.2 or in the final paragraph: A schematic view similar with the one from the previous versions of the manuscript (April 2017, on which I have sent feedback at the time) (see above) may still be useful for readers that are new to the field of mitochondria physiology, but are starting to employ such measurements in their work.
Nicoleta: I think this is very good.
It clarifies and summarises the terminology and what it means in an experimental setting.
In 3.3 page 20 we may need to add more detail, may be an example of additional normalization that could be employed.
EG response:
I agree. I just asked one of our co-authors to consider an example of normalization which elicited a good discussion during the past MiPschool.
Nicoleta:
I have attached the scheme from previous version for your reference. The Ideea is to have something visual and simple that integrates the new 'terms'.
EG response: Now your comment is clear. And you put me to work – what do you think of the text accompanying the figure?
I do suggest to include a short paragraph on the crosstalk between mitochondria and the cells via Ca, and other signaling pathways which may affect mitochondrial activity in the conclusion part.
Although it is beyond the scope of the review it puts it in a wider physiological context.
Here are my minor comments on the manuscript. .., but I hope that you find them useful. As I comment in the abstract, I am not sure to what extent prior knowledge of the existing terminology can be assumed. However, it is clearly aimed at a readership already active in the field.
EG: Would this be correct: „Do students expect researchers of bioenergetics explain Mitchell's theory of chemiosmotic energy transformation?”
David: I thought that the original ... was ok... or "Do students expect researchers of bioenergetics to explain Mitchell's theory of chemiosmotic energy transformation?" would be fine - it depends on the type of expectation
There are sentences that seem to not connect well, maybe because they were written by different authors. For instance, the last paragraph of the conclusion, seems to be more suitable for an introduction, rather than for a conclusion. The content of the text, in our opinion, is very good, very clear and useful for an harmonization of terms. The major terminology issues about mitochondrial states are addressed, and the text sets the paradigm for the upcoming parts. As mentioned in the ms, there will be the need of a "part 2", totally dedicated to mitochondrial enzyme nomenclature. We hope we are able to contribute with more critic comments on that subject, as it is more related to our expertise thus far, and a more active role on the endeavor.
  • 2018-08-16 Leo Sazanov:
Yes, will be happy to contribute - I think some changes to Fig. 1 are needed.
  • 2018-08-16 Yau-Huei Wei:
I agree with you that it is a very important concept in mitochondrial physiology. However, there are misunderstandings and misuse of this term.
EG:
Due to feedback by other co-authors asking for a short and more explanatory section and figure on coupling states, I added new Fig. 6 and text, specifically shown on the website, such that it is not necessary to screen the entire pdf for news.

Here two points:

"respiration state” in Abstract: here ETS is required, and the abstract is modified to make it more clear.
“When considering the Gibbs energy of a system, G [J], is divided ..” – it is really when dividing
Yau-Huei Wei:
Thank you for your reply to my suggested revision of the manuscript that you drafted nicely to review important parameters that have been widely used in characterizing the quality and function of isolated mitochondria. I am sure that this is a valuable paper and reference for life scientists, researchers in pharmaceutical companies and clinicians.
One comment that I do have based on the preliminary reading that I did over my morning coffee is that for someone like me who views all of this as more of a means to an end (the end being understanding physiology and pathophysiology on a more macroscopic level) instead of an end unto itself, I find that when I am struggling to understand something it is because it lacks a context for me – either from a methodological standpoint (if you add X and Y to the reaction mixture, the results represent Z) or a physiological standpoint (adding X and Y and getting Z represents these in vivo conditions). Context is not necessary for someone like yourself and many of the co-authors who live in the world of mitochondrial physiology, but in order to gain a broader acceptance of the revised terminology and concepts, that additional context may be necessary.
File:OXPHOS system.jpg
Fig. 1. The mitochondrial respiratory system. In oxidative phosphorylation the electron transfer system (A) is coupled to the phosphorylation system (B). See Eqs. 4 and 5 for further explanation. Modified from (A) Lemieux et al (2017) and (B) Gnaiger (2014).
EG response: Abstract modified according to your suggestion.
Pi* added to Fig. on OXPHOS. “Energy production“? – I do not like it (1st law of thermodynamics?). I like the ‘exogenous’ uncoupler, versus endogenous uncoupling.


