The voltammetry of cytochrome c oxidase immobilized in lipid bilayer membranes on gold electrodes and amperometric data of cytochrome c reacting at these electrodes under flow conditions are reported. A submonolayer of octadecyl mercaptan formed on electrodeposited silver anchors and becomes a part of the lipid bilayer membrane on the gold electrode. The supported lipid bilayer membrane containing cytochrome c oxidase is formed during a deoxycholate dialysis procedure. Slow scan rate cyclic voltammograms (20 mV/s) taken at the oxidase-modified electrodes show well-defined anodic waves. Fast scan rate cyclic voltammograms (200 mV/s) taken at the oxidase-modified electrodes show well-defined anodic and cathodic waves. Cyclic voltammograms taken at the oxidase-modified electrodes under 0.1 mM sodium cyanide show an increase (ca. 300%) in electrode capacitance and well-defined anodic and cathodic waves irrespective of scan rate. The voltammetric data are consistent with electron transfer of cytochrome c oxidase coupled with changes in nonfaradaic current and possibly diffusion of cytochrome c oxidase in a lipid multilayer structure. Quartz crystal microbalance data of cytochrome c binding to lipid bilayer membranes containing no cytochrome c oxidase under flow conditions are presented.

Cytochrome c oxidase immobilized in lipid bilayer membranes on gold electrodes mediates electron transfer between reduced cytochrome c in solution and the electrode. Under flow conditions, fixed potential amperometry of anaerobic solutions of reduced cytochrome c shows changes in the rate of enzyme mediated electron transfer from cytochrome c to oxidase modified electrodes. These data are consistent with literature that describes both activation of the resting oxidase state to the pulsed oxidase state and subsequent decay of the pulsed oxidase state to the resting oxidase state.

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Scanning force microscopic images of cytochrome c oxidase immobilized within an electrode-supported lipid bilayer membrane are reported. These images represent the first direct evidence that the microscopic architecture of this important model system effectively mimics the microstructure proposed for the inner mitochondrial membrane. The images reveal (1) that the oxidase is present as monomers and small aggregates within the supported membrane and (2) that the oxidase constitutes ∼20% of the lipid bilayer membrane. The latter finding allows an estimation of the lower limit for the minimum turnover rate (∼0.5 electrons/s) required for the transition of the oxidase from its resting to pulsed kinetic state.

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