Abstract
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.Graphical Fructose dehydrogenase (FDH) consists of three subunits, but only two are involved in the electron transfer process: (I) 2e−/2H+ fructose oxidation, (II) internal electron transfer (IET), (III) direct electron transfer (DET) through 2 heme c; FDH activity either in solution or when immobilized onto an electrode surface is enhanced about 2.5-fold by adding 10 mM CaCl2 to the buffer solution, whereas MgCl2 had an “inhibition” effect. Moreover, the additions of KCl or NaCl led to a slight current increase
Highlights
In the last 30 years, studies of direct electron transfer (DET) reactions between redox enzymes and electrodes have attracted much interest to understand the background reaction mechanism [1, 2] and gain sufficient basic knowledge to be able to establish the basis and to improve the performance of third generation biosensors and enzymatic fuel cells (EFCs) [3,4,5,6]
Several of the redox enzymes mentioned above, such as sulfite oxidase (SOx), alcohol PQQ dehydrogenase (ADH), cellobiose dehydrogenase (CDH), and fructose dehydrogenase (FDH) are composed of at least two different domains, where one domain is the catalytically active domain containing a bound cofactor such as flavin adenine dinucleotide (FAD), PQQ, or MOCO, and a second domain, a cytochrome containing heme c or heme b, acting as an electron transfer domain or in other words Bas a built in mediator^ [31] connecting the redox enzyme to its natural electron acceptor, or when immobilized onto the surface of an electrode to the electrode if the enzyme is properly orientated on its surface
The response current rapidly decreased when increasing the pH above 4.5, assessing no DET activity starting at pH 7
Summary
In the last 30 years, studies of direct electron transfer (DET) reactions between redox enzymes and electrodes have attracted much interest to understand the background reaction mechanism [1, 2] and gain sufficient basic knowledge to be able to establish the basis and to improve the performance of third generation biosensors and enzymatic fuel cells (EFCs) [3,4,5,6]. Several of the redox enzymes mentioned above, such as SOx, ADH, CDH, and FDH are composed of at least two different domains, where one domain is the catalytically active domain containing a bound cofactor such as FAD, PQQ, or MOCO, and a second domain, a cytochrome containing heme c or heme b, acting as an electron transfer domain or in other words Bas a built in mediator^ [31] connecting the redox enzyme to its natural electron acceptor, or when immobilized onto the surface of an electrode to the electrode if the enzyme is properly orientated on its surface. The DET mechanism for membrane bound FDH has not yet been elucidated [45] and FDH has attracted a growing interest regarding all factors that can influence the DET reaction (e.g., pH, cations, ionic strength, etc.) [46,47,48]
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