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Evolution-Age-Gender-Lifestyle-Environment: mitochondrial fitness mapping

The protonmotive force and respiratory control

OXPHOS-coupled energy cycles. From Gnaiger 2014 MitoPathways.

Scope of MITOEAGLE publications: respiratory control

Target a broad audience – also the new generation
List of terms including historical terms; abbreviations (mtDNA, mt to abbreviate mitochondr*); OXPHOS capacity versus State 3 (discuss saturating ADP/Pi .. concentrations)
Scientific terminology should be general and platform independent - meet the demands of all working groups.

Mitochondrial respiratory control: a conceptual perspective on coupling states

MITOEAGLE recommendations Part 1.

MITOEAGLE Terminology Committee - first draft prepared by Erich Gnaiger (work in progress 2017-04-18 - ...)

Journal: Int J Biochem Cell Biol (W Koopman will be the new editor); Open Access is a requirement

Abstract

Clarity of concepts and consistency of nomenclature is a signature of the quality of a research area across its specializations, aimed at facilitating translational communication and teaching. The expanding field of mitochondrial respiratory physiology can benefit from the harmonization of nomenclature on mitochondrial respiratory states and control parameters. Peter Mitchell’s protonmotive, chemiosmotic force across the inner mitochondrial membrane, Δp_mt, establishes the link between electron transfer and phosphorylation of ADP to ATP, and between the chemical (pH difference, ΔpH) and electric (mt-membrane potential difference, ΔΨ_mt) components of energy transformation. This unifying concept provides the framework upon which a consistent terminology on mitochondrial physiology and bioenergetics can be based. IUPAC guidelines are followed for general terms of physical chemistry, extended by concepts of nonequilibrium thermodynamics and open systems. The differential nomenclature of classical bioenergetics (numerical differentiation of experimental protocol-linked respiratory States 1, 2, 3, 4 and 5) is incorporated into a concept-driven constructive terminology to address the basic meaning of a respiratory state and direct attention from the experimental ‘how’ to the concentual ‘why’. LEAK states are evaluated to study resting respiration, L, compensating mainly for the proton leak. OXPHOS capacity, P, is measured at saturating levels of ADP and inorganic phosphate to obtain kinetic references values for diagnostic applications. The ETS state differentiates the oxidative capacity of the electron transfer system, E, from OXPHOS capacity, revealing the limitation of P by the phosphorylation system. Development of databases on mitochondrial respiratory control requires application of strictly defined terms for comparison of respiratory states.

Introduction

Every study of mitochondrial function and disease in human tissues and cells is faced with evolution, age, gender, lifestyle and environment (EAGLE) as essential background conditions characterizing the individual patient, subject, study group, species, tissue or – to some extent - cell line. This range of factors is too wide to be accommodated in any single project on mitochondrial respiration. Only a large and well coordinated network can manage to generate the necessary type, quality and number of consistent data to address the complexity of EAGLE. The global MITOEAGLE network is a strategic innovation to develop harmonization protocols towards generating a rigorously monitored database on mitochondrial respiratory function. A quality control and data management system (QMS) is necessary to interrelate results obtained in a large number of studies, to interpret pathological phenotypes, and to set results into the multidimensional context of EAGLE.

Reliability and comparability of quantitative results depends on accuracy of measurement under well defined conditions. To relate results of studies carried out by different research groups further requires reporting in a conceptually meaningful framework, assigning values expressed in common units (the measurements) to specific attributes (the rows in a table). If the attribute is well defined and understood (‘well defined experimental conditions’), it does not matter which names or symbols are given to the rows in the table of a database. But if terminology is vague or ambiguous when characterizing the type of result, then comparison of even the most accurate data can lead to confusion and may turn valuable signals into wasteful noise. Clarification of some fundamental terms used in studies of mitochondrial respiratory control, therefore, represents a basic component of a QMS. Even if standardization of nomenclature remains a goal of the optimist and is out of reach in the real world, harmonization of the technical jargon will improve the awareness of the intricate meaning of divergent scientific vocabulary. The MITOEAGLE Terminology Committee aims at accomplishing the ambitious goal to harmonize, unify and thus simplify the terminology in the field of mitochondrial physiology.

