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LEAK respiration

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LEAK respiration

Description

L.jpg LEAK respiration or LEAK oxygen flux, L, compensating for proton leak, proton slip, cation cycling and electron leak, is measured as mitochondrial respiration in the LEAK state, in the presence of reducing substrate(s), but absence of ADP (theoretically, absence of inorganic phosphate presents an alternative), or after enzymatic inhibition of the phosphorylation system. The LEAK state is the non-phosphorylating resting state of intrinsic uncoupled or dyscoupled respiration when oxygen flux is maintained mainly to compensate for the proton leak at a high chemiosmotic potential, when ATP synthase is not active. In this non-phosphorylating resting state, the electrochemical proton gradient is increased to a maximum, exerting feedback control by depressing oxygen flux to a level determined mainly by the proton leak and the H+/O ratio. In this state of maximum protonmotive force, LEAK respiration is higher than the LEAK component in state P (OXPHOS capacity). > MiPNet article

Abbreviation: L

Reference: Gnaiger 2012 MitoPathways, Gnaiger 2009 Int J Biochem Cell Biol


MitoPedia methods: Respirometry 


MitoPedia topics: "Respiratory state" is not in the list (Enzyme, Medium, Inhibitor, Substrate and metabolite, Uncoupler, Sample preparation, Permeabilization agent, EAGLE, MitoGlobal Organizations, MitoGlobal Centres, ...) of allowed values for the "MitoPedia topic" property. Respiratory state"Respiratory state" is not in the list (Enzyme, Medium, Inhibitor, Substrate and metabolite, Uncoupler, Sample preparation, Permeabilization agent, EAGLE, MitoGlobal Organizations, MitoGlobal Centres, ...) of allowed values for the "MitoPedia topic" property. 

File:LEAK CI+II.jpg
LEAK respiration corresponds to resting, non-phosphorylating electron transfer with a shortcircuit of the proton cycle across the inner mt-membrane due to intrinsic uncoupling or dyscoupling. 2[H] indicates the reduced hydrogen equivalents of CHO substrates and electron transfer to oxygen. H+out are protons pumped out of the matrix phase. Proton leaks (a property of the inner mt-membrane) dissipate energy of translocated protons, and proton slip prevents full translocation of protons across the inner mt-membrane (a property of the proton pumps). Measurement of LEAK respiration is possible in intact cells by inhibition of the phosphorylation system and in mt-preparations supported by an ETS-competent substrate state, exemplifed as CI+II-linked substrate supply. Modified after Gnaiger 2012 MitoPathways.

LEAK respiration: a concept-linked terminology of respiratory states

Publications in the MiPMap
Gnaiger E (2014) LEAK respiration: a concept-linked terminology of respiratory states. Mitochondr Physiol Network 2014-07-04.

» Gnaiger 2012 MitoPathways

OROBOROS (2014) MiPNet

Abstract: L.jpg Mitochondrial respiratory states have been defined originally by Chance and Williams (1955) as a sequence (from 1 to 5) of titrations and transitions in a respiratory protocol, including State 4 as a LEAK state of respiration obtained after exhaustion of the added ADP. The second state (State 2) is induced by addition of 'high ADP'. Confusion persists in the current literature as to the meaning of State 2, which can be resolved by a transition from a specific protocol-linked to a generalized concept-linked terminology.


O2k-Network Lab: AT Innsbruck Gnaiger E


Labels:




Regulation: Coupling efficiency;uncoupling  Coupling state: LEAK, ETS"ETS" is not in the list (LEAK, ROUTINE, OXPHOS, ET) of allowed values for the "Coupling states" property. 

HRR: Theory 



The LEAK state

LEAK states of respiration are frequently called State 4, State 4o, basal state, or inactive state. Importantly, State 2 is not a LEAK state according to the classical definition.


Respiration compensating for the proton leak is the main component of LEAK respiration. If proton leak were the only component involved, it would make sense to simply write leak respiration for the compensatory oxygen flux. Proton slip and cation cycling, however, are also involved to a larger or smaller extent to stimulate LEAK respiration. The upper case 'LEAK', therefore, should make us aware that this is more accurately considered as an acronym, and cannot be taken as a definitive delineation of the stimulatory mechanism in the LEAK state of respiration.

  • Note: The distinction between LEAK and ETS capacity helps to clarify the important difference between uncoupled or dyscoupled respiration (LEAK) and noncoupled respiration (ETS capacity).


Protocols for measurement of LEAK respiration

The LEAK state can be induced experimentally in various ways, which may yield idential estimates of LEAK respiration, or may show deviations that help to critically assess the proper protocol to be applied in specific cases:

a) LEAK state with ATP

LEAK respiration in the presence of ATP, LT, and absence of ATPase activity.

