Mitochondrial Pathways and Respiratory Control 2012 - the Blue Book

 

Gnaiger E (2012) Mitochondrial Pathways and Respiratory Control. An Introduction to OXPHOS Analysis. Mitochondr Physiol Network 17.18. OROBOROS MiPNet Publications, Innsbruck: 64 pp.

 

3rd edition: 2400 prints, ISBN: 978-3-9502399-6-6 

Colour print, soft cover.  -  Open Access >> download pdf  >> References and notes





Contents

1. OXPHOS Analysis ........................................................................................................

7

2. MitoPathways to Complex I


    Respiratory Substrate Control with Pyruvate, Malate and Glutamate ............................... 

19

3. MitoPathways to Complex II

    Glycerophosphate Dehydrogenase and ETF .....................................................................

26

4. MitoPathways to Complexes I+II


    Convergent Electron Transfer at the Q-junction................................................................

29

5. Respiratory States


    Coupling Control and Coupling Control Ratios ..................................................................

44

6. Conversions of Metabolic Fluxes.................................................................................

51

A1. Respiratory Coupling States and Coupling Control Ratios ...................................... 

54

A2. Substrates, Uncouplers and Inhibitors .................................................................... 

56

References ......................................................................................................................

59

www.bioblast.at/index.php/Gnaiger_2012_MitoPathways


Bioblast Wiki ...................................................................................................................

61

The OROBOROS   -   Feeding on Negative Entropy ......................................................... 

63



Summary

Mitochondrial Pathways and Respiratory Control

 

The present introduction to the analysis of oxidative phosphorylation (OXPHOS analysis) combines concepts of bioenergetics and biochemical pathways related to mitochondrial core energy metabolism. This provides the basis for the design of substrate-uncoupler-inhibitor titration (SUIT) protocols in novel O2k-assays established since publication in 2007 of ‘Mitochondrial Pathways’ (The Blue Book, 1st ed; Fig. 1).

 

Application of SUIT protocols for OXPHOS analysis is a component of metabolic phenotyping (Fig. 2). OXPHOS analysis extends conventional bioenergetics to the level of mitochondrial physiology for functional diagnosis in health and disease. SUIT protocols with substrate combinations are now established in studies with isolated mitochondria, permeabilized cells and permeabilized muscle fibres and tissue homogenates using high-resolution respirometry (HRR). The OROBOROS Oxygrap-2k (O2k) represents a unique instrument for HRR. No other platform is suited for application of SUIT protocols due to a restricted number of serial titrations available in multiwell systems or insufficient signal stability and restricted oxygen capacity in small chambers. 

 

A Mitochondrial Physiology Network has evolved as (i) a network of scientists linked by applications of the O2k (WorldWide MiPNet: www.oroboros.at/?MiPNet), and (ii) a format of publications available on the OROBOROS website, which summarizes protocols, introductory guidelines and discussions of concepts (MiPNet Protocols: www.oroboros.at/?O2k-Protocols). (iii) Beyond the restricted space for references selected in the printed edition, O2k-Publications are listed as supplementary information that is updated continuously on the Bioblast wiki (www.bioblast.at/index.php/O2k-Publications). The number of O2k-Publications increases rapidly (presently >800) with extending areas of biomedical and clinical applications of the OROBOROS Oxygraph-2k. (iv) A glossary is presented on the Bioblast ‘MitoPedia’ website with emphasis on developing a consistent terminology in mitochondrial physiology (www.bioblast.at/index.php/MitoPedia_Glossary:_Terms_and_abbreviations).

 

‘MitoPathways’ has become an element of high-resolution respirometry and mitochondrial physiology. A mosaic evolves by combining the elements into a picture of modern mitochondrial respiratory physiology.

 

I thank all contributors to the Mitochondrial Physiology Network for their cooperation and feedback. In particular, I want to acknowledge the experimental contributions by the authors and co-authors of various publications emerging from international cooperations. Without the team of OROBOROS INSTRUMENTS, including the partners in electromechanical engineering (Oxygraph-2k; Philipp Gradl, WGT Elektronik, Kolsass, Austria) and software development (DatLab; Lukas Gradl, software security networks, Innsbruck, Austria) the experimental advances on ‘MitoPathways’ would not have been possible.

 

For references and notes see Bioblast online information:

www.bioblast.at/index.php/Gnaiger_2012_MitoPathways

 

 

Innsbruck, July 2007 - Dec 02 2012                                          Erich Gnaiger



Chapter 1. OXPHOS Analysis

  

The protoplasm is a colony of bioblasts. Microorganisms and granula are at an equivalent level and represent elementary organisms, which are found wherever living forces are acting, thus we want to describe them by the common term bioblasts.

Richard Altmann (1894)

 

Oxidative phosphorylation (OXPHOS) is a key element of bioenergetics, extensively studied to resolve the mechanisms of energy transduction in the mitochondrial electron transfer system and analyze various modes of mitochondrial (mt) respiratory control in healt and disease. OXPHOS flux control is exerted by (i) coupling of electron transfer to proton translocation and ATP synthesis mediated by the chemiosmotic, proton motive force, and uncoupling by proton leaks; (ii) substrates and catalytic capacities of respiratory complexes, carriers, transporters, and mt-matrix enzymes of core energy metabolism; (iii) kinetic regulation by concentrations of ADP, inorganic phosphate, oxygen and reduced substrates feeding electrons into the electron transfer system; (iv) specific inhibitors such as NO and H2S.

 

1. Chemiosmotic Coupling

 

Peter Mitchell’s chemiosmotic coupling theory explains the fundamental mechanism of mitochondrial and microbial energy transformation, marking Richard Altmann’s ‘bioblasts’ as the systematic unit of bioenergetics and of the human symbiotic ‘supraorganism’ with microbial–mammalian co-metabolic pathways. The transmembrane chemiosmotic force or electrochemical proton potential, Δp, has a chemical component (proton gradient) and electrical component (membrane potential). Δp provides the link between electron transfer and phosphorylation of ADP to ATP (Fig. 1.1).

 

Fig. 1.1. Energy transformation in coupled fluxes, J, and forces, F and Δp, of oxidative phosphorylation. 2[H] indicates the reduced hydrogen equivalents of CHO substrates and electron transfer to oxygen. JH+out is coupled output flux. Proton leaks dissipate energy of trans-located protons from low pH in the positive P-phase to the negative N-phase.


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