‘Every professional group develops its own technical jargon for talking about matters of critical concern. .. People who know a word can share that idea with other members of their group, and a shared vocabulary is part of the glue that holds people together and allows them to create a shared culture’ (Miller 1991).

Constructive versus differential terminologies

Five classical states of mitochondrial respiration and cytochrome redox states have been introduced by Chance and Williams (1955; 1956). These respiratory states are explained by a protocol with isolated mitochondria (imt) in a closed respirometric chamber:

State 1 is obtained after addition of imt to isotonic respiration medium containing inorganic phosphate but no adenylates, specifically no ADP, and no fuel substrates (CHNO).

State 2 is induced by addition of a high level of ADP, which stimulates transiently respiration on the basis of endogenous fuel substrates, followed by a low respiratory activity limited by fuel substrate availability.

State 3: Respiratory fuel substrates are added to initiate State 3. Respiration is stimulated while the ADP level is still high and supports coupled energy transformation in oxidative phosphorylation.

State 4: If the imt preparation is of high quality, devoid of ATPase activity and well coupled, gradual depletion of ADP by phosphorylation to accumulating ATP leads to a sudden decline of oxygen uptake in the transition from State 3 to State 4, when a maximum mt-membrane potential and the ATP/ADP ratio are maintained. State 4 respiration is limited by physiological uncoupling (mainly the intrinsic proton leak). A repeated titration of high ADP can restore active respiration in State 3.

State 5 is the anoxic state after the aerobic-anoxic transition, with zero respiration in a closed respirometric system. Ideal closed respirometers without any backdiffusion of oxygen into the chamber are difficult to obtain in practice, whence this steady state (State 5) is characterized by the oxygen-limited respiration compensating for the oxygen backdiffusion at near-zero oxygen levels.

‘It is essential to define both the substrate and ADP levels in order to identify the steady-state condition of the mitochondria during the experiment’ (Chance and Williams, 1956).

It has been suggested to extend the differential nomenclature (States 1 to 5) by a concept-driven constructive terminology on coupling states (Gnaiger 2009). In a constructive theory, the meanings are expressed by terms which carry information on the concept they represent (Miller 1991). The concept-driven terminology must be (i) general and not restricted to a particular experimental protocol with imt in a closed oxygraph, (ii) applicable to any mt-preparation, (iii) describe and guide studies with any experimental platform using a wide variety of experimental protocols, and (iv) extend the focus of classical bioenergetics on coupling mechanisms and coupling sites of oxidative phosphorylation to perspectives on mitochondrial physiology and pathology with diagnostic applications. This does not lead simply to a system of synonyms, but to a conceptual differentiation between the classical respiratory states which have been entirely sufficient to describe a specific bioenergetic protocol on one hand, and the extended constructive framework of the theory of mitochondrial physiology on the other hand.

Capacities of the electron transfer system, oxidative phosphorylation and resting LEAK respiration (ETS, OXPHOS, LEAK) and four-compartmental OXPHOS model. (i) Capacity of the ETS module, E, in the noncoupled state, generating the protonmotive force, Δp_mt. OXPHOS capacity, P, is partitioned into (ii) the dissipative LEAK component, L (disspation of Δp_mt), and (iii) the free OXPHOS capacity, ≈P=P-L (energy conversion driven by Δp_mt). If ≈P is limited by the capacity of the phosphorylation system, then (iv) the apparent ETS excess capacity, ExP=E-P, is available to drive coupled processes other than phosphorylation without competing with ATP production. Free divided by total ETS capacity, ≈E/E, is the ETS coupling efficiency. Free divided by total OXPHOS capacity, ≈P/P, is the OXPHOS coupling efficiency. From Gnaiger 2014 MitoPathways.

Three fundamental coupling states of mitochondrial preparations

ETS, state E: Noncoupled state at optimal uncoupler concentration for maximum oxygen flux as a measure of electron transfer system (ETS) capacity, in the presence of fuel substrates and oxygen. The abbreviation State 3u is used frequently in bioenergetics, to indicate the state of maximum respiration, E, without sufficient emphasis on the fundamental difference between state P (OXPHOS capacity; coupled, with an uncoupled component) and state E (ETS capacity, noncoupled).

