Weiss, SA, Bushby, RJ, Evans, SD and Jeuken, LJ (2010) A study of cytochrome bo3 in a tethered bilayer lipid membrane. Biochim. Biophys. Acta 1797:1917-23
An assay has been developed in which the activity of an ubiquinol oxidase from Escherichia coli, cytochrome bo(3) (cbo(3)), is determined as a function of the hydrophobic substrate ubiquinol-10 (UQ-10) in tethered bilayer lipid membranes (tBLMs). UQ-10 was added in situ, while the enzyme activity and the UQ-10 concentration in the membrane have been determined by cyclic voltammetry. Cbo(3) is inhibited by UQ-10 at concentrations above 5-10 pmol/cm(2), while product inhibition is absent. Cyclic voltammetry has also been used to characterise the effects of three inhibitors; cyanide, inhibiting oxygen reduction; 2-n-Heptyl-4-hydroxyquinoline N-oxide (HQNO), inhibiting the quinone oxidation and Zn(II), thought to block the proton channels required for oxygen reduction and proton pumping activity. The electrochemical behaviour of cbo(3) inhibited with HQNO and Zn(II) is almost identical, suggesting that Zn(II) ions inhibit the enzyme reduction by quinol, rather than oxygen reduction. This suggests that at Zn(II) concentration below 50µM the proton release of cbo(3) is inhibited, but not the proton uptake required to reduce oxygen to water.
Biocatalysis/drug effects; Cytochromes/antagonists & inhibitors; Cytochromes/metabolism; Dose-Response Relationship, Drug; Electrochemical Techniques/instrumentation; Electrochemical Techniques/methods; Electrodes; Escherichia coli Proteins/antagonists & inhibitors; Escherichia coli Proteins/metabolism; Hydrophobic and Hydrophilic Interactions; Hydroxyquinolines/pharmacology; Kinetics; Lipid Bilayers/chemistry; Lipid Bilayers/metabolism; Oxidation-Reduction/drug effects; Oxidoreductases/metabolism; Sodium Cyanide/pharmacology; Substrate Specificity; Ubiquinone/analogs & derivatives; Ubiquinone/chemistry; Ubiquinone/metabolism; Zinc Sulfate/pharmacology
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