Department of Chemistry and Drug Technologies, Sapienza University of Rome, Piazzale Aldo Moro 5, 00185, Rome, Italy.
Department of Analytical Chemistry/Biochemistry, Lund University, P.O. Box 124, 221 00, Lund, Sweden.
Anal Bioanal Chem. 2018 May;410(14):3253-3264. doi: 10.1007/s00216-018-0991-0. Epub 2018 Mar 22.
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 CaCl, a current increase of up to ≈ 240% was observed, probably due to an intra-complexation reaction between Ca and the aspartate/glutamate residues found at the interface between the dehydrogenase domain and the cytochrome domain of FDH. Contrary to CaCl, addition of MgCl 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 CaCl was added. However, by further increasing the concentration of CaCl up to 50 mM and 100 mM, a decrease in the absorbance with a slight inhibition effect was observed for the highest CaCl concentration. Addition of MgCl 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 CaCl 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 Abstract 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 CaCl to the buffer solution, whereas MgCl had an "inhibition" effect. Moreover, the additions of KCl or NaCl led to a slight current increase.
我们通过使用安培法、分光光度法和圆二色性（CD）报告了 pH 值和一价/二价阳离子对果糖脱氢酶（FDH）的催化电流响应、内部电子转移（IET）和结构的影响。在石墨电极上进行安培测量，当在接触缓冲液中改变二价/一价阳离子的浓度时，研究了对果糖响应电流的影响。在存在 10 mM CaCl 的情况下，观察到电流增加了高达 ≈240%，这可能是由于 Ca 与 FDH 的脱氢酶结构域和细胞色素结构域之间的界面处的天冬氨酸/谷氨酸残基之间的内络合反应所致。与 CaCl 相反，添加 MgCl 没有显示出任何特殊影响，而添加单价阳离子（Na 或 K）导致最大响应电流略有线性增加。为了补充安培测量，在存在 1 个电子非质子受体细胞色素 c 或 2 个电子-质子受体 2,6-二氯靛酚（DCIP）的均相条件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 时，可以观察到吸光度显着增加高达 200%。然而，通过进一步将 CaCl 的浓度增加至 50 mM 和 100 mM，观察到吸光度略有下降，并且在最高 CaCl 浓度下观察到轻微的抑制作用。添加 MgCl 或单价阳离子出人意料地对电子向电子受体的转移没有影响。与细胞色素 c 相反，在所测试的阳离子中，没有一种似乎影响催化速率。为了将安培和分光光度测量的结果相关联，已经进行了 CD 实验，结果表明当 CaCl 的浓度增加至 50 mM 时，FDH 的结构发生了很大变化，此时酶分子开始聚集，可能由于在酶表面发生螯合反应而阻碍了底物进入活性位点，该反应发生在酶表面的谷氨酸/天冬氨酸残基上。 果糖脱氢酶（FDH）由三个亚基组成，但只有两个参与电子转移过程：（I）2e/2H 果糖氧化，（II）内部电子转移（IET），（III）通过 2 个血红素 c 直接电子转移（DET）；将 10 mM CaCl 添加到缓冲溶液中可将 FDH 在溶液中的活性或固定在电极表面上的活性分别提高约 2.5 倍，而 MgCl 具有“抑制”作用。此外，添加 KCl 或 NaCl 会导致电流略有增加。