OXPHOS, state P: In mitochondrial physiology and pathology, maximal mitochondrial respiration in the coupled state is measured for quantitative determination of OXPHOS capacity, P (Gnaiger 2009). State P is the ADP-activated state at maximum oxygen flux as a measure of the capacity for oxidative phosphorylation (OXPHOS), supported by saturating ADP and inorganic phosphate concentrations and in the presence of fuel substrates and oxygen. The definition of State 3 lacks a fundamental attribute of OXPHOS capacity. 'High ADP' in State 3 is a concentration of ADP specifically selected to allow measurememt of a State 3 to State 4 transition of isolated mitochondria in a closed respirometric system. Starting at oxygen levels near air saturation, the 'high ADP' concentration added must be low enough to allow phosphorylation to ATP at a coupled oxygen consumption that does not lead to oxygen depletion during the transition to State 4. In contrast, OXPHOS capacity requires evaluation of kinetically saturating ADP concentrations, which are usually an order of magnitude higher than 'high ADP' in State 3.

LEAK, state L: Resting state of non-phosphorylating respiration when oxygen flux is maintained mainly to compensate for the proton leak in the absence of ATP synthesis, either due to absence or depletion of ADP at a maximum ATP/ADP ratio, or due to inhibition of phosphorylation by inhibitor such as oligomycin or atractyloside, in the presence of fuel substrates and oxygen. State 4 represents an overestimation of LEAK respiration if ATPase activity prevents final accumulation of ATP and maintains a continuous stimulation of respiration by recycled ADP. This can be tested by inhibition of phosphorylation (oligomycin).

In kinetic studies an infinite number of intermediary steady states can be established (i) between LEAK an OXPHOS, using ADP-regenerating enzyme systems or steady-state ADP injections, (ii) between LEAK and ETS or OXPHOS and ETS, by uncloupler titrations, and (iii) between any of the three fundamental coupling states and ROX, by inhibitor titrations.

Partitioning of respiration in various coupling states

Gnaiger 2014 MitoPathways

ROX: Residual oxygen consumption (ROX) is not characteristic of a coupling state, but represents a baseline for correction of mitochondrial respiration in the presence of oxygen and the absence of fuel substrates or after addition of inhibitors of the electron transfer system. Total respiration is thus partitioned into a component functionally linked to the electron transfer system and a component not linked to the ETS (ROX). ROX is frequently considered to be equivalent to non-mitochondrial respiration, which ignores the fact that mitochondria contain enzymes which catalyse oxygen-consuming reactions not linked to the ETS and OXPHOS.

Partitioning of LEAK respiration: LEAK respiration can be partitioned into patial oxygen consumption compensating for (1) proton leaks, (2) proton slip, (3) cation cycling, and (4) electron leak.

Free OXPHOS capacity, ≈P: ≈P is the partial oxygen consumption which is strictly coupled to phosphorylation of ADP to ATP, ~P, when partitioning OXPHOS capacity into a coupled and uncoupled component.

LEAK respiration in the OXPHOS state, L_P: Since OXPHOS is partially coupled, intrinsic uncoupling and dyscoupling contribute to the control of flux in the OXPHOS state. Oxygen consumption in the OXPHOS state, therefore, is partitioned into the free OXPHOS capacity, ≈P, and nonphosphorylating LEAK respiration, L_P. It is frequently assumed that LEAK respiration, L, as measured in the LEAK state, overestimates the LEAK component of respiration, L_P, as measured in the OXPHOS state, particularly if the protonmotive force is not adjusted to equivalent levels in L and L_P. However, if the LEAK component increases with enzyme turnover during P, the low enzyme turnover during L may counteract the effect of the higher Δp_mt (Garlid et al 1993).

P = ≈P + L_P.

