We have developed a simple native-like surface-tethered membrane system to investigate the activity of cbo(3) (cytochrome bo(3)), a terminal oxidase in Escherichia coli. The tethered membranes consist of E. coli inner-membrane extracts mixed with additional E. coli lipids containing various amounts of the cbo(3) substrate UQ-10 (ubiquinol-10). Tethered membranes are formed by self-assembly from vesicles on to gold electrodes functionalized with cholesterol derivatives. cbo(3) activity was monitored using CV (cyclic voltammetry) with electron transfer to cbo(3) mediated by UQ-10. The apparent K(m) for oxygen with this system is 1.1+/-0.4 microM, in good agreement with values reported in the literature for whole-cell experiments and for purified cbo(3). Increasing the concentration of lipophilic UQ-10 in the membrane leads to an increase in cbo(3) activity. The activity of cbo(3) with long-chain ubiquinones appears to be different from previous reports using short-chain substrate analogues such as UQ-1 in that typical Michaelis-Menten kinetics are not observed using UQ-10. This native-like membrane model thus provides new insights into the interaction of transmembrane enzymes with hydrophobic substrates which contrasts with studies using hydrophilic UQ analogues.

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.

The inner membrane of Escherichia coli, over-expressing an ubiquinol oxidase, cytochrome bo 3 (cbo 3 ), was tethered in a planar configuration to a gold electrode. Electron transfer to cbo 3 was achieved via native ubiquinol-8 or added ubiquinol-10 and impedance spectroscopy was used to characterise the diffusion properties of the ubiquinol/ubiquinone in the tethered membrane system. Spectra were obtained at varying DC potentials covering the potential window in which the voltammetric catalytic wave of cbo 3 is visible. These spectra were compared to those obtained after addition of a potent inhibitor of cbo 3 , cyanide, and the difference in impedance was analysed using a derived equivalent circuit, which is similar to that of Open Finite-Length Diffusion (OFLD) or the finite Warburg circuit, but with the boundary conditions modified to account for the fact that ubiquinol reoxidation is limited by enzyme activity. Analysis of the impedance spectra of the tethered membrane system gave kinetic parameters that are consistent with values obtained using cyclic voltammetry. Importantly, the diffusion rate of ubiquinone (10 −13 -10 −12 cm 2 /s) was found to be orders of magnitude lower than accepted values for lateral diffusion (10 −8 -10 −7 cm 2 /s). It is hypothesised that this result represent perpendicular diffusion of quinone across the membrane, corresponding to a 'flip' time between 0.05 and 1 s.

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Quinone oxidoreductases are a class of membrane enzymes that catalyse the oxidation or reduction of membrane-bound quinols/quinones. The conversion of quinone/quinol by these enzymes is difficult to study because of the hydrophobic nature of the enzymes and their substrates. We describe some biochemical properties of quinones and quinone oxidoreductases and then look in more detail at two model membranes that can be used to study quinone oxidoreductases in a native-like membrane environment with their native lipophilic quinone substrates. The results obtained with these model membranes are compared with classical enzyme assays that use water-soluble quinone analogues.