flavin-adenine-dinucleotide and potassium-phosphate

Redox processes in nano-TiO(2)-flavin adenine dinucleotide (TiO(2)-FAD) layer-by-layer assembled films on ITO substrate electrodes are investigated and compared in contact to aqueous electrolyte media (for dilute and saturated electrolyte) and in contact to solid humidified salt electrolyte (for extreme salt levels and different types of salts). Under these unusual conditions an aqueous microphase present at the gas | salt | electrode interface allows voltammograms to be obtained and redox processes to be analysed. It is demonstrated that the 2-electron 2-proton reduction of FAD can be used as reporter redox system to determine local pH at the electrode | gas | salt interface as pH 15, 12, 7 for contacts to K(3)PO(4), K(2)HPO(4), and KH(2)PO(4), respectively. Exposure to gases such as carbon dioxide is shown to lead to unexpected changes in surface pH. In the future, bio-electrochemical microphase processes under halophilic conditions could be useful for air-quality and rapid gas sensing devices.

Topics: Carbon Dioxide; Electrochemical Techniques; Electrodes; Electrolytes; Flavin-Adenine Dinucleotide; Gases; Hydrogen-Ion Concentration; Nanocomposites; Oxidation-Reduction; Phosphates; Potassium Compounds; Protons; Salts; Titanium

Single steps in the catalytic cycle of pyruvate oxidase from Lactobacillus plantarum have been characterized kinetically and mechanistically by stopped-flow in combination with kinetic solvent isotope effect studies. Reversible substrate binding of pyruvate occurs with an on-rate of 6.5 x 10(4) M(-1) s(-1) and an off-rate of pyruvate of 20 s(-1). Decarboxylation of the intermediate lactyl-ThDP and the reduction of FAD which consists of two consecutive single electron-transfer steps from HEThDP to FAD occur with rates of about k(dec) = 112 s(-1) and k(red) = 422 s(-1). Flavin radical intermediates are not observed during reduction, and kinetic solvent isotope effects are absent, indicating that electron transfer and protonation processes are not rate limiting in the overall reduction process. Reoxidation of FADH(2) by O(2) to yield H(2)O(2) takes place at a pseudo-first-order rate of about 35 s(-1) in air-saturated buffer. A comparable value of about 35 s(-1) was estimated for the phosphorolysis of the acetyl-ThDP intermediate at phosphate saturation. In competition with phosphorolysis, enzyme-bound acetyl-ThDP is hydrolyzed with a rate k = 0.03 s(-1). This is the first report in which the reaction of enzyme-bound acetyl-ThDP with phosphate and OH(-) is monitored directly by FAD absorbance changes using the sequential stopped-flow technique.

Topics: 2,6-Dichloroindophenol; Buffers; Catalysis; Decarboxylation; Deuterium Oxide; Flavin-Adenine Dinucleotide; Kinetics; Lactobacillus; Oxidation-Reduction; Phosphates; Potassium Compounds; Pyruvate Oxidase; Pyruvic Acid; Solvents; Spectrophotometry; Substrate Specificity