Cytochrome c control efficiency

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Cytochrome c control efficiency

Description

The cytochrome c control factor expresses the control of respiration by externally added cytochrome c, c, as a fractional change of flux from substrate state CHO to CHOc. In this flux control factor (FCF_c), CHOc is the reference state with stimulated flux; CHO is the background state with CHO substrates, upon which c is added, FCF_c = (J_CHOc-J_CHO)/J_CHOc. » MiPNet article

Abbreviation: FCF_c

Reference: Gnaiger 2013 Abstract MiP2013

MitoPedia methods: Respirometry 

MitoPedia topics: "Respiratory control ratio" 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 control ratio"Respiratory control ratio" 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., Substrate and metabolite 

Cytochrome c control: a test for outer mt-membrane integrity

Gnaiger E (2014) Cytochrome c control: a test for outer mt-membrane integrity. Mitochondr Physiol Network 2014-04-21.

OROBOROS (2014) MiPNet

Abstract: The cytochrome c control factor, FCF_c, provides an index for the outer mt-membrane integrity.

• O2k-Network Lab: AT Innsbruck Gnaiger E

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HRR: Theory, Protocol"Protocol" is not in the list (Oxygraph-2k, TIP2k, O2k-Fluorometer, pH, NO, TPP, Ca, O2k-Spectrophotometer, O2k-Manual, O2k-Protocol, ...) of allowed values for the "Instrument and method" property. 

O2k-SOP 

The outer mitochondrial membrane integrity can be evaluated by stimulation of respiration by exogenously added cytochrome c and expressing the normalized c-effect as the flux control factor, FCF_c.

Determination of cytochrome c loss

Addition of cytochrome c (10 µM final concentration) may stimulate OXPHOS capacity or ETS capacity and thus provides evidence for the occurrence of cytochrome c loss.

Cytochrome c test

When using cytochrome c as a quality control for permeabilised muscles from various species, which cytochrome c should we use (a wide range of types of cytochrome c is available from Sigma-Aldrich) and at which concentration?

We apply routinely cytochrome c from Sigma C 7752. It would be interesting to compare the consequence of application of different sources of cytochrome c.

In a detailed discussion on the dependence of respiration in isolated mitochondria and permeabilized cardiac fibers, we showed for the first time showing that cytochrome c kinetics is different when studying a segment of the ETS (CII-linked: succinate+rotenone) versus the isolated step of cytochrome c oxidase (CIV; ascorbate+TMPD+antimycin A). For CII-linked respiration, an exernal cytochrome c concentration of 10 µM yields kinetic saturation (monophasic hyperbolic), but kinetics is biphasic for CIV and 10 µM is not saturating. ,

Importantly, cytochrome c increases the chemical background oxygen flux in the presence of ascorbate and TMPD, dependent on oxygen concentration and cytochrome c concentration, and appropriate chemical background corrections are required. , Without ascorbate and TMPD, added cytochrome c is stable.

Evaluation of the cytochrome c effect, when respiration is slightly unstable: Mark respiration just before cytochrome c addition and after. Take these to values to calculate the increase of respiration due to cytochrome c addition.

At which step of the protocol should cytochrome c be added?

If a stimulatory effect of cytochrome c is observed, respiratory capacities measured before cytochrome c addition might be cytochrome c limited and therefore underestimated. This provides an argument to add cytochrome c at an early state of the protocol.

It is important not to add cytochrome c in a LEAK state: There is always an unexplained activation of respiration, unrelated to the injury of the outer mt-membrane. Add cytochrome c only after activation by ADP.

Cytochrome c release

Cytochrome c release induced by sample preparation

A preparation induced damage of the outer mitochondrial membrane and as a result subsequent loss of cytochrome c can be detected by a stimulation of respiration after the addition of cytochrome c. The preparation induced damage can also affect the respiratory complexes. Therefore, experimental runs showing a preparation induced cytochrome c release should be excluded from the final data set. In perfectly prepared muscle fibers cytochrome c should have no stimulatory effect on maximum respiratory activity, in liver biopsies a small effect is observed, even in carefully prepared samples.

Cytochrome c release induced by treatment

Treatment-triggered cytochrome c release, e.g. cell death induction, has to be distinguished from preparation induced damage. If cytochrome c is released as a result of apoptosis induction, this is a biological phenomenon and a relevant result.

Interpretation of the cytochrome c control factor

Cytochrome c release throughout the mitochondrial population in a tissue in vivo may induce a pathophysiological limitation of respiratory capacity, since the released cytochrome c accumulates in the cytosol at a low (non-saturating) concentration. If all mitochondria in a particular cell release their cytochrome c, then the cytosolic concentration increases to a level nearly saturating respiration (Brown and Buritaite #Ref). If the pathology involves a change of mt-membrane properties which render the mitochondria more vulnerable to experimental damage during tissue preparation, then the cytochrome c effect is to be interpreted differently. Such a pathophysiological modification is specifically revealed by the respirometric approach to OXPHOS analysis with SUIT protocols.