Gnaiger 2018 MiPschool Tromso A1

Mitochondrial states and rates: 1. Electron transfer pathways and respiratory control. 2. Coupling control.

Link: MitoEAGLE

Gnaiger E (2018)

Event: MiPschool Tromso-Bergen 2018

The MitoEAGLE project aims at establishing a quantitative database on mitochondrial (mt) respiratory physiology. In this context the necessity for harmonizing the terminology has become increasingly apparent. Substrate-uncoupler-inhibitor titrations (SUIT) are applied to experimentally control electron transfer pathways in mitochondrial preparations. Complementary to pathway control states (PCS), coupling control states (CCS: ET, OXPHOS, LEAK) are defined in mt-preparations, and the corresponding respiratory rates are of diagnostic significance [1]. Strategically designed SUIT protocols reveal a diversity of mt-respiratory control patterns and pathway additivity depending on species, organs, cell types, and pathophysiological states, as a hallmark of the transition from bioenergetics to mitochondrial physiology [2]. A rationale for categorizing PCS helps in selecting SUIT protocols according to the specific research question or diagnostic aim, and is essential for interpreting experimental results [3].

Figure 1 summarizes selected PCS, categorized according to fuel substrate types and the complexity of mitochondrial pathway types at different electron transfer- (ET-) pathway levels. ET-pathway levels are linked to ET-substrate types. The single enzyme step of Complex IV is at level 1. ET-pathway level 2 is stimulated by duroquinol (DQ) feeding electrons into Complex III (CIII) with further electron transfer to CIV and O₂. ET-pathway level 3 feeds electrons from succinate to CII, and glycerophosphate (Gp) to GpDH directly upstream of the Q-junction. Electron transfer from type 4 substrates (N) feeds electrons into the N-junction from dehydrogenases and enzyme systems directly upstream of NADH and CI. The requirement of a combined operation of the F-junction and N-junction puts type F substrates to level 5 of pathway integration. F-junction substrates are fatty acids involved in β-oxidation, generating (enzyme-bound) FADH₂, the substrate of electron transferring flavoprotein (CETF). In contrast, FADH₂ is the product of CII. A N-linked co-substrate (typically malate is required, and FAO can be inhibited completely by inhibition of Complex I (CI). Under physiological conditions, combinations of the fuel substrate types extend the complexity of PCS, exerting additive or competitive effects on respiratory capacity [2-5]. Analysis of combined NS- versus single N- and S-pathway capacities yields information on pathway interactions and channeling through supercomplex assemblies [4], and leads to a re-evaluation of apparent excess capacities of CIV [5].

Biochemical cell ergometry aims at measurement of J_O2,max (compare V_O2,max in exercise ergometry of humans and animals) of cell respiration linked to phosphorylation of ADP to ATP. The corresponding OXPHOS-capacity is based on saturating concentrations of ADP, [ADP]*, and inorganic phosphate, [P_i]*, available to the mitochondria. This is metabolically opposite to experimental uncoupling of respiration, which yields noncoupled ET-capacity. Contrasting the concept-driven terminology on CCS (LEAK, OXPHOS, ET) from the historical terminology in bioenergetics (States 1 to 5) provides insights into the aims and rigorous quality control of diagnostic mitochondrial physiology [1]. Corresponding to the respiratory coupling states, the respiratory rates are distinguished as L, P, and E. On a statistical basis, the classical respiratory acceptor control ratio (RCR = State 3/State 4 respiration) has to be replaced by the biochemical coupling efficiency, defined as OXPHOS coupling efficiency, j_≈P = (P-L)/P = 1-L/P, or ET-coupling efficiency, j_≈E = (E-L)/E = 1-L/E, which are equivalent at zero excess E-P capacity (ExP = E-P = 0) (Figure 2 [4]).

We invite scientists and students to support our effort to prepare joint publications for implementing a consistent terminology on respiratory states, to ‘facilitate effective transdisciplinary communication, education, and ultimately further discovery’ and advance the quality and impact of mitochondrial physiology [1].

• Bioblast editor: Gnaiger E

Affiliations and support

1. D. Swarovski Research Lab, Dept. Visceral, Transplant Thoracic Surgery, Medical Univ Innsbruck
2. Oroboros Instruments, Innsbruck, Austria

Contribution to COST Action CA15203 MitoEAGLE, supported by COST (European Cooperation in Science and Technology), and K-Regio project MitoFit.

References

1. MitoEAGLE preprint 2018-09-04(41) Mitochondrial respiratory states and rates: Building blocks of mitochondrial physiology Part 1. - www.mitoeagle.org/index.php/MitoEAGLE_preprint_2018-02-08
2. 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«
3. Doerrier C, Garcia-Souza LF, Krumschnabel G, Wohlfarter Y, Mészáros AT, Gnaiger E (2018) High-Resolution FluoRespirometry and OXPHOS protocols for human cells, permeabilized fibers from small biopsies of muscle, and isolated mitochondria. Methods Mol Biol 1782:31-70. - »Bioblast link«
4. 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«
5. 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 7:2840. - »Bioblast link«
6. Gnaiger E (2015) On coupling control of mitochondrial respiration. What is wrong with the RCR? - Gnaiger 2015 Abstract MiPschool Cape Town 2015 II

Figures

Figure 1. ET-pathway control states are defined in mitochondrial preparations complementary to coupling control states. From http://www.bioblast.at/index.php/Electron_transfer-pathway_state

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Figure 2. Respiratory acceptor control ratio as a function of OXPHOS coupling efficiency, j_≈P. RCR is the State 3/State 4 flux ratio [4], equal to P/L if State 3 is at saturating [ADP] and [P_i]. RCR from 1.0 to infinity is highly non-linear in the typical experimental range of RCR 3 to 10: when j_≈P increases from 0.8 to 0.9, RCR doubles from 5 to 10. RCR increases to infinity at the limit of j_≈P=1.0. Statistical analyses of RCR±SD require linearization by transformation to j_≈P (from ref. 4; see ref. 6 for further details).

Labels: MiParea: Respiration, mt-Awareness 

Coupling state: LEAK, OXPHOS, ET  Pathway: F, N, S, Gp, DQ, CIV, NS, Other combinations 

Event: Oral