Complex IV

high-resolution terminology - matching measurements at high-resolution

Complex IV

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. C_IV 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: C_IV

Reference: MiPNet06.06

MitoPedia methods: Respirometry 

MitoPedia topics: Enzyme 

HRR protocol: Complex IV assay

Ascorbate and TMPD

Ascorbate (As; 2 mM) and TMPD (Tm; 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 C_IV reveal an apparent excess capacity of C_IV. 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 and O2k-Manual:

• MiPNet06.06 ChemicalBackground
• http://www.oroboros.at/?O2k-DatLab-FluxAnalysis
• http://www.oroboros.at/?Protocols_Cell_HRR (Section 11)
• Chemicals: http://www.oroboros.at/?Protocols_titrations

Publications:

• Intact 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 fibres: Kuznetsov_2004_Am_J_Physiol_Heart_Circ_Physiol, Lemieux_2011_Int_J_Biochem_Cell_Biol