Talk:The protonmotive force and respiratory control

Work flow

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 after (A) Lemieux et al (2017) and (B) Gnaiger (2014).

• 2017-08-15 New Fig. 1 in Version 15 (E Gnaiger).

• 2017-08-15 Tony Moore:

• 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)." E Gnaiger

• 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."

• F_O2 versus F

Added after Eq. 1: ".. and a chemical part incorporating the Faraday constant"

• 2017-08-15 Beata Velika: 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.
• 2017-08-14 E Gnaiger 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.

• 2017-08-14 Guy Brown:

"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!"

• 2017-08-14 Masashi Tanaka: Corrections and comments (see below).
• 2017-08-13 Brian Irving: The review is coming together nicely. Much more advanced than a couple weeks ago. Great job. I added a few comments.
• 2017-08-12 E Gnaiger 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 E Gnaiger)

• 2017-08-12 Feedback and suggestions on Version 10 by A Molina.
• 2017-08-11 Feedback and suggestions on Version 9 by J Iglesias-Gonzalez.
• 2017-08-11 HK Lee: ‘Control’ should be defined. The chapter on protonmotive force is difficult to read.

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). - (added by E Gnaiger)

• 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] = P_out [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 E Gnaiger)

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 E Gnaiger)

• 2017-08-08 to 09: Updated versions by E Gnaiger, 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, ∆p_mt, is not a force (Cohen et al 2008). The fundamental forces of physics are distinguished from motive forces (e.g. ∆p_mt) 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, F_tr, involved in an exergy transformation, defined as the partial derivative of Gibbs energy, ∂_trG, per advancement, d_trξ, of the transformation, tr (the isomorphic motive unit in the transformation): F_tr = ∂_trG/d_trξ (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, P_tr = ∂_trG/dt [W=J∙s­^-1], is closely linked to the dissipation function (Prigogine 1967) and is the product of flow, I_tr=d_trξ∙dt-1 [xtr∙s^-1] times generalized force, F_tr = ∂_trG/∂_trξ [J∙xtr^-1] (Gnaiger 1993b).

• 2017-08-02 Updated ms summarizing WG1 input by J Iglesias-Gonzalez.
• 2017-08-29 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.
• 2017-04-21 PX Petit: I find the text clearly exposed even if the publicity for Mitoeagle is too much pronounced (but that is a choice).
• 2017-04-18 Version 1 circulated to MITOEAGLE by E Gnaiger.
• 2017 Mar 21-23: Barcelona MITOEAGLE Workshop: WG1 – presentations and group discussions.

2017-08-14 Guy Brown

Response by E Gnaiger

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. On "Control and regulation": I do not understand your question.
5. tr: consider spelling this out in all following symbols.
6. 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.
7. 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.”
8. Your excellent comments on detail have all been incorporated in Version 14.
9. "∆p_mt - 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.
10. “The protonmotive force is maximum in the LEAK state” - I agree with your comment – is “elevated” better?
11. "Forces and flows in physics and irreversible thermodynamics: Why?" – Can we ignore IUPAC recommendations?

2017-08-14 Masashi Tanaka

Response by E Gnaiger

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 J_m,O2peak of 45 to 60 nmol·s^-1·g^-1 (Gnaiger 2014).”

2017-08-12 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 Hong Kyu Lee

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;

• 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.
• 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.

2017-08-01 Javier Iglesias-Gonzalez

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.

2017-04-21 Patrice Petit

I find the text clearly exposed even if the publicity for Mitoeagle is to much pronounced (but that is a choice).

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

Sincerely yours

Patrice - Petit PX