Excess E-P capacity, ExP: ExP is the difference of the ETS capacity and OXPHOS capacity, ExP = E-P. At ExP > 0, the capacity of the phosphorylation system exerts a limiting effect on OXPHOS capacity. In addition, ExP depends on coupling efficiency, since P aproaches E at increasing uncoupling. When partitioning ETS capacity, a OXPHOS-linked component (intrinsig LEAK respiration plus strictly coupled respiration) is distinguished from ExP as the apparent excess capacity above the OXPHOS-linked component.

Free ETS capacity, ≈E: ≈E is the ETS capacity corrected for LEAK respiration, ≈E = E-L. ≈E is the respiratory capacity potentially available for phosphorylation of ADP to ATP and ion transport. Oxygen consumption in the ETS state, therefore, is partitioned into the free ETS capacity, ≈E, and LEAK respiration, L_P, compensating for proton leaks, slip and cation cycling:

E = ≈E + L_P.

Rates and states

Units (important for a database); analogous to electic terms: Flow [C.s-1]; Flux [C.s-1.m-2]; Rate (?)

Metabolic fluxes and steady-state variables are measured in defined mitochondrial respiratory states. Strictly, steady states can be obtained only in open systems, in which internal transformations (e.g. oxygen consumption or enthalpy change of chemical reactions) are instantaneously compensated by external flows (oxygen supply or heat flow across the system boundaries), such that changes do not occur in the system but only in the environment. Mitochondrial respiratory states measured in closed systems may satisfy the criteria of pseudo-steady states for limited periods of time, when the changes occurring in the system (e.g. oxygen concentration; ADP concentration; mt-membrane potential) do not exert significant effects on the metabolic fluxes (respiration, phosphorylation of ADP to ATP). Such pseudo-steady states require saturation levels of substrates to be maintained and thus depend on the kinetics of the processes under investigation.

Intact cells versus mitochonddrial preparations

Normalization of flux and flow

Conclusions

With a perspective to extend the present recommendations on coupling control (part 1) to pathway control of mitochondrial respiration (part 2), substrate-uncoupler-inhibitor-titration (SUIT) protocols, and harmonization of experimental procedures, MITOEAGLE will be a gateway and milestone to better diagnose mitochondrial respiratory defects which are linked to various age-related health risks, including cardiovascular and degenerative diseases, such as type 2 diabetes, neurodegenerative diseases (Alzheimer’s, Parkinson’s, Huntington’s), and several types of cancer.

