Electron transfer mechanism of cytochrome c at the oil/water interface as a biomembrane model.

The electron transfer (ET) between cytochrome c (Cyt c) in water (W) and 1,1'-dimethylferrocene (DiMFc) in 1,2-dichloroethane (DCE) was studied. The cyclic voltammograms obtained for the interfacial ET under various conditions could be well reproduced by digital simulation based on the ion-transfer (IT) mechanism, in which the ET process occurs not at the DCE/W interface but in the W phase nearest the interface. In this mechanism, the current signal is due to the IT of DiMFc(+) as the reaction product. On the other hand, the measurement of the double-layer capacity showed that Cyt c is adsorbed at the DCE/W interface. However, the contribution from the adsorbed proteins to the overall ET is considered to be small because of the thicker reaction layer in the IT mechanism. These findings would offer a useful suggestion for the behaviors of Cyt c in vivo.

Electron transfer mediated by membrane-bound d-fructose dehydrogenase adsorbed at an oil/water interface.

The catalytic activity of a membrane-bound enzyme, d-fructose dehydrogenase (FDH), at the polarized oil/water (O/W) interface was studied. Multisweep cyclic voltammetry and ac voltammetry were carried out to show the irreversible adsorption of FDH at the interface. Using the thusly prepared FDH-adsorbed O/W interface, clear steady-state catalytic current was observed in amperometry and cyclic voltammetry, where 1,1'-dimethylferrocenium ion (DiMFc(+), electron acceptor) and d-fructose (substrate) were added to the O and W phases, respectively. The observed catalytic current was then analyzed by using two mechanisms. In mechanism (A), the heme c site of FDH, where DiMFc(+) is reduced, was assumed to be located in the O-phase side of the interface. The intramolecular electron transfer in FDH should be affected by the Galvani potential difference of the interface (Δ(O)(W)ϕ). However, the theoretical equations derived for the catalytic current could not reproduce the experimental data. In mechanism (B), the heme c site was assumed to be in the W-phase side. In this case, Δ(O)(W)ϕ should affect the interfacial distribution of DiMFc(+). This mechanism could reproduce well the observed potential dependence of the catalytic current.

Electrochemical consideration on the optimum pH of bilirubin oxidase.

Steady-state current-potential curves were obtained for the direct electron transfer (DET) of bilirubin oxidase (BOD) at a highly oriented pyrolytic graphite electrode, and the theoretical analysis based on nonlinear regression enabled us to determine the formal redox potential (E degrees') of BOD in a wide pH range of 2.0 to 8.5. Cyclic voltammetric measurements were also performed for substrates, including p-phenylenediamine (PPD), o-aminophenol (OAP), and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), and their E degrees ' values or the anodic peak potentials (for OAP) were determined at various pH values. The difference in the redox potentials between BOD and substrates (DeltaE degrees') showed a maximum at pH 6.5 to 8.0, pH 6.5 to 8.0, and pH 3.5 to 4.5 for PPD, OAP, and ABTS, respectively. These pH ranges should be thermodynamically most favorable for the electron transfer between BOD and the respective substrates. In practice, the pH ranges showing a maximum DeltaE degrees' corresponded well with the optimum pH values for the O(2) reduction activity of BOD: pH 6.5 to 7.5, pH 8.0 to 8.5, and pH 4.0 for PPD, OAP, and ABTS, respectively. Thus, it was suggested that DeltaE degrees ' should be one of the primary factors determining the activity of BOD with the substrates.

Electrochemical extraction of proteins by reverse micelle formation.

The transfer of proteins by the anionic surfactant bis(2-ethylhexyl) sulfosuccinate (AOT) at a polarized 1,2-dichloroethane/water (DCE/W) interface was investigated by means of ion-transfer voltammetry. When the tetrapentylammonium salt of AOT was added to the DCE phase, the facilitated transfer of certain proteins, including cytochrome c (Cyt c), ribonuclease A, and protamine, could be controlled electrochemically, and a well-defined anodic wave for the transfer was obtained. At low pH values (e.g., pH 3.4), the anodic wave was usually well-separated from the wave for the formation of protein-free (i.e., unfilled) reverse micelles. The anodic wave for the protein transfer was analyzed by applying the theory for facilitated transfer of ions by charged ligands and then supplying information regarding the number of AOT anions reacting with one protein molecule and the total charge carried by the protein transfer. However, controlled-potential electrolyses performed for the transfer of Cyt c, which is red, revealed that the protein-AOT complexes were unstable in DCE and liable to aggregate at the interface when the pH of the W phase was 3.4. At pH 7.0, when formation of unfilled reverse micelles occurred simultaneously, the protein-AOT complexes appeared to be stabilized, probably via fusion with unfilled reverse micelles.