The iron-amino acid interactions Fe-O(Glu/Asp), Fe-N(His), and Fe-S(Cys) are the three major iron-ligand bonds in proteins. To compare their properties in proteins, we used atomic force microscopy (AFM)-based single-molecule force spectroscopy to investigate a superoxide reductase (Fe(III