LEAK state with ATP: LT (classical State 4 in isolated mitochondria) after phosphorylation of ADP to ATP is completed, or when a high concentration of ATP is added in the absence of ATP (Gnaiger 2000 Proc Natl Acad Sci U S A).

In contradiction to the original definition of State 2 (ROX), yet with reference to Chance and Williams (1956), 'State 2' has later been used for describing this functionally different state of LEAK respiration:

State 2: substrate added, respiration low due to lack of ADP. .. the controlled respiration prior to addition of ADP, which is strictly termed “state 2”, is functionally the same as state 4, and the latter term is usually used for both states’ (Nicholls & Ferguson 1992).

Thus State 2 was re-defined as functionally the same as State 4. State 2 (Chance and Williams 1955, 1956), however, is substrate-limited residual oxygen consumption at high ADP (ROXD), whereas LN and LT (State 4) are LEAK states in the absence of adenylates (LN: no ADP, no ATP) or presence of ATP (LT).

To overcome the termonological confusion persisting in the scientific literature, the respiratory coupling states of LEAK respiration, OXPHOS capacity and ETS capacity are distinguished from residual oxygen consumption (ROX; Gnaiger 2009).


b) LEAK state with oligomycin

LEAK respiration induced by inhibition of ATP synthase by oligomycin, LOmy.

LEAK state with oligomycin: LOmy (in isolated mitochondria or other mitochondrial preparations, and intact cells).


c) LEAK state without adenylates

LEAK respiration without adenylates, LN.

LEAK state without adenylates: LN (in isolated mitochondria or other mitochondrial preparations, using a protocol different from the classical State 2-3-4 sequence).

Sequential addition of (1) mitochondria, (2) ADP, and (3) reduced substrates is the basis of the original State 1-2-3 definitions of respiratory states (Chance and Williams 1955 part III, 1956), where State 2 is zero respiration or residual oxygen consumption in the absence of substrate. An alternative protocol is well established, as shown e.g. by the classical Fig. 5A (Chance and Williams 1955 part I): 600 µM ADP is added after a state described as ‘Aerobic mitochondria plus succinate’. That state was never defined as ‘State 2’ by Brit Chance. Later Estabrook (1967) made this protocol more popular, with addition of substrate before any ADP or ATP was added.

In this alternative protocol, a respiratory LEAK state is induced in isolated mitochondria, permeabilized tissues, or permeabilized cells, adding the mitochondrial preparation to respiration medium containing inorganic phosphate (State 1), then adding reduced substrate (no external adenylates). This second state (Estabrook 1967) is a non-phosphorylating LEAK state, LN (N for no adenylates; Gnaiger 2009), when substrate-saturated respiration compensates for the proton leak (mainly) in the absence of ADP.


Related terms in MitoPedia

State 4 versus State 2

Static head

State 4 is frequently referred to as 'static head' of isolated mitochondria. Equivalence requires testing, if at State 4 (in a protocol defined by Chance and Williams 1955) ATPase activity is actually zero, such that respiration at State 4 is not partially stimulated by partial recycling of ATP to ADP. In the latter case, State 4 respiration would be higher than LEAK respiration and thus higher than respiration at static head.

OXPHOS-coupled energy cycles. Source: The Blue Book

Respiratory coupling states

P.jpg OXPHOS, P

E.jpg ETS, E

R.jpg ROUTINE, R

L.jpg LEAK, L


References

  1. Caplan SR, Essig A (1983) Bioenergetics and linear nonequilibrium thermodynamics. The steady state. Harvard Univ. Press, Cambridge. 435 pp.
  2. Chance B, Williams GR (1955) Respiratory enzymes in oxidative phosphorylation. I. Kinetics of oxygen utilization. J Biol Chem 217: 383-93.
  3. Chance B, Williams GR (1955) Respiratory enzymes in oxidative phosphorylation. III. The steady state. J Biol Chem 217: 409-27.
  4. Chance B, Williams GR (1956) The respiratory chain and oxidative phosphorylation. Adv Enzymol 17: 65-134.
  5. Estabrook R (1967) Mitochondrial respiratory control and the polarographic measurement of ADP:O ratios. Methods Enzymol 10: 41-7.
  6. Gnaiger E (1993a) 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.
  7. Gnaiger E (1993b) Nonequilibrium thermodynamics of energy transformations. Pure & Appl. Chem. 65: 1983-2002.
  8. Gnaiger E (2009) Capacity of oxidative phosphorylation in human skeletal muscle. New perspectives of mitochondrial physiology. Int J Biochem Cell Biol 41: 1837–45. PMID: 19467914
  9. Gnaiger E (2012) Mitochondrial pathways and respiratory control. An introduction to OXPHOS analysis. 3rd ed. Mitochondr Physiol Network 17.18. OROBOROS MiPNet Publications, Innsbruck: 64 pp. >> Open Access
  10. Nicholls DG, Ferguson SJ (2002) Bioenergetics 3. Academic Press, London. 287 pp.