Cytochrome c Oxidase Immobilized in Stable Supported Lipid Bilayer Membranes

Abstract

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.

Electrochemistry of Metalloproteins Attached through Functional Self‐Assembled Monolayers on Gold and Ferromagnetic Electrodes

“…The voltammograms have a very different shape and feature single-electron anodic and cathodicp rocesses at + 130 and 50 mV (vs. SCE)w ith DE p = 80 mV ( Figure 6A). This redox behavior,h owever,d iffers from the existing reports, [18] whichc ould be the result of different workingelectrodes and coupling molecules betweent he working electrode and the redox probe.Ad iffusionless redox process was confirmed by the linear dependence (R 2 = 0.98-0.99) of the anodic and cathodic currents as af unction of the scan rates ( Figure 6B). The numerical values relevant to the scanrate dependence of the potential peak-to-peak separation value andt he half-waver edox potentialf or mixed SAM/Cyt C oxidaseo na nA u-coated Ni working electrode are shown in Ta ble S2.T he ratio of anodic to cathodic current density (I pa /I pc ) is independent of the scan rate and approaches one, which implies the same charge formation for both the reduced and oxidized species( Figure S10).…”

Recent advances in the electrochemistry and spectroelectrochemistry of membrane proteins

“…This dialysis is commonly used to reconstitute the oxidase into vesicles (Hinkle et al , 1972 ) with its cytochrome c binding site preferentially oriented outward. The stability of the membrane was later improved by adding a layer of electrodeposited silver onto the gold surface (Burgess et al , 1998 ). Sulfur binds more strongly at silver surfaces than gold, and the thiol hydrocarbon tail is believed to adopt a nearly normal orientation on such surfaces.…”

Use of Surface Enhanced Infrared Absorption Spectroscopy (SEIRA) to probe the functionality of a protein monolayer

“…13,14 Second, the orientated and surface-attached protein is reconstituted into a lipid environment by in situ dialysis ( Figure 3b). [15][16][17] This concept ensures orientated immobilization as well as reconstitution into the native matrix, which enhances the stability of the membrane protein and restores full functionality. Figure 4 displays a time-dependent series of adsorption spectra of detergent-solubilized cytochrome c oxidase (CcO) via the affinity of the genetically introduced His-tag to the Ni-NTA modified gold surface.…”