An Interdisciplinary Study of Lysozyme Interactions with Hexacyanoferrate(III)/(II) Ions.

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.