The inner membrane of Escherichia coli, overexpressing an ubiquinol oxidase, cytochrome bo3 (cbo3), was "tethered" in a planar configuration to a gold electrode. Electron transfer to cbo3 was achieved via native ubiquinol-8 or added ubiquinol-10, and impedance spectroscopy was used to characterize the diffusion properties of the ubiquinol/ubiquinone in the tethered membrane system. Spectra were obtained at varying direct current (DC) potentials covering the potential window in which the voltammetric catalytic wave of cbo3 is visible. These spectra were compared to those obtained after addition of a potent inhibitor of cbo3, cyanide, and the difference in impedance was analyzed 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) cm2/s) was found to be orders of magnitude lower than accepted values for lateral diffusion (10(-8)-10(-7) cm2/ s). It is hypothesized that this result represent perpendicular diffusion of quinone across the membrane, corresponding to a "flip" time between 0.05 and 1 s.