  • 2017-08-15
New Fig. 1 in Version 15 (EG).
  • What happens when the ETS is branched at the oxidase level - need to define what "uncoupled" is!
Added text: "Such uncoupling is different from switching to mitochondrial pathways which involve less than three coupling sites with electron entry into the Q-junction bypassing Complex I (Fig. 1; including a bypass of CIV by alternative oxidases, not shown). This may be considered as a switch of gears (stoichiometry) rather than uncoupling (loosening the stoichiometry)." EG
  • the symbol »P: Ambiguous and likely to cause confusion! In my view you need to also define that you have changed this to »P/O2 and not O indicating you are referring to the full 4 electron reduction of oxygen!
Added to the text!
  • How are you going to define the ROX state in tissues that have had additional oxidase added i.e. AOX following Gene therapy.
AOX has to be inhibited, too, to measure ROX, since AOX is part of the (genetically modified) electron transfer system. See original text: "ROX is measured either in the absence of fuel substrates or after blocking the electron supply to cytochrome c oxidase and alternative oxidases."
  • FO2 versus F
Added after Eq. 1: ".. and a chemical part incorporating the Faraday constant"


  • 2017-08-14EG added to Version 14 many suggestions of GC Brown, and
Coupled versus bound processes: Since the chemiosmotic theory explains the mechanism of coupling in OXPHOS, it may be interesting to ask if the electrical and chemical parts of proton translocation are coupled processes. This is not the case according to the definition of coupling given above (in the manuscript). It is not possible to physically uncouple the electrical and chemical processes, which are only theoretically partitioned as electrical and chemical components (Eq. 1) and can be measured separately. If partial processes (fluxes, forces) are non-separable, i.e. cannot be uncoupled, then these are not coupled but are defined as bound processes. The electrical and chemical part of Eq. 1 are tightly bound partial forces of the protonmotive force.
  • "I have very fond memories of the MiP summer schools.I can’t help much with this, but I enclose some thoughts.In general, the purpose is a bit unclear, and it rambles over a variety of areas, without clear definitions, recommendations or focus. If I were doing it, I would half the length and sharpen the focus, and make the definitions and recommendations crystal clear. But I realise that doing this within a large consortium is not easy!"
Response by EG: As always, I fully appreciate your sharp and to-the-point level of discussion. Like me, many of us consider you as a teacher. Therefore, I respond with full appreciation that you ‘help us with this’. And yes: the large consortium has by now a history of dedicated meetings with good discussions, resulting in an evolutionary approach that needs to be appreciated without the claim of full-power selective optimization. I thank you so much for your detailed feedback. Many suggestions that you made are implemented in the new version. In addition my comments are summarized here:
  1. Abstact: This was written before the group-dynamics changed the focus of the ms. With more and more questions on ‘clarification’, ‘definition of terms’, the attempt to summarize some simple recommendations shifted to ‘educational’. So far, more ‘explanatory’ was the result, without reassertion if this was also more ‘educational’. We need the help of experts in science writing who are firm with the basic concepts.
  2. I agree (without necessarily speaking of other contributors) that we should reduce ‘crazy’ recommendations on abbreviations. It is a cancerous phenomenon of scientific literature, and we better stay away of too much of the same. imt etc are not even used in later sections, thus we can get rid of it.
  3. I suggest an exception of the above agreement toavoid abbreviations: mt. Would you suggest that we recommend to use ‘mitochondrial DNA’ and skip mtDNA?
  4. tr: consider spelling this out in all following symbols.
