Electrochemical investigations of the reaction mechanism and kinetics between NADH and riboflavin immobilised on amorphous zirconium phosphate

Electrochemical investigations of the reaction mechanism and kinetics betwe en riboflavin immobilised on zirconium phosphate (ZPRib) in carbon paste an d NADH showed results yielding reliable information about aspects on the me chanism of the electron transfer reaction between the flavin and NADH. The formal potential (E-o') of the adsorbed riboflavin was -220 mV versus SCE a t pH 7.0. A shift about 250 mV towards a more positive potential compared w ith its value in solution was assigned to the interaction between the basic nitrogen of riboflavin and the acid groups of ZP. The invariance of the E- o' with the pH of the contacting solution and the effect of different buffe r constituents were attributed to the protection effect of ZP over the ribo flavin. The electrocatalytic oxidation of NADH at the electrode was investi gated using cyclic voltammetry and rotating disk electrode methodology usin g a potential of -50mV versus SCE. The heterogeneous electron transfer rate constant, k(obs), was 816 M-1 s(-1) and the Michaelis-Menten constant, K-M , was 1.8 mM (confirming a charge transfer complex intermediate in the reac tion) for an electrode with a riboflavin coverage of 6.8 x 10(-10) mol cm(- 2). This drastic increase in the reaction rate between NADH and the immobil ised riboflavin was assigned to the shirt of the E-o'. A surprising effect with addition of calcium or magnesium. ion to the solution was also observe d. The E-o' was shifted to -150 mV versus SCE and the reaction rate for NAD H oxidation increased drastically.