Ricerc@Sapienza

We report on the influence of pH and monovalent/divalent cations on the catalytic current response, internal electron transfer (IET), and structure of fructose dehydrogenase (FDH) by using amperometry, spectrophotometry, and circular dichroism (CD). Amperometric measurements were performed on graphite electrodes, onto which FDH was adsorbed and the effect on the response current to fructose was investigated when varying the pH and the concentrations of divalent/monovalent cations in the contacting buffer. In the presence of 10 mM CaCl2, a current increase of up to ≈ 240% was observed, probably due to an intra-complexation reaction between Ca2+ and the aspartate/glutamate residues found at the interface between the dehydrogenase domain and the cytochrome domain of FDH. Contrary to CaCl2, addition of MgCl2 did not show any particular influence, whereas addition of monovalent cations (Na+ or K+) led to a slight linear increase in the maximum response current. To complement the amperometric investigations, spectrophotometric assays were carried out under homogeneous conditions in the presence of a 1-electron non-proton-acceptor, cytochrome c, or a 2-electron-proton acceptor, 2,6-dichloroindophenol (DCIP), respectively. In the case of cytochrome c, it was possible to observe a remarkable increase in the absorbance up to 200% when 10 mM CaCl2 was added. However, by further increasing the concentration of CaCl2 up to 50 mM and 100 mM, a decrease in the absorbance with a slight inhibition effect was observed for the highest CaCl2 concentration. Addition of MgCl2 or of the monovalent cations shows, surprisingly, no effect on the electron transfer to the electron acceptor. Contrary to the case of cytochrome c, with DCIP none of the cations tested seem to affect the rate of catalysis. In order to correlate the results obtained by amperometric and spectrophotometric measurements, CD experiments have been performed showing a great structural change of FDH when increasing the concentration CaCl2 up to 50 mM, at which the enzyme molecules start to agglomerate, hindering the substrate access to the active site probably due to a chelation reaction occurring at the enzyme surface with the glutamate/aspartate residues.