  5. Section 2.2. “You need a section here, outlining why the classical nomenclature is not sufficient. This is important!“ - Thanks, there should be more detail in the first introductory sentences.
  6. LEAK state - thanks, would you agree on this: “A state of mitochondrial respiration when oxygen flux is maintained at saturating levels of oxygen and respiratory substrates, and zero ATP-turnover without addition of any experimental uncoupler, as an estimate of the maximal proton leak rate.”
  7. Your excellent comments on detail have all been incorporated in Version 14.
  8. "∆pmt - not clear why you are using mt here." Protonmotive force could be used for any membrane, but in the context of this manuscript, confusion is very unlikely." – But think of all the literature not taking into account the plasma membrane potential. - Later versions: "∆pmt changed to "∆pH+
  1. “The protonmotive force is maximum in the LEAK state” - I agree with your comment – is “elevated” better?
  2. "Forces and flows in physics and irreversible thermodynamics: Why?" – Can we ignore IUPAC recommendations?
I have added several corrections and comments to the manuscript, mainly according to the small handout that I received at the MiPschool.
I was interested in the ROUTINE state, but the section 2.3 for Intact cells versus mitochondrial preparations has been deleted.
After coming back from Obergurgl, I was interested in the patch-clamp analysis of mitochondria.
I noticed that Bertholet et al applied a patch-clamp method to analyze UCP1-positive and UCP1-negative beige adipocytes.
They detected "Creatine Cycling” in mitochondria from UCP1-negative beige adipocytes (Fig. 7 D/E).
Response by EG:
  1. On Fig. 1A: Perhaps we should simplify it for the present purpose, since the chapter on ‘pathway control’ has been shifted away form the present Part 1 to another manuscript (Part 2), where these issues should be discussed and controversies resolved in full detail.
  2. Intact cell respiration was also shifted to another future manuscript.
  3. I added ‘intermembrane space’ to Fig. 1B.
  4. V[dot]O2max: I fully agree, I just do not know how to add the dot in Microsoft Word. In the old style, it was simple to just put such a dot on paper.
  5. Definition of V: It is unfortunate that V[dot]O2max has been introduced, but we cannot expect to change the sport science symbols (they should change from volume to amount of substance for metabolic oxygen consumption). And we cannot change V. Therefore V[dot] needs to be distinguished from the other definitions of V. I added: “.. whereas maximum mass-specific oxygen flux, V[dot]O2max or V[dot]O2peak, is constant across a large range of individual body mass (Weibel and Hoppeler 2005). V[dot]O2peak of human endurance athletes is 60 up to 80 ml O2·min-1·kg-1 body mass, converted to Jm,O2peak of 45 to 60 nmol·s-1·g-1 (Gnaiger 2014).”


  • 2017-08-12 EG
added to Version 12
Proton leak: Proton leak is the process in which protons are translocated across the inner mt-membrane in the direction of the downhill protonmotive force without coupling to phosphorylation. The proton leak flux depends on ∆pmt and is a property of the inner mt-membrane. Proton slip: Proton slip is the process in which protons are only partially translocated by a proton pump and slip back to the original compartment. The proton slip is a property of the proton pump and depends on the turnover rate of the proton pump. - (added by EG)
  • 2017-08-12
Feedback and suggestions on Version 10 by Anthony Molina.
I managed to set aside some time to work on the manuscript. My suggested revisions and comments are embedded in the document using the “track changes” mode and margin comments.
  • On »P (»P/O ratio) and «P: Can a stronger statement be made here? I like the suggested symbols.
  • On ADP concentration: It may be useful to discuss the potential confusion between high ADP and saturating ADP. The arbitrariness of some commonly used protocols is a problem in the field, particularly when using plate-based systems for measuring respiration.


