Grabowska Ola, Żamojć Krzysztof, Kloska Anna, Niedziałkowski Paweł, Samsonov Sergey A, Wyrzykowski Dariusz
Faculty of Chemistry, University of Gdańsk, Wita Stwosza 63, 80-308 Gdańsk, Poland.
Faculty of Biology, University of Gdańsk, Wita Stwosza 59, 80-308 Gdańsk, Poland.
Int J Mol Sci. 2025 Sep 2;26(17):8511. doi: 10.3390/ijms26178511.
In this article, the binding interactions of lysozyme with hexacyanoferrate(III)/(II), i.e., [Fe(CN)] and [Fe(CN)] ions, have been characterised using steady-state fluorescence spectroscopy (SF), isothermal titration calorimetry (ITC), circular dichroism spectroscopy (CD), cyclic voltammetry (CV), and molecular-dynamics-based computational approaches. Studies have shown that under experimental conditions (10 mM cacodylate buffer, pH 7, 298.15 K), complexes with a 1:1 stoichiometry are formed. Four distinct regions on the lysozyme surface patches with the potential to bind hexacyanoferrate(III)/(II) were identified and described. Thermodynamic parameters revealed that the interactions are predominantly governed by electrostatic and van der Waals forces. These interactions enhance the electron transfer kinetics of the [Fe(CN)] system. The secondary structure of the protein is not affected by these interactions. Enzyme activity studies demonstrated that the affinity of lysozyme for the substrate remained unchanged regardless of whether free lysozyme or the lysozyme-[Fe(CN)] complex was present in the test sample. Finally, biological tests performed on both Gram-positive (, ) and Gram-negative (, ) bacteria confirmed the results of the biochemical analysis, indicating that [Fe(CN)] ions do not block the active site of the enzyme and do not interfere with its activity.
在本文中，已使用稳态荧光光谱法（SF）、等温滴定量热法（ITC）、圆二色光谱法（CD）、循环伏安法（CV）以及基于分子动力学的计算方法，对溶菌酶与六氰合铁（III）/（II），即[Fe(CN)]⁴⁻和[Fe(CN)]³⁻离子的结合相互作用进行了表征。研究表明，在实验条件下（10 mM二甲胂酸盐缓冲液，pH 7，298.15 K），会形成化学计量比为1:1的配合物。已鉴定并描述了溶菌酶表面斑块上四个有潜力结合六氰合铁（III）/（II）的不同区域。热力学参数表明，这些相互作用主要受静电和范德华力支配。这些相互作用增强了[Fe(CN)]⁴⁻/[Fe(CN)]³⁻体系的电子转移动力学。蛋白质的二级结构不受这些相互作用的影响。酶活性研究表明，无论测试样品中存在的是游离溶菌酶还是溶菌酶 - [Fe(CN)]配合物，溶菌酶对底物的亲和力均保持不变。最后，对革兰氏阳性菌（金黄色葡萄球菌、枯草芽孢杆菌）和革兰氏阴性菌（大肠杆菌、铜绿假单胞菌）进行的生物学测试证实了生化分析的结果，表明[Fe(CN)]⁴⁻/[Fe(CN)]³⁻离子不会阻断酶的活性位点，也不会干扰其活性。
