Direct measurement of cyclic current-voltage responses of integral membrane proteins at a self-assembled lipid-bilayer-modified electrode: cytochrome f and cytochrome c oxidase.

Abstract

Direct cydic voltage-current responses, produced in the absence of redox mediators, for two detergentsolubiized integral membrane proteins, spinach cytochromef and beef heart cytochrome c oxidase, have been obtained at an optically transparent indium oxide electrode modified with a self-assembled lipid-bilayer membrane. The results indicate that both proteins interact with the lipid membrane so as to support quasi-reversible electron transfer redox reactions at the semiconductor electrode. The redox potentials that were obtained from analysis of the cyclic "voltammograms," 365 mV for cytochrome f and 250 and 380 mV for cytochrome c oxidase (vs. normal hydrogen electrode), compare quite well with the values reported by using conventional titration methods. The ability to obtain direct electrochemical measurements opens up another approach to the investigation of the properties of integral membrane redox proteins.It is well known that the use of direct electrochemical methods to obtain thermodynamically correct values for the redox potentials of electron-transfer proteins has proven to be rather difficult as a consequence of strong interactions between these proteins and the electrode surface, which often lead to adsorption and structural alteration (1-7). Attempts to circumvent this problem have resulted in the development of a variety of strategies for chemically modifying metal and semiconductor surfaces so as to provide an electron transfer pathway that maintains protein native structure and eliminates strong binding (1, A major conclusion that we arrived at on the basis of our above-mentioned experiments on the use of lipid-bilayermodified electrodes to carry out direct electrochemical measurements on redox proteins was that specific, but relatively weak, adsorption processes involving electrostatic or hydrophobic forces were an important component of the ability of these systems to function in mediating protein electron transfer reactions. It seemed reasonable to us, then, that integral membrane proteins, which normally are embedded in phospholipid bilayers, should also find such electrode coatings a hospitable environment, allowing them to directly participate in electron transfer reactions with an underlying metal or semiconductor surface. Our experiments have, indeed, borne this out, and we report below cyclic voltagecurrent measurements of the redox reactions between a lipid-bilayer-modified electrode and two integral membrane proteins: cytochrome f and cytochrome c oxidase.Cytochrome f is a heme c-containing component of the chloroplast b6fcomplex that is involved in the photosynthetic transport of electrons from plastoquinol to photosystem I via plastocyanin (49). It has been isolated in a monomeric, water-soluble form from Spirulina (a cyanobacterium) and from spinach (as an octameric aggregate), rape, and turnip (higher plants) (50, 51). Structural evidence obtained with the spinach protein suggests that a 20-residue segment between residues 251 and 270 of the 285-residue protein may be invol...

Recent advances in the electrochemistry and spectroelectrochemistry of membrane proteins

“…The first immobilization of bovine cytochrome c oxidase ( cco ) within a phosphatidylcholine lipid bilayer freely suspended on a semiconductor i ndium tin oxide electrode was reported in the early 1990s (Salamon et al , 1993 ). The reconstitution of the enzyme in the phospholipid membrane was performed by detergent dilution of concentrated solutions of the enzyme.…”

“…Cco reconstituted either within a supported lipid bilayer on an ITO electrode (Salamon et al , 1993 ) or within a hybrid bilayer assembled on a gold electrode (Cullison et al , 1994 ) also exhibited a DET. The values of the redox potentials suggest a communication with the Cu A and cyt-a 3 cofactors respectively.…”

Native E. coli inner membrane incorporation in solid-supported lipid bilayer membranes

“…In general, proteins are either purified and reconstituted into lipid vesicles, which are then used to form SBLMs, 19 or alternatively, lipid bilayers are formed and then are incubated with the desired proteins. 28,29 Incorporating proteins in these ways is effective when only relatively small amounts of a single protein are needed. However, as the number of proteins required for an active system increases, the process becomes more complex.…”

Enhanced Peroxidase Activity of Hemoglobin in a DNA Membrane and Its Application to an Unmediated Hydrogen Peroxide Biosensor

“…[4][5][6] Others and we have found that many eT proteins are strongly membrane-associated, and a certain kind of organized molecular structure in the membrane is necessary for their efficient eT. [7][8][9][10][11] Although DNA is widely known as the preferred gene-storage medium, it also has a well characterized structure, which can be used as a useful material to create well defined structures on molecular level. 12 Therefore, the interaction between DNA and proteins has become one of the principal biological interactions, and heme protein-DNA complexes have attracted much recent interest.…”