  • 2017-08-11
Feedback and suggestions on Version 9 by Javier Iglesias-Gonzalez.
  • I wish you would elaborate more on the respiratory 'control', I always wondered how and why this term is adopted, instead of other terms, such as mitochondrial functional characteristics or functional anatomy.
  • Addition to the MS by EG: Control and regulation: The terms metabolic control and regulation are frequently used synonymously, but are distinguished in metabolic control analysis (Fell 1997). Respiratory control may be exerted by (1) ATP demand (Fig. 2), (2) fuel substrate, pathway competition and oxygen availability (starvation and hypoxia), (3) the protonmotive force, redox states, flux-force relationships, coupling and efficiency, (4) mitochondrial enzyme activities and allosteric regulation by adenylates, phosphorylation of regulatory enzymes, Ca2+ and other ions including pH, (5) inhibitors (e.g. NO or intermediary metabolites, such as oxaloacetate), (6) enzyme content, concentrations of cofactors and conserved moieties) such as adenylates, NADH/NAD+, coenzyme Q, cytochrome c); (7) metabolic channeling by supercomplexes, (8) mitochondrial density and morphology (fission and fusion), (9) hormone levels, gender, life style (influencing all control mechanisms listed before), and (10) genetic or acquired diseases causing mitochondrial dysfunction (for reviews see Brown 1992; Gnaiger 1993a, 2009; 2014; Morrow et al 2017).
  • I was really happy to find a section "Size-specific quantities", where you wrote "The well-established scaling law in respiratory physiology reveals a strong interaction of oxygen consumption and body mass by the fact that mass-specific basal metabolic rate (oxygen flux) does not increase proportionally and linearly with body mass, whereas maximum mass-specific oxygen flux, VO2max, is constant across a large range of body mass (Weibel and Hoppeler 2005)." However, I understand the mass-specific (basal) metabolic rate (oxygen flux) decrease proportionally if not linearly with body mass.
  • I found the discussion on the protonmotive force very challenging. I wonder if you could make it simpler for more wide audiences.
  • 2017-08-10 Version 10:
Integration of suggestions and corrections by T Komlodi and A Meszaros.
Phosphorylation, »P: Although phosphorylation in the context of OXPHOS is clearly defined as phosphorylation of ADP to ATP, potentially involving substrate-level phosphorylation as part of the tricarboxylic acid cycle (succinyl-CoA ligase), in the matrix (phosphoenylpyruvate carboxykinase) and in the cytosol (pyruvate kinase, phosphoglycerate kinase). ADP is formed in the adenylate kinase reaction, 2 ADP <--> ATP + AMP. In isolated mitochondria high adenylate kinase related ATP production can be detected in the presence of ADP and without respiratory substrates (Komlódi and Tretter 2017). On the other hand, the term phosphorylation is used in the general literature in many different contexts (phosphorylation of enzymes, etc.). This justifies consideration of a symbol more discriminative than P as used in the P/O ratio (phosphate to atomic oxygen ratio), where P indicates phosphorylation of ADP to ATP or GDP to GTP. We propose the symbol »P for the energetic uphill direction of phosphorylation coupled to catabolic reactions, and likewise the symbol «P for the corresponding downhill reaction (Fig. 2).
Coupling and efficiency: In an energy transformation, tr, coupling occurs between processes, if a coupling mechanism allows work to be performed on the endergonic or uphill output process (work per unit time is power; dW/dt [J/s] = Pout [W]; with a positive partial Gibbs energy change) driven by the exergonic or downhill input process (with a negative partial Gibbs energy change). At the limit of maximum efficiency of a completely coupled system, the (negative) input power equals the (positive) output power, such that the total power equals zero at an efficiency of 1. If the coupling mechanism is disengaged, the output process becomes independent of the input, and both proceed in their downhill direction (Fig. 2). - (added by EG)