References

Altmann R (1894) Die Elementarorganismen und ihre Beziehungen zu den Zellen. Zweite vermehrte Auflage. Verlag Von Veit & Comp, Leipzig:160 pp, 34 Tafeln. - »Bioblast link«
Chance B, Williams GR (1955) Respiratory enzymes in oxidative phosphorylation. I. Kinetics of oxygen utilization. J Biol Chem 217:383-93. - »Bioblast link«
Chance B, Williams GR (1955) Respiratory enzymes in oxidative phosphorylation. III. The steady state. J Biol Chem 217:409-27. - »Bioblast link«
Chance B, Williams GR (1956) The respiratory chain and oxidative phosphorylation. Adv Enzymol17:65-134. - »Bioblast link«
Cohen ER, Cvitas T, Frey JG, Holmström B, Kuchitsu K, Marquardt R, Mills I, Pavese F, Quack M, Stohner J, Strauss HL, Takami M, Thor HL (2008) Quantities, Units and Symbols in Physical Chemistry, IUPAC Green Book, 3rd Edition, 2nd Printing, IUPAC & RSC Publishing, Cambridge. - »Bioblast link«
Estabrook R (1967) Mitochondrial respiratory control and the polarographic measurement of ADP:O ratios. Meth Enzymol 10:41-7. - »Bioblast link«
Galluzzi L, Vitale I, Abrams JM, Alnemri ES, Baehrecke EH, Blagosklonny MV, Dawson TM, Dawson VL, El-Deiry WS, Fulda S, Gottlieb E, Green DR, Hengartner MO, Kepp O, Knight RA, Kumar S, Lipton SA, Lu X, Madeo F, Malorni W, Mehlen P, Nuñez G, Peter ME, Piacentini M, Rubinsztein DC, Shi Y, Simon HU, Vandenabeele P, White E, Yuan J, Zhivotovsky B, Melino G, Kroemer G (2012) Molecular definitions of cell death subroutines: recommendations of the Nomenclature Committee on Cell Death 2012. Cell Death Differ 19:107-20. - »Bioblast link«
Garlid KD, Semrad C, Zinchenko V (1993) Does redox slip contribute significantly to mitochondrial respiration? In: Schuster S, Rigoulet M, Ouhabi R, Mazat J-P (eds) Modern trends in biothermokinetics. Plenum Press, New York, London:287-93. - »Bioblast link«
Gnaiger E (1993) Nonequilibrium thermodynamics of energy transformations. Pure Appl Chem 65:1983-2002. - »Bioblast link«
Gnaiger E (1993) Efficiency and power strategies under hypoxia. Is low efficiency at high glycolytic ATP production a paradox? In: Surviving Hypoxia: Mechanisms of Control and Adaptation. Hochachka PW, Lutz PL, Sick T, Rosenthal M, Van den Thillart G (eds) CRC Press, Boca Raton, Ann Arbor, London, Tokyo:77-109. - »Bioblast link«
Gnaiger E (2009) Capacity of oxidative phosphorylation in human skeletal muscle. New perspectives of mitochondrial physiology. Int J Biochem Cell Biol 41:1837-45. - »Bioblast link«
Gnaiger E (2014) Mitochondrial pathways and respiratory control. An introduction to OXPHOS analysis. 4th ed. Mitochondr Physiol Network 19.12. OROBOROS MiPNet Publications, Innsbruck:80 pp. - »Bioblast link«
Gnaiger E, Méndez G, Hand SC (2000) High phosphorylation efficiency and depression of uncoupled respiration in mitochondria under hypoxia. Proc Natl Acad Sci U S A 97:11080-5. - »Bioblast link«
International Union of Biochemistry and Molecular Biology: Recommendations for terminology and databases for biochemical thermodynamics. - »Open Access«
Hatefi Y, Haavik AG, Fowler LR, Griffiths DE (1962) Studies on the electron transfer system. XLII. Reconstitution of the electron transfer system. J Biol Chem 237:2661-9. - »Bioblast link«
International Union of Biochemistry (1981) Symbolism and terminology in enzyme kinetics. - »Open Access«
Lemieux H, Blier PU, Gnaiger E (2017) Remodeling pathway control of mitochondrial respiratory capacity by temperature in mouse heart: electron flow through the Q-junction in permeabilized fibers. Sci Rep (in press). - »Preprint Bioblast link«
Miller GA (1991) The science of words. Scientific American Library New York:276 pp. - »Bioblast link«
Mitchell P (1961) Coupling of phosphorylation to electron and hydrogen transfer by a chemi-osmotic type of mechanism. Nature 191:144-8. - »Bioblast link«
Mitchell P (1966) Chemiosmotic coupling in oxidative and photosynthetic phosphorylation. Glynn Research Ltd, Bodmin:192 pp. - The Grey Book 1.
Mitchell P (1968) Chemiosmotic coupling and energy transduction. Glynn Research Ltd, Bodmin:111 pp. - The Grey Book 2.
Mitchell P, Moyle J (1967) Respiration-driven proton translocation in rat liver mitochondria. Biochem J 105:1147-62. - »Bioblast link«

Mitochondrial respiratory control: pathway states in mt-preparations and intact cells

MITOEAGLE recommendations Part 2.

The mitochondrial respiratory system
Substrates and inhibitors
Switch to pathway-related nomenclature instead of enzyme-linked terminology (N/NS/S versus CI/CI+II/CII)

Action

» WG1 Action - WG1 MITOEAGLE protocols, terminology, documentation: Standard operating procedures and user requirement document: Protocols, terminology, documentation

