Zeenit 650p

Metal ion binding assay was performed as previously described (Si et al. 2017b (link)). Briefly, for removing all binding ions, purified Fur protein (300 μM) was added to the solution containing 50 mM Tris, 25 mM diethylene triamine pentaacetic acid, and 10% glycerol at pH 7.4. After incubation for 1 h on ice, the protein solution was dialyzed three times with buffer (50 mM Tris, 10% glycerol, pH 8.0) at 4 °C. For reconstitution with metal ions, the resulting apo-Fur protein (100 μM) was added to 500 μM of the desired divalent-metal ions (Fe²⁺ or Mn²⁺) and incubated on ice for 30 min, with Milli-Q water for preparing ions solution as the control. These solutions were dialyzed again to remove unbound metal ions and the metal ions bound to the protein were analyzed using atomic absorption spectroscopy (ZEEnit 650P; Analytik Jena, Jena, Germany).

Metal-free Mca and reconstituted Mca with the desired metal ions were prepared as previously described [22] (link). Briefly, purified protein (100 µM) was added to the solution containing 25 mM Tris, 25 mM diethylene triamine pentaacetic acid, and 10% glycerol, at pH 7.5 and put on ice. After 1 h, the protein solution was dialyzed with buffer (25 mM Tris, 10% glycerol, pH 7.5) at 4°C. The concentration of residual metal ions was determined by atomic absorption spectrometry (ZEEnit 650P, Analytik Jena, Germany). For reconstitution with metal ion, apo-Mca (10 µM) was added to a stoichiometric concentration of the desired divalent-metal ions (Co²⁺, Fe²⁺, Mn²⁺, Ni²⁺ and Zn²⁺) and put on ice for 30 min. To determine the optimal metal/protein ratio, apo-Mca was incubated with various concentrations of the metal ions (0–25 µM) for 30 min on ice prior to activity assays. These solutions were dialyzed again as mentioned above to remove unbound metal ions and then analyzed by atomic absorption spectrophotometry.