Extensive quantities: An extensive quantity increases proportional with system size. The magnitude of an extensive quantity is completely additive for non-interacting subsystems, such as mass or flow expressed per defined system. The magnitude of these quantities depends on the extent or size of the system (Cohen et al 2008).
Size-specific quantities: ‘The adjective specific before the name of an extensive quantity is often used to mean divided by mass’ (Cohen et al 2008). A mass-specific quantity (e.g. mass-specific flux is flow divided by mass of the system) is independent of the extent of non-interacting homogenous subsystems. Tissue specific quantities are of fundamental interest in comparative mitochondrial physiology, where specific refers to the type rather than mass of the tissue. The term specific, therefore, must be clarified further, such that tissue mass-specific (e.g. muscle mass-specific) quantities are defined. - (added by EG)
  • 2017-08-08 to 09:
Updated versions by [Gnaiger E|EG]], with Sections 3.2. Normalization: flows and fluxes and 3.3. Conversion: oxygen, protons, ATP
Forces and flows in physics and irreversible thermodynamics: According to definition in physics, a potential difference and as such the protonmotive force, ∆pmt, is not a force (Cohen et al 2008). The fundamental forces of physics are distinguished from motive forces (e.g. ∆pmt) of statistical and irreversible thermodynamics. Complementary to the attempt towards unification of fundamental forces defined in physics, the concepts of Nobel laureates Lars Onsager, Erwin Schrödinger, Ilya Prigogine and Peter Mitchell (even if expressed in apparently unrelated terms) unite the diversity of ‘isomorphic’ flow-force relationships, the product of which links to the dissipation function and Second Law of thermodynamics (Prigogine 1967; Schrödinger 1944). A motive force is the change of potentially available or ‘free’ energy (exergy) per isomorphic motive unit (force=exergy/motive unit; in integral form, this definition takes care of isothermal and non-isothermal processes). A potential difference is, in the framework of flow-force relationships, an isomorphic force, Ftr, involved in an exergy transformation, defined as the partial derivative of Gibbs energy, ∂trG, per advancement, dtrΟ, of the transformation, tr (the isomorphic motive unit in the transformation): Ftr = ∂trG/dtrΟ (Gnaiger 1993a,b). This formal generalization represents an appreciation of the conceptual beauty of Peter Mitchel’s innovation of the protonmotive force against the background of the established paradigm of the electromotive force (emf) defined at the limit of zero current (Cohen et al 2008).
Coupling, efficiency and power: In energetics (ergodynamics) coupling is defined as an exergy transformation fuelled by an exergonic (downhill) input process driving the advancement of an endergonic (uphill) output process. The (negative) output/input power ratio is the efficiency of a coupled energy transformation. Power, Ptr = ∂trG/dt [W=J∙s­-1], is closely linked to the dissipation function (Prigogine 1967) and is the product of flow, Itr=dtrΟ∙dt-1 [xtr∙s-1] times generalized force, Ftr = ∂trG/∂trΟ [J∙xtr-1] (Gnaiger 1993b).
  • 2017-08-08: Updated paragraph by [Gnaiger E|EG]] on ‚P/O‘
JV,O2 is coupled at mitochondrial steady states to proton cycling, J∞H+ = JH+out = JH+in (Fig. 1). JH+out [pmol∙s-1∙ml-1] is converted into an electric flux (per volume), Jel [”C∙sÂŹ1∙mlÂŹ1=”A∙mlÂŹ1] = JH+out [pmol∙s-1∙ml-1]∙F [C∙mol-1]∙10-6. F is the Faraday constant (96,485.3 C∙mol-1). At a JH+out/JO2 ratio or H+out/O2 of 20 (H+out/O2=10), a volume-specific oxygen flux of 100 pmol∙s-1∙ml-1 would correspond to a proton flux of 2,000 pmol H+out∙sÂŹ1∙mlÂŹ1 or volume-specific current of 193 ”A∙mlÂŹ-1.
Jel [”A∙mlÂŹ1] = JH+out∙F∙10-6 [pmol∙s-1∙ml-1∙”C∙pmol-1](1.1)
Jel [”A∙mlÂŹ1] = JV,O2∙(H+out/O2)∙F∙10-6 [pmol∙s-1∙ml-1∙”C∙pmol-1](1.2)

