Gnaiger 2018 MiPschool Tromso A1

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Erich Gnaiger
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 JO2,max (compare VO2,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, [Pi]*, available to the mitochondria. This is metabolically opposite to experimental uncoupling of respiration, which yields noncoupled ET-capacity. The concept-driven terminology on CCS (LEAK, OXPHOS, ET) provides insights into the aims and rigorous quality control of diagnostic mitochondrial physiology [1]. With emphasis on conceptual differences, harmonization is achieved with the historical terminology in bioenergetics (States 1 to 5). 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 only at an excess E-P capacity equal to zero (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 and rates, 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.


  1. Gnaiger E, Aasander Frostner E, Abdul Karim N, Abumrad NA, Acuna-Castroviejo D, Adiele RC et al (2019) Mitochondrial respiratory states and rates. MitoFit Preprint Arch doi:10.26124/mitofit:190001. - »Bioblast link«
  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) The functional design of the electron transfer system (ETS) and mitochondrial respiratory control. - Gnaiger 2015 Abstract MiPschool Cape Town 2015
  7. Gnaiger E (2015) On coupling control of mitochondrial respiration. What is wrong with the RCR? - Gnaiger 2015 Abstract MiPschool Cape Town 2015 II


SUIT-catg FNSGpCIV.jpg
Figure 1. ET-pathway control states are defined in mitochondrial preparations complementary to coupling control states (from ; see ref. 6 for further details).

MiP2014 Gnaiger Figure2.jpg
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 [Pi]. 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. 7 for further details).


A: Why are various substrate combinations applied in protocols for mitochondrial respiration?
  1. Can glutamate (G) be used without malate (M), and how do you interprete G-OXPHOS capacity?
  2. Can pyruvate (P) be used without M, and how do you interprete P-OXPHOS capacity?
  3. Can M be used alone, and how do you interprete M-OXPHOS capacity?
  4. Can a fatty acid (F) be used alone, and why is a low concentration of M suggested for F-OXPHOS capacity?
  5. Which role does mt-malic enzyme (mtME) play in mitochondrial pathway control using the above substrates?
  6. How do F-OXPHOS and F-ET capacities change upon titration of Rot?
B: Compare the significance of using succinate (S) alone or in combination with rotenone (Rot).
  1. Does S supplied alone to mitochondrial preparations establish a physiologically relevant state?
  2. Which metabolic problems occur when S is supplied alone to mitochondrial preparations, and do these problems occur generally in mt-preparations?
  3. Should S or Rot be titrated first when using intact cells or mt-preparations?
C: Select a SUIT protocol and interprete patterns of mitochondrial respiratory control:
  1. Which SUIT protocol(s) do you use for evaluation of a defect by dyscoupling?
  2. You suspect a specific defect of fatty acid oxidation (F-OXPHOS capacity). - Which SUIT protocol do you select?
  3. PM-ET capacity is down. How do you proof that a Complex I (CI)defect is responsible?
  4. OXPHOS capacity is down with PM as substrates (N-pathway), but PM-ET capacity is unchanged. - Where is the injury?
  5. PM-ET and S-ET capacities are similar. Adding these mathematically yields a higher value compared to the measurement of ET-capacity when these substrates are provided in combination (PMS-ET capacity). - How do you interpret such a result?
  6. PM-ET capacity is high, F-ET capacity is moderate, but addition of F after PM in the noncoupled state does not stimulate respiration. - What is the significance?
D: What is the difference between State 3 and OXPHOS capacity?
  1. How do you measure OXPHOS capacity?
  2. Give a definition of State 3 and OXPHOS capacity.
  3. What is the difference between a respiratory state and a respiratory rate?
  4. Why is the distinction between State 3 and OXPHOS capacity important?
E: Which definitions can you provide on metabolic efficiency?
  1. How are biochemical flux efficiency and thermodynamic efficiency of OXPHOS related?
  2. Is efficiency important in biological evolution?
  3. Is efficiency important in our society?

Labels: MiParea: Respiration, mt-Awareness 

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

Event: A1, Oral  MitoEAGLE