The peptide-tethered lipid membrane as a biomimetic system to incorporate cytochrome c oxidase in a functionally active form

Peptide-supported lipid bilayers are investigated as a new class of solidsupported membranes tethered to the support by a peptide spacer. They are referred to as peptide tethered lipid membranes (tBLMs), formed by the fusion of liposomes with a thiopeptide-lipid monolayer chemisorbed on a gold support. Peptide tBLMs are designed as a biomimetic system to investigate integral membrane proteins. As an example, cytochrome c oxidase (COX) from bovine heart is incorporated into the preformed peptide tBLM by dilution of the solubilised protein below the critical micellar concentration. The formation of the lipid film as well as the incorporation of the protein were monitored by surface plasmon resonance spectroscopy and surface plasmon fluorescence spectroscopy. COX is activated by adding the reduced form of cytochrome c to the air-saturated buffer solution. Using electrochemical techniques, such as square wave voltammetry (SWV) and chronoamperometry (CA), the direct electron transfer between COX and the gold electrode is observed as well as proton transport from the inside to the outside across the lipid bilayer. Proton transport is then further investigated using impedance spectroscopy, although the electrode is shown to be only partially (70%) covered with a bilayer while defect domains with only a monolayer of peptide or peptide-lipid coexist (approx. 30%). Proton transport carried out by the COX is shown to be voltage dependent. This transport is indicated as a resistance in parallel to the resistance of the lipid film. As a consequence, the total resistance decreases as a function of the concentration of cytochrome c and increases again either by removal of the substrate or by addition of cyanide as an inhibitor of COX. The conductance in the presence of the activated enzyme correlates with the known turnover rate of COX. These experiments demonstrate the possibility to assess the activity of integral membrane proteins incorporated in peptide tBLMs using electrochemical techniques. The system could thus be promising for screening as well as biosensor applications.