In the OXPHOS state or at high [ADP], JH+in would in turn drive a phosphorylation flux, JV,»P, of 500 pmol∙sÂŹ1∙mlÂŹ1 at a H+in/»P ratio of 3.7 (nH+; Fig. 2B). For NADH- and succinate-linked respiration, the mechanistic »P/O2 ratio is calculated at 20/3.7 and 12/3.7 (Eq 2), or 5.4 and 3.3 (equivalent to »P/O of 2.7 and 1.6; Watt et al 2010), in direct agreement with the measured »P/O ratio for succinate of 1.58 ± 0.02 (Gnaiger et al 2000), »P/O2 = (H+out/O2)/( H+in/»P)(2) In summary, JV,»P [pmol∙s-1∙ml-1] = JV,O2∙(H+out/O2)/( H+in/»P)(3.1) JV,»P [pmol∙s-1∙ml-1] = JV,O2∙(»P/O2)(3.2)

Gnaiger E, MĂ©ndez G, Hand SC. High phosphorylation efficiency and depression of uncoupled respiration in mitochondria under hypoxia. Proc Natl Acad Sci USA 2000b;97:11080-5. Watt IN, Montgomery MG, Runswick MJ, Leslie AG, Walker JE. Bioenergetic cost of making an adenosine triphosphate molecule in animal mitochondria. Proc Natl Acad Sci U S A 2010;107:16823-7.

Many thanks for your very valuable comments. The nomenclature on ETS complexes (including not only CI to CIV) versus mt-enzymes versus the gene-linked terminology should indeed be discussed thoroughly. I will add your comments under your name to the website for discussion. The pathway control states have been removed from our first part and will be the focal topic of Part 2. Perhaps Fig. 2A should be simplified for Part 1, to avoid the terms CGpDH and CETF.
  • 2017-08-02
Updated ms summarizing WG1 input by J Iglesias-Gonzalez.
  • 2017-07-29
Version 3 on website: http://www.mitoeagle.org/index.php/Mitochondrial_respiratory_control:_MITOEAGLE_recommendations_1
  • 2017-07-28 to 29:
Obergurgl MITOEAGLE Workshop: WG1 – group discussion and edits based on printed Version 2.
Concerning:
  • State 1: depending on the fact that mitochondrial extract of isolated mitochondria is crude or purified (for exemple on Percoll gradients or sucrose gradients), the isolated mitochondria have still somes endogenous substrats or not that makes a pulse at the start during their early dissipation. Could this be taken into consideration or being indiscted for the users.
  • State 4: I do not find this very well writen since after the addition of ADP (state 3) the mitochondrial membrane potential drop and reacquire its high value (at the state 4) when all the ADP has been transformed in ATP has been used? am I wrong. So this shoul be writen clearly.
Why escaping to state about CR (respiratory control) and ADP/O measurements and definitions?
Again, I perfectly understand the reference to the bioblast link by since we do not live outside teh real world the reference are usually referred to pubmed also... so we should refer to both...
For te publication, the suggestion is nice but by tradition our journals are more....
  • BBA general subjects or bioenergetic: 6554 occurrences
  • FEBS J: 2911 FEBS letters 2563 (so 1+2 = 5474 Occurrences)
  • Journal of Biological Chemistry: 7730 occurrences
My personnal preference will go To BBA general subjects... But should be decided in commun
  • 2017-04-18
Version 1 circulated to MITOEAGLE by E Gnaiger.
  • 2017 Mar 21-23:
Barcelona MITOEAGLE Workshop: WG1 – presentations and group discussions.


