The protonmotive force and respiratory control

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COST Action CA15203 (2016-2021): MitoEAGLE
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 Terminology Group: respiratory control

• Target a broad audience – introduce the new generation of investigators
• 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 Group

• First draft and corresponding author: E. Gnaiger
• Contributing co-authors (alphabetical, to be extended): M.G. Alves, R. Brown, A.J. Chicco, J. Collins, L. Crisostomo, T. Dias, G. Distefano, C. Doerrier, H. Dubouchaud, P. Garcia-Roves, L.F. Garcia-Souza, E. Gnaiger, K.T. Hellgren, C.L. Hoppel, J. Iglesias-Gonzalez, B.A. Irving, S. Iyer, G. Keppner, T. Komlodi, A. Krajcova, G. Krumschnabel, M. Markova, A.T. Meszaros, A. Molina, A.L. Moore, C.M. Palmeira, K. Nozickova, K. Siewiera, O. Sobotka, P. Stankova, Z. Sumbalova, B. Velika

• Contributing co-authors: Confirming to have read the final manuscript, possibly to have made suggestions for improvement, and to agree to implement the recommendations into future manuscripts, presentations and teaching materials.

• Supporting co-authors: in preparation
• Journal: Int J Biochem Cell Biol (as discussed at MITOEAGLE Barcelona 2017); Open Access is a requirement.

• Version 1: 2017-04-18; last update (Version 10): 2017-08-10

Work in progress: - Updated: 2017-08-10 - » Versions

‘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).

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 conceptual ‘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 reference 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.

States and rates

• Force, F_tr: A generalized force (intensive quantity) in thermodynamics or ergodynamics is the partial Gibbs (Helmholtz) energy change per advancement of a transformation (tr).
• Intensive quantity
• Internal flow, I_int
• Open system
• Oxygen flow, I_O2
• Oxygen flux, J_O2
• Respiratory state

Normalization: fluxes and flows

• Units (important for a database); analogous to electic terms: Flow [C.s-1]; Flux [C.s-1.m-2]; Rate (?)
• Extensive quantity
• External flow, I_ext
• Mitochondrial marker
• Specific quantity

List of selected terms and symbols

• Coupling control factor, CCF
• Coupling control ratio, CCR
• Coupled respiration
• Dyscoupled respiration
• Isolated mitochondria, imt
• Mitochondria, mt (Greek mitos: thread; chondros: granule) are small structures within cells, which function in cell respiration as powerhouses or batteries. Mitochondria belong to the bioblasts of Richard Altmann (1894). Abbreviation: mt, as generally used in mtDNA. Singular: mitochondrion (bioblast); plural: mitochondria (bioblasts).
• Mitochondrial inner membrane, mt-im
• Mitochondrial outer membrane, mt-om
• Mitochondrial matrix, mt-matrix
• Mitochondrial membrane potential, mtMP, Δψ_mt
• Mitochondrial preparations, mtprep are isolated mitochondria (imt), tissue homogenate (thom), mechanically or chemically permeabilized tissue (permeabilized fibres, pfi) or permeabilized cells (pce). In these preparations the cell membranes are either removed (imt and smtp) or mechanically (thom) and chemically permeabilized (pfi), while the mitochondrial functional integrity and to a large extent the mt-structure is maintained.
• Mitochondrial respiration
• Noncoupled respiration
• Oligomycin, Omy
• Permeabilized tissue or cells, pti, pce
• Phosphorylation system
• Proton leak
• Proton pump
• Proton slip
• Protonmotive force, Δp_mt
• Tissue homogenate, thom
• Uncoupler, U

Mitochondrial respiratory control: pathway states in mt-preparations

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)

Mitochondrial respiratory control: cell respiration

MITOEAGLE recommendations Part 3.

Intact cells versus mitochondrial preparations

• Intact cells, ce
• Basal respiration
• Cell respiration
• Resting metabolic rate
• ROUTINE state, state R: ROUTINE respiration of intact, viable cells is regulated according to physiological activity, at intracellular non-saturating ADP levels. R increases under various conditions of activation. When incubated in culture medium, cells maintain a ROUTINE level of activity, R (ROUTINE mitochondrial respiration; corrected for residual oxygen consumption due to oxidative side reactions). ROUTINE activity may include aerobic energy requirements for cell growth and is thus fundamentally different from the definition of basal metabolic rate (BMR). When incubated for short experimental periods in a medium devoid of fuel substrates, the cells respire solely on endogenous substrates at the corresponding state of ROUTINE activity, e_R (e, endogenous substrate supply).

It is difficult to stimulate living cells to maximum OXPHOS activity, since ADP and inorganic phosphate do not equilibrate across intact plasma membranes, and thus saturating concentrations of these metabolites can hardly be achieved in living cells. LEAK and ETS states, however, can be induced in viable cells with application of inhibitors of the phosphorylation system and uncouplers, respectively, due to the fact that cell membranes are highy permeable for these substances. External fuel substrates are taken up by living cells to various extents, and intracellular metabolism of exogenous and endogenous substrates supports mitochondrial respiration with a physiological substrate supply. In contrast, mt-preparations depend on the external supply of fuel substrates which support the electron transfer system with reducing equivalents. ETS competence of external substrates is required for all coupling states of mt-preparations (L, P, E) and depends on (i) transport of substrates across the inner mt-membrane or oxidation by dehydrogenases located on the outer face of the inner mt-membrane (e.g. glycerophosphate dehydrogenase complex, CGpDH), (ii) oxidation in the mt-matrix (TCA cycle dehydrogenases and other matrix dehydrogenases, e.g. mtGDH) or on the inner face of the inner mt-membrane (succinate dehydrogenase, CII), (iii) oxidation of substrates without accumulation of inhibitory endproducts (e.g. oxaloacetate inhibiting succinate dehydrogenase; NADH and oxaloacetate inhibiting malate dehydrogenase), and (iv) electron transfer through the membrane-bound ETS (mETS). Endproducts must be either easily exported from the matrix across the inner mt-membrane (e.g. malate formed from succinate via fumarate), or metabolized in the TCA cycle (e.g. malate-derived oxaloacetate forming citrate in the presence of external pyruvate&malate).

Mitochondrial respiratory control: coupling control ratios and control factors

MITOEAGLE recommendations Part 4.

• ETS coupling efficiency, j_≈E: j_≈E = ≈E/E = (E-L)/E = 1-L/E
• LEAK control ratio, L/E

• Excess E-P capacity factor, j_ExP: j_Exp = (E-P)/E = 1-P/E
• OXPHOS control ratio, P/E

• OXPHOS coupling efficiency, j_≈P: j_≈P = ≈P/P = (P-L)/P = 1-L/P
• L/P coupling control ratio, L/P
• Respiratory acceptor control ratio, RCR: RCR = State 4/State 3

• netOXPHOS control ratio, ≈P/E: ≈P/E = (P-L)/E

• Uncoupling control ratio, UCR: UCR = E/R

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