» WG1 Project application

» Pre-publication: Mitochondrial respiratory control states

» MitoPedia: Respiratory control ratios

» MitoPedia: SUIT

» 2017-07 MiPschool Obergurgl 2017

» 2017-03 MITOEAGLE Barcelona 2017

» 2016-11 MITOEAGLE 2016 Verona IT

@@ Line 24: / Line 24: @@
 :::: Every study of mitochondrial function and disease in human tissues and cells is faced with '''e'''volution, '''a'''ge, '''g'''ender, '''l'''ifestyle and '''e'''nvironment (EAGLE) as essential background conditions characterizing the individual patient, subject, study group, species, tissue or – to some extent - cell line. This range of factors is too wide to be accommodated in any single project on mitochondrial respiration. Only a large and well coordinated network can manage to generate the necessary type, quality and number of consistent data to address the complexity of EAGLE. The global MITOEAGLE network is a strategic innovation to develop harmonization protocols towards generating a rigorously monitored database on mitochondrial respiratory function. A quality control and data management system (QMS) is necessary to interrelate results obtained in a large number of studies, to interpret pathological phenotypes, and to set results into the multidimensional context of EAGLE.
 :::: Reliability and comparability of quantitative results depends on accuracy of measurement under well defined conditions. To relate results of studies carried out by different research groups further requires reporting in a conceptually meaningful framework, assigning values expressed in common units (the measurements) to specific attributes (the rows in a table). If the attribute is well defined and understood (‘well defined experimental conditions’), it does not matter which names or symbols are given to the rows in the table of a database. But if terminology is vague or ambiguous when characterizing the type of result, then comparison of even the most accurate data can lead to confusion and may turn valuable signals into wasteful noise. Clarification of some fundamental terms used in studies of mitochondrial respiratory control, therefore, represents a basic component of a QMS. Even if standardization of nomenclature remains a goal of the optimist and is out of reach in the real world, harmonization of the technical jargon will improve the awareness of the intricate meaning of divergent scientific vocabulary. The MITOEAGLE Terminology Committee aims at accomplishing the ambitious goal to harmonize, unify and thus simplify the terminology in the field of mitochondrial physiology.
+:::::::::::: ''‘Every professional group develops its own technical jargon for talking about matters of critical concern. .. People who know a word can share that idea with other members of their group, and a shared vocabulary is part of the glue that holds people together and allows them to create a shared culture’'' (Miller 1991).
-=== Respiratory coupling states: from bioenergetics to mitochondrial physiology ===
+==== Constructive versus differential terminologies ====
-::::::: ''‘Every professional group develops its own technical jargon for talking about matters of critical concern. .. People who know a word can share that idea with other members of their group, and a shared vocabulary is part of the glue that holds people together and allows them to create a shared culture’'' (Miller 1991).
-==== Constructive versus differential terminologies ====
 :::: Five classical states of mitochondrial respiration and cytochrome redox states have been introduced by Chance and Williams (1955; 1956). These respiratory states are explained by a protocol with isolated mitochondria (imt) in a closed respirometric chamber:
 ::::* '''[[State 1]]''' is obtained after addition of imt to isotonic respiration medium containing inorganic phosphate but no adenylates, specifically no ADP, and no fuel substrates (CHNO).
 ::::* '''[[State 2]]''' is induced by addition of a high level of ADP, which stimulates transiently respiration on the basis of endogenous fuel substrates, followed by a low respiratory activity limited by fuel substrate availability.
 ::::* '''[[State 3]]''': Respiratory fuel substrates are added to initiate State 3. Respiration is stimulated while the ADP level is still high and supports coupled energy transformation in oxidative phosphorylation.
