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.

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我们通过使用安培法，分光光度法和圆二色性（CD）报告了pH和一价/二价阳离子对催化电流响应，内部电子转移（IET）和果糖脱氢酶（FDH）结构的影响。在吸附了FDH的石墨电极上进行安培测量，并在改变pH和接触缓冲液中二价/单价阳离子的浓度时研究了对果糖响应电流的影响。在10 mM CaCl ₂存在下，观察到电流增加约≈240％，这可能是由于Ca ²⁺与在脱氢酶结构域和细胞色素之间的界面处发现的天冬氨酸/谷氨酸残基之间的内部络合反应所致。FDH的域。与氯化钙相反_{如图2所示}，MgCl _2的添加没有显示任何特别的影响，而单价阳离子（Na ⁺或K ⁺）的添加导致最大响应电流的线性增加。为了进行电流分析研究，在均质条件下分别在1电子非质子受体，细胞色素c或2电子质子受体2,6-二氯吲哚酚（DCIP）的存在下，进行了分光光度测定。在细胞色素c的情况下，当添加10 mM CaCl ₂时，可以观察到吸光度显着增加，最高可达200​​％。但是，通过进一步增加CaCl ₂的浓度最高50 mM和100 mM时，对于最高的CaCl ₂浓度，观察到吸光度降低且具有轻微的抑制作用。令人惊讶地，MgCl ₂或一价阳离子的添加​​对电子转移至电子受体没有影响。与细胞色素c的情况相反，使用DCIP时，未测试的阳离子似乎都不会影响催化速率。为了使通过安培法和分光光度法测得的结果相互关联，已进行了CD实验，表明当增加CaCl ₂的浓度时，FDH的结构发生了很大变化。 高达50 mM时，酶分子开始聚集，阻碍底物进入活性位点，这可能是由于酶表面与谷氨酸/天冬氨酸残基发生的螯合反应所致。

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