Please integrate the statements below into the work flow

Out of chronological context, many statements loose context and are not clear. For instance, section numbers have changed over time, etc. ~ Gnaiger Erich (talk) 20:47, 7 September 2017 (CEST)
I think that this is a great service to the field. I have made some brief comments in the attached.
This looks great. Thanks for the edits.
I went through your interesting review which for me also enlightened new aspects on the mitochondrial chemophysical properties. I completely agree with the need to establish a common language and standard criteria in the field of mitochondria, so that different studies can be compared and abnormalities associated with diseases will be defined and maybe used in the future as biomarkers. I introduced several changes and asked for several clarifications by comments and notes in the text. As I am not an expert in fluxes and flows, forces and rates my input to these parts is limited.
I do suggest to include a short paragraph on the crosstalk between mitochondria and the cells via Ca, and other signaling pathways which may affect mitochondrial activity in the conclusion part. Although it is beyond the scope of the review it puts it in a wider physiological context.
First, I want to thank you for sharing the manuscript. Since I am more a "mitochondrial geneticist" than a mitochondrial physiologist, I found the manuscript on mitochondrial respiratory control very clear and well written, and it will be very helpful for my students (and also for myself actually)!! I have very few edits (attached is the manuscript).
I applaud you and the co-authors on an excellent first step toward (re)defining terminology and concepts on mitochondrial respiratory control. While the definitions and explanations are very carefully stated, I agree with previous comments that the overall length and level of detail might limit how well the key concepts and terms are grasped and adopted by what we hope will be a broad readership of physiologists and biochemists.
Happy to be a co-author and provide critical feedback on this important paper. Just glancing at it now – it is in pretty good shape.
I added in a few comments on the attached PDF. I think it looks great and I’m glad to have been a part of the process. 'This bubble feels a bit disjointed from the rest of the paper. I'm not sure that it's necessary since it's never referred back to.'
As for our concern, this version can get our approval. However, we think the text can be improved in the aspect of linguistic coherence.
  • David G Nicholls:
Thank you for the invitation to contribute to your review. However, there is too great a distance between your in-depth approach and the minimalistic ‘need-to-know’ proton circuitry that I have promulgated for the past 40 years or so, and so I must decline. Good luck with the review.
I appreciate the opportunity to contribute to this joint review. Please give me a few days to examine the most recent version – if I feel that I understand the material well enough to comment and/or approve, I will be happy to do so and join the co-author list.
Just glancing at it, it seems to be in a great shape and have improved significantly since the last time I read it.
Many thanks for your kind offer, putting together a review on Mitochondrial respiratory control is such a great idea and would be of great value to the community. I’d be delighted to be a part and support this review.
Please find the attached word document with some minor suggestions for your consideration.
Find attached my comments after reading the manuscript. There is a lot of work behind the content of this manuscript. Some parts will be more difficult to follow for scientists new to the field of mitochondrial physiology but the information supply is correct, careful and hopefully an starting point to start unifying terminology.
  • Garry Buettner
I fully appreciate pushing the community to a common language and lab approaches so that data gathered can be useful far beyond the specific question of an experiment.
Your editing to include our thoughts is wonderful. I offer a few suggestions and minor edits.
I agree with you and the purpose and philosophy that the paper pursues.
I think it looks really good! It does get very complex at times, but so is the underlying information that is to be communicated.
I’ve just finished with the last version (9) of the paper. I really like how it looks like now and I have let some of our PhD students to read (as a test for a beginner) and she enjoyed a lot the paper. I added very few suggestions which I attached in the word document.
  • Brian Irving: The review is coming together nicely. Much more advanced than a couple weeks ago. Great job. I added a few comments.
I attached the PDF file with my comments. The paper is excellent material for all oxygraphists and bioenergetics. It is also in perfect agreement with the scientific viewpoints of our laboratory.
In general this is very well written text!
The manuscript is attached with some suggestions included.
  • Hong Kyu Lee:The chapter on protonmotive force is difficult to read.
Thank you for your generous invitation to become an author of this historical paper. This is a great honor to me. I have some comments to make;
Here is my revision and suggestions to the manuscript. Thank you for putting all that work together in a very helpful review for mitochondrial scientists.
I enjoy reading the review and found it incredibly helpful. Some section, however, are a little harder to follow and not easy to read, mostly because the amount of formulas and units.  ::::I felt like the main message is lost in the details. I added my comments directly in the document.
I really enjoyed the manuscript - clear definitions that are about meaning of processes not just numbers.
About Table - super idea!
As a whole the manuscript reads easily. I have just few comments/suggestions. Please note that I worked on the version 24’ / 2017-08-25.
  • Morten Scheibye-Knudsen
Regarding the journal, if you get a large consensus which includes a large contingency of authors I guess this could be of interest for a higher level journal because it could act as a reference point. Perhaps even something like Cell Metabolism. - I have attached a version with a some suggestions as you can see. It is obviously a great reference manuscript for mitochondrial investigations and I have only a few corrections/comments.
I have just caught up with v17, which is looking very good indeed
Overall, I think it is a valuable position statement, which neatly conveys the need for a universal nomenclature on respiration states (i.e. not restricted to a particular protocol) whilst giving necessary context to the historical states. Thank you for bringing this all together.
Again, thank you very much for the invitation in this excellent and seminal work. I am sending the PDF file with some comments and additions.
I find the text clearly exposed even if the publicity for Mitoeagle is to much pronounced (but that is a choice).
first of all I would like to thank you for this really nice initiative. See attached my annotated comments, and I hope it will further strenghten the manuscript.
I have read the MS, thank's for putting things together. I have added my comments in the pdf attached. I like the newly added part on mitochondria in generell. The part on protonmotive force is ambitious.
I think this is a very important project, and it will be fantastic to have this resource (and your future MitoEAGLE work) available to mitochondrial physiologists. I’m happy to add my name as a co-author if it is helpful. I don’t have much to add to the manuscript, its already very clear and I agree whole-heartedly with nearly all of what is written. I have a few editorial comments for you to consider below.
I would like to offer a few comments to the manuscript, which I found to be very good. :::: I always appreciate your thoughtfulness and approach to science.
I have read joint review, it is very nice manuscript, and very useful for teaching. I really like the chapter Normalization: flows and fluxes, its very good expalined.
I have carefully red version 20 and in my opinion is a fantastic peace of work. Up to this moment I did not have any recommendation for changes or improvements. I will follow the new versions. It is a honor for me to participate in this project.
I really enjoyed reading the manuscript.It is great.