 ::::* '''[[State 4]]''': If the imt preparation is of high quality, devoid of ATPase activity and well coupled, gradual depletion of ADP by phosphorylation to accumulating ATP leads to a sudden decline of oxygen uptake in the transition from State 3 to State 4, when a maximum mt-membrane potential and the ATP/ADP ratio are maintained. State 4 respiration is limited by physiological uncoupling (mainly the intrinsic proton leak). A repeated titration of high ADP can restore active respiration in State 3.
 ::::* '''[[State 5]]''' is the anoxic state after the aerobic-anoxic transition, with zero respiration in a closed respirometric system. Ideal closed respirometers without any backdiffusion of oxygen into the chamber are difficult to obtain in practice, whence this steady state (State 5) is characterized by the oxygen-limited respiration compensating for the oxygen backdiffusion at near-zero oxygen levels.
 :::::::::::: ''‘It is essential to define both the substrate and ADP levels in order to identify the steady-state condition of the mitochondria during the experiment’'' (Chance and Williams, 1956).
@@ Line 41: / Line 45: @@
 [[File:EPL-free and excess.jpg|right|300px|thumb|Capacities of the electron transfer system, oxidative phosphorylation and resting LEAK respiration (ETS, OXPHOS, LEAK) and four-compartmental OXPHOS model. (i) Capacity of the ETS module, ''E'', in the noncoupled state, generating the protonmotive force, Δ''p''<sub>mt</sub>. OXPHOS capacity, ''P'', is partitioned into (ii) the dissipative LEAK component, ''L'' (disspation of Δ''p''<sub>mt</sub>), and (iii) the free OXPHOS capacity, ''≈P=P-L'' (energy conversion driven by Δ''p''<sub>mt</sub>). If ''≈P'' is limited by the capacity of the phosphorylation system, then (iv) the apparent ETS excess capacity, ''ExP=E-P'', is available to drive coupled processes other than phosphorylation without competing with ATP production. Free divided by total ETS capacity, ''≈E/E'', is the ETS coupling efficiency. Free divided by total OXPHOS capacity, ''≈P/P'', is the OXPHOS coupling efficiency. From [[Gnaiger 2014 MitoPathways]].]]
 ==== Three fundamental coupling states of mitochondrial preparations ====
 ::::* '''[[ETS capacity |ETS, state ''E'']]''': Noncoupled state at optimal uncoupler concentration for maximum oxygen flux as a measure of electron transfer system (ETS) capacity, in the presence of fuel substrates and oxygen. The abbreviation State 3u is used frequently in bioenergetics, to indicate the state of maximum respiration, ''E'', without sufficient emphasis on the fundamental difference between state ''P'' (OXPHOS capacity; coupled, with an uncoupled component) and state ''E'' (ETS capacity, noncoupled).
 ::::* '''[[OXPHOS capacity |OXPHOS, state ''P'']]''': In mitochondrial physiology and pathology, maximal mitochondrial respiration in the coupled state is measured for quantitative determination of OXPHOS capacity, ''P'' (Gnaiger 2009). State ''P'' is the ADP-activated state at maximum oxygen flux as a measure of the capacity for oxidative phosphorylation (OXPHOS), supported by saturating ADP and inorganic phosphate concentrations and in the presence of fuel substrates and oxygen. The definition of State 3 lacks a fundamental attribute of OXPHOS capacity. 'High ADP' in State 3 is a concentration of ADP specifically selected to allow measurememt of a State 3 to State 4 transition of isolated mitochondria in a closed respirometric system. Starting at oxygen levels near air saturation, the 'high ADP' concentration added must be low enough to allow phosphorylation to ATP at a coupled oxygen consumption that does not lead to oxygen depletion during the transition to State 4. In contrast, OXPHOS capacity requires evaluation of kinetically saturating ADP concentrations, which are usually an order of magnitude higher than 'high ADP' in State 3.
 ::::* '''[[LEAK respiration |LEAK, state ''L'']]''': Resting state of non-phosphorylating respiration when oxygen flux is maintained mainly to compensate for the proton leak in the absence of ATP synthesis, either due to absence or depletion of ADP at a maximum ATP/ADP ratio, or due to inhibition of phosphorylation by inhibitor such as oligomycin or atractyloside, in the presence of fuel substrates and oxygen. State 4 represents an overestimation of LEAK respiration if ATPase activity prevents final accumulation of ATP and maintains a continuous stimulation of respiration by recycled ADP. This can be tested by inhibition of phosphorylation (oligomycin).
