Description
Complex IV or cytochrome c oxidase is the terminal oxidase of the mitochondrial ETS, 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 by David Keilin.
Abbreviation: CIV
Reference: MiPNet06.06
MitoPedia methods:
Respirometry
MitoPedia topics:
Enzyme
HRR protocol: Complex IV assay
Ascorbate and TMPD
Ascorbate (2 mM) and TMPD (0.5 mM) are added to mitochondrial preparations. It is important to add ascorbate before TMPD to avoid uncontrollable autoxidation. Ascorbate maintains TMPD in a reduced state. Electrons flow from TMPD to cytochrome c.
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 ETS 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).
Correction for chemical background
Due to autooxidation of ascorbate and TMPD, a chemical correction has to be applied on flux. Autooxidation is higher in the presence than in the absence of cytochrome c, and is dependent on 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). Cyanide is OK except if you use pyruvate as a substrate. Pyruvate particularly at high oxygen levels reverses the inhibition by cyanide (see Cyanide).
References
Protocols:
- http://www.oroboros.at/?Chemical_background
- http://www.oroboros.at/?Protocols_Cell_HRR (Section 11)
- Chemicals: http://www.oroboros.at/?Protocols_titrations
Publications:
- Intact/permeabilized cells: Renner_2002_Mol_Biol_Rep, Renner_2003_Biochim_Biophys_Acta
- Isolated mitochondria: Gnaiger_2002_Biochem_Soc_Trans
- Permabilized fibres: Kuznetsov_2004_Am_J_Physiol_Heart_Circ_Physiol, Lemieux_2011_Int_J_Biochem_Cell_Biol