A study of cytochrome bo3 in a tethered bilayer lipid membrane

Abstract

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.

Figure 1

A schematic representation of the tBLM used in this work.

Figure 2

(Top) Cyclic voltammograms of a tBLM containing cbo₃ at increasing UQ-10 coverage (determined separately using cyclic voltammograms at 1 V/s). Electrode area is ~ 0.25 cm² (Top, Insert) The UQ-10 coverage (determined using cyclic voltammograms at 1 V/s) as a function of UQ-10 added to the electrolyte from a DMSO stock solution. (Bottom) (Closed symbols) Normalised activity of cbo₃ as a function of UQ-10 coverage for three different experiments (squares, triangles and circles). The UQ-10 coverage (bottom axis) is determined by determination of the UQ-10 peak area from voltammograms measured at 1 V/s. (Open symbols) Comparison with previously published data[13] in which UQ-10 is cosolubilised in vesicles prior to tBLM formation and the UQ-10 coverage (top axis) is determined at 10 mV/s after inhibition of cbo₃ with NaCN. The top and bottom axis have different scales due to observed differences in UQ-10 coverage when determined at 1 V/s and 10 mV/s (see text).

Figure 3

(Top) Cyclic voltammograms for cytochrome bo₃ inhibited with (NaCN) at 0μM, 5μM, 10μM, 20μM, 30μM and 90μM, (HQNO) at 0μM, 40μM, 60μM, 80μM and 190μM and (ZnSO₄) at 0μM, 25μM, 50μM and 125μM. The QU-10 concentration in the membrane is between 5 and 10 pmol/cm². The electrode area is 0.25 cm² (Middle) Baseline corrected voltammograms of the data shown in Top. The baseline was taken from the cyclic voltrammogram measured at the highest concentration of inhibitor. (Middle, insert) Extracted data for IC₅₀ determination. Error bars are standard deviations determined from at least three experiments. (Bottom) First derivatives of the data shown in Middle.

Figure 4

Schematic representation of the tBLM and the rates that influence the currents measured with cyclic voltammetry.