Description

[[Description::Complex IV or cytochrome c oxidase is the terminal oxidase of the mitochondrial Electron transfer pathway, reducing oxygen to water, with reduced cytochrome c as a substrate. CIV is frequently abbreviated as COX or CcO. It is the 'ferment' (Atmungsferment) of Otto Warburg, shown to be related to the cytochromes discovered by David Keilin.]]

Abbreviation: Has abbr::CIV

Reference: [[Info::MiPNet06.06]]

MitoPedia topics: Enzyme

HRR protocol: Complex IV assay

Electron flow through Complex IV (CIV / cytochrome c oxidase, COX) is measured in intact mitochondria after inhibition of Complex III by antimycin A and addition of tetramethyl-p-phenylenediamine TMPD (Tm), and of ascorbate (As). Correction for autooxidation of TMPD and ascorbate, which is strongly oxygen-dependent, is a routine procedure in high-resolution respirometry. For that we recommend the evaluation of chemical background using inhibitors of CIV (such as cyanide, azide) after ascorbate and TMPD titrations.

Since CIV is a proton pump of the electron transfer-pathway, CIV-linked respiration (L, P, and E) can be measured in different coupling states. Measurement of CIV activity requires uncoupler titrations to eliminate any potential control by the phosphorylation system, and a cytochrome c test to avoid any limitation by cytochrome c release.

Ascorbate and TMPD

Ascorbate (As; 2 mM) and TMPD (Tm; 0.5 mM) are added to mitochondrial preparations. TMPD is a COX specific electron donor, while ascorbate ensures that the TMPD is reduced and continues to donate electrons to build a linear rate of CIV activity. After sequential titration of ascorbate and TMPD (with or without added cytochrome c - autooxidation is higher in the presence of cytochrome c.), the total oxygen flux increases due to (i) COX activity and (ii) autooxidation of ascorbate and TMPD as a function of oxygen pressure. It is important to add ascorbate before TMPD to avoid uncontrollable autoxidation.

Cytochrome c oxidase (CIV) in intact mitochondra has a biphasic kinetics for TMPD (Gnaiger_2002_Biochem_Soc_Trans). Due to the high K'_m of the low-affinity phase, TMPD cannot be added at saturating concentrations, since then autoxidation of TMPD would be strikingly high. The actually chosen TMPD concentration, therefore, is a compromise. Inhibitor titrations with cyanide or azide of Electron transfer pathway pathway flux versus the single step of CIV reveal an apparent excess capacity of CIV. The optimum TMPD concentration, therefore, is adjusted to obtain a reaction velocity corresponding to the apparent excess capacity (Gnaiger_1998_J Exp Biol).

Cyanide cannot be used when pyruvate is titrated because it inhibits cyanide at high oxygen levels (see Cyanide).

Correction for chemical O2 background

After full inhibition of CIV the large effect of autooxidation is responsible for the remaining oxygen flux. For CIV activity determination, the signal derived from this autooxidation is subtracted from the oxygen flux measured before CIV inhibition. This provides a complete internal calibration of the chemical background, as a function of oxygen concentration.

The oxygen dependence is linear above c. 50 µM O₂, but biphasic (linear + hyperbolic) below 50 µM O₂. If you maintain oxygen in the linear dependence above 50 µM, you can use the automatic instrumental background correction in DatLab to also take care of the chemical background correction (in the ‘Edit Experiment’ window the instrumental background parameters are replaced by the combined background parameters).

References

O2k-Procedure: MiPNet06.06 Chemical O2 background

Publications:

Living cells are inadequate for CIV measurement with As+Tm - use permeabilized cells: Renner_2002_Mol_Biol_Rep, Renner_2003_Biochim_Biophys_Acta
Isolated mitochondria: Gnaiger_2002_Biochem_Soc_Trans
Permabilized fibers: Kuznetsov_2004_Am_J_Physiol_Heart_Circ_Physiol, Lemieux_2011_Int_J_Biochem_Cell_Biol

SUITbrowser question: CIV

The SUITbrowser can be used to find SUIT protocols to assess CIV activity, alongside answering other research questions.

List of publications: Complex IV;cytochrome c oxidase

»Publications: Complex IV single step

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{{#ask: Pathways::CIV |?Was published in year=Year |?Has title=Reference |?Coupling states=Coupling |?Pathways=Pathway |?Preparation=Preparation |?Mammal and model=Organism |?Tissue and cell=Tissue;cell |format=broadtable |limit=5000 |offset=0 |sort=Was published in year |order=descending }}

»Abstracts: Complex IV single step

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{{#ask: Pathways::CIV |?Was submitted in year=Year |?Has title=Reference |?Coupling states=Coupling |?Pathways=Pathway |?Preparation=Preparation |?Mammal and model=Organism |?Tissue and cell=Tissue;cell |format=broadtable |limit=5000 |offset=0 |sort=Was submitted in year |order=descending }}