Takamasa Sagara

Bioelectrocatalysis at electrodes coated with alcohol dehydrogenase, a quinohemoprotein with heme c serving as a built-in mediator

Alcohol dehydrogenase (ADH), a bacterial membrane-bound protein containing pyrroloquinoline quinone (PQQ) and heme c was held by adsorption on electrodes of gold, silver, glassy carbon, or pyrolytic graphite. All the electrodes with adsorbed ADH produced anodic currents which oxidized ethanol, in which the adsorbed ADH catalysed the electrolysis of ethanol. The electrocatalysis behavior could be described by a theoretical equation for bioelectrocatalysis at an enzyme-coated electrode, and was characterized by two quantities, the Michaelis constant Km, and maximum current density Imax/A. Using electroreflectance measurements with an ADH-coated gold electrode it was revealed that electron transfer occurred between heme c of the adsorbed ADH and the electrode. On the basis of these results, the reaction mechanism of the bioelectrocatalysis is discussed and oriented adsorption of ADH is proposed with the heme c moiety being in close contact with the electrode and with the PQQ moiety, the site reacting with the substrate, facing toward the solution.

Regulation of the redox order of four hemes by pH in cytochrome C3 from D. vulgaris Miyazaki F

The assignment of 1H-NMR signals of the heme methyl and propionate groups of cytochrome c3 of D. vulgaris Miyazaki F was performed. The heme assignment was revised for hemes 2 and 3 (sequential heme numbering). Namely, heme 4 is mainly reduced at first with hemes 1, 2 and 3 following it in this order. The p2H titration of heme methyl signals in four macroscopic oxidation states was performed in the p2H range of 5.2 to 9.0. While the heme methyl resonances in the fully oxidized state showed just small changes with p2H, most resonances in the intermediate oxidation states revealed clear p2H dependence. In particular, the methyl resonances of heme 1 shifted significantly in the acidic region. Then, the chemical shifts of beta-CH2 (next to the carboxyl group) of all propionate groups in the fully oxidized state were observed at various p2H in the range of 4.5 to 9.0. Only the propionate group at C-13 (IUPAC-IUB nomenclature) of heme 1 showed a clear change in this p2H range, its titration curve being similar to those of the methyl resonances of heme 1 in the intermediate oxidation states. pKa of the propionate group was 5.95 +/- 0.05. Analysis of the microscopic formal redox potentials was carried out for the observations at p2H 5.2, 7.1 and 9.0. The redox potentials of heme 1 showed the most remarkable p2H dependence, resulting in the change of the order of the redox potentials of four hemes. A significant change was also found in the interacting potential between hemes 1 and 2. In the light of the p2H-titration experiments, the propionate at C-13 of heme 1 was identified as the most plausible ionizable group responsible for the p2H dependence of microscopic redox potentials of heme 1 in the acidic region.

Electroreflectance study of hemin adsorbed on a pyrolytic graphite electrode surface and its coadsorption with methylene blue

The electrode reaction of hemin (Hm) adsorbed on an a–b plane pyrolytic graphite (BPG) electrode was investigated by using the potential-modulated UV-visible reflectance method (ER method). It was found that the orientation of the Hm molecule is not flat on the a–b plane of the BPG electrode. Coadsorption of Hm with methylene blue (MB) was also studied. There is no significant interaction between Hm and MB at the BPG electrode. The formal potentials of Hm and MB adsorbed on the BPG electrode are close together and the simultaneous electrode reactions of these dyes give rise to a single voltammetric wave, which is impossible to resolve by traditional voltammetric techniques. The ER method enabled us to resolve these two reactions. A difference was found between the displacement of the adsorbed MB by Hm and the displacement of Hm by MB. It was suggested that the adsorption of one Hm molecule on the MB-preadsorbed BPG electrode requires the displacement of five adjacent MB molecules.

Heterogeneous electron-transfer rate measurements of cytochrome c3 at mercury electrodes

Abstract The electron-transfer rate constant of cytochrome c 3 at mercury electrodes in aqueous solution was measured by the galvanostatic double pulse method at 25°C. The resulting rate constant and transfer coefficient were 1.2 cm s −1 and 0.48, respectively. The measured rate constant agreed well with that estimated using Marcus theory and the homogeneous self-exchange rate measured by NMR.

Electrode reaction of the soluble domain of the membrane-bound hydrogenase from Desulfovibrio vulgaris, strain Miyazaki F

Abstract The electrode reaction of the soluble domain of membrane-bound hydrogenase from Desulfovibrio vulgaris , Miyazaki F, was studied at mercury and six other solid electrodes. A cathodic wave at −0.30 V (vs. NHE) at pH 7.0 observed for hydrogenase adsorbed on a mercury electrode is attributed to the reduction of the active center of hydrogenase, the iron-sulfur cluster. This reduction process involves proton transfer. Once reduced, the hydrogenase loses its reoxidation activity at the mercury electrode. Hydrogenase adsorbed on a mercury electrode catalyzes the hydrogen production reaction, whose onset potential at pH 7.0 is 120 mV more positive than the reversible hydrogen electrode potential. The catalytic activity disappears when hydrogenase is thiocarboxymethylated, while the reduction activity of the active center is retained.

Electroreflectance study of redox reactions of methylene blue incorporated into polypyrrole coated on a pyrolytic graphite electrode

Abstract The redox reactions of methylene blue (MB) incorporated into polypyrrole (PPy) on pyrolytic graphite (BPG) electrodes were investigated by the potential-modulated electroreflectance (ER) method. The incorporation of MB into the PPY film was achieved by either electropolymerization of pyrrole in the presence of MB or polarization of the PPy-coated electrode at negative potentials in an MB solution. The ER spectrum of MB incorporated into the PPy-film, which was similar to that of MB adsorbed directly to the BPG electrode, suggested that MB in the PPy film diffused out during deaeration of the electrolyte solution and only MB adsorbed on the BPG electrode surface reacted. In the presence of MB in both the PPy film and solution phase, the measured ER spectrum was the sum of the ER spectra of MB adsorbed on the BPG electrode surface and MB in both the PPy film and solution phase. Each of the two spectra was detected separately using the phase-shift method.