Steady-state kinetic analysis was performed on the reaction between d-fructose and ferricyanide with the quinohemoprotein fructose dehydrogenase from Gluconobacter species. The d-fructose oxidation dependence on the ferricyanide concentration resulted in a series of parallel reciprocal plots, and the reaction was assumed to proceed by a ping-pong type of mechanism. A reciprocal plot of the reduction of ferricyanide at saturating concentration of d-fructose gave a break which was considered to appear as a result of the two active centers, namely PQQ and heme e functioning. A scheme of action is proposed and the bimolecular rate constant of the d-fructose oxidation, the k cat for PQQ and the electron transfer rate between the PQQH2 and heme c are calculated and account for 2.2 ± 0.4.104 M−1s−1, (93 ± 14) and (162 ± 7) s−1, respectively.