Please find attached PDF file with my first comments comments and proposals of additions.

I know, that it is non physiological situation (in swollen mitochondria) but it is possible to measure in plant mitochondria (e.g. potato tuber) a cytochrome c dependent, antimycin-A resistant respiration thanks to the NADH-cytochrome c reductase. This phenomenon was described between 1970 and 1980.

Later there is no paper directly connecting an outer mitochondrial membrane enzyme with the complex IV. What do you think? Can we mention about this? Such phenomenon was not described for liver or hart mitochondria.

EG:
It would be helpful to widen the perspective to plant mitochondria.
Wieckowski Mariusz:
I think that it is a mistake and hexokinase should be deleted. Reading this sentence I have a feeling that hexokinase among other mentioned here enzymes is DIRECTLY involved in the phosphorylation of ADP. It is well known that e.g based on the paper of Nelson and Kabir (BBA; 1985) that in mitochondria from hepatoma adenylate kinase provides about 50% of ATP that IS USED by Hexokinase. I do not know the metabolic situation in which hexokinase phosphorylates ADP. I propose to delete hexokinase.
EG:
Hexokinase: Perhaps we need a figure for explanation. In my understanding the mitochondrial hexokinase reaction serves a function in metabolic channeling analogous to mitochondrial creatine kinase. What do you think? This could be an extension to Fig. 1B.
  • Yau-Huei Wei: I have completed reading and revising this manuscript (attached). I appreciate your enthusiasm and great effort in preparing this manuscript.
This sounds like a very exciting Project! Congratulations for that terrific milestone!
I have to say that I found it very interesting, and useful. Enclosed you will find some minor comments (that you will find as sticky notes in the pdf file).

The manuscript flows well although I need to tell you that in some sections (3.2, and 3.3) I have no critical input, and more biophysical knowledge than the one I have would be required to do so.

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