@@ Line 48: / Line 55: @@
 ==== Partitioning of respiration in various coupling states ====
+:::: [[Gnaiger 2014 MitoPathways]]
 ::::* '''[[Residual oxygen consumption |ROX]]''': Residual oxygen consumption (ROX) is not characteristic of a coupling state, but represents a baseline for correction of mitochondrial respiration in the presence of oxygen and the absence of fuel substrates or after addition of inhibitors of the electron transfer system. Total respiration is thus partitioned into a component functionally linked to the electron transfer system and a component not linked to the ETS (ROX). ROX is frequently considered to be equivalent to non-mitochondrial respiration, which ignores the fact that mitochondria contain enzymes which catalyse oxygen-consuming reactions not linked to the ETS and OXPHOS.
 ::::* '''Partitioning of LEAK respiration''': LEAK respiration can be partitioned into patial oxygen consumption compensating for (1) proton leaks, (2) proton slip, (3) cation cycling, and (4) electron leak.
-::::* '''Partitioning of OXPHOS capacity''': Since OXPHOS is partially coupled, intrinsic uncoupling and dyscoupling contribute to the control of flux in the OXPHOS state. Oxygen consumption in the OXPHOS state, therefore, is partitioned into the free OXPHOS capacity, ''≈P'', strictly coupled to phosphorylation, ''~P'', and nonphosphorylating LEAK respiration, ''L<sub>P</sub>''. It is frequently assumed that LEAK respiration, ''L'', as measured in the LEAK state, overestimates the LEAK component of respiration, ''L<sub>P</sub>'', as measured in the OXPHOS state, particularly if the protonmotive force is not adjusted to equivalent levels in ''L'' and ''L<sub>P</sub>''. However, if the LEAK component increases with enzyme turnover during ''P'', the low enzyme turnover during ''L'' may counteract the effect of the higher Δ''p''<sub>mt</sub> (Garlid et al 1993).
+::::* '''[[Free OXPHOS capacity |Free OXPHOS capacity, ''≈P'']]''': ''≈P'' is the partial oxygen consumption which is strictly coupled to phosphorylation of ADP to ATP, ''~P'', when partitioning OXPHOS capacity into a coupled and uncoupled component.
+::::* '''LEAK respiration in the OXPHOS state, ''L<sub>P</sub>''''': Since OXPHOS is partially coupled, intrinsic uncoupling and dyscoupling contribute to the control of flux in the OXPHOS state. Oxygen consumption in the OXPHOS state, therefore, is partitioned into the free OXPHOS capacity, ''≈P'', and nonphosphorylating LEAK respiration, ''L<sub>P</sub>''. It is frequently assumed that LEAK respiration, ''L'', as measured in the LEAK state, overestimates the LEAK component of respiration, ''L<sub>P</sub>'', as measured in the OXPHOS state, particularly if the protonmotive force is not adjusted to equivalent levels in ''L'' and ''L<sub>P</sub>''. However, if the LEAK component increases with enzyme turnover during ''P'', the low enzyme turnover during ''L'' may counteract the effect of the higher Δ''p''<sub>mt</sub> (Garlid et al 1993).
 :::::::::::::::::::: ''P'' = ''≈P'' + ''L<sub>P</sub>''.
+::::* '''[[Excess E-P capacity |Excess ''E-P'' capacity, ''ExP'']]''': ''ExP'' is the difference of the ETS capacity and OXPHOS capacity, ''ExP'' = ''E-P''. At ''ExP'' > 0, the capacity of the phosphorylation system exerts a limiting effect on OXPHOS capacity. In addition, ''ExP'' depends on coupling efficiency, since ''P'' aproaches ''E'' at increasing uncoupling. When partitioning ETS capacity, a OXPHOS-linked component (intrinsig LEAK respiration plus strictly coupled respiration) is distinguished from ''ExP'' as the apparent excess capacity above the OXPHOS-linked component.
+::::* '''[[Free ETS capacity |Free ETS capacity, ''≈E'']]''': ''≈E'' is the ETS capacity corrected for LEAK respiration, ''≈E'' = ''E-L''. ''≈E'' is the respiratory capacity potentially available for phosphorylation of ADP to ATP and ion transport. Oxygen consumption in the ETS state, therefore, is partitioned into the free ETS capacity, ''≈E'', and LEAK respiration, ''L<sub>P</sub>'', compensating for proton leaks, slip and cation cycling:
+:::::::::::::::::::: ''E'' = ''≈E'' + ''L<sub>P</sub>''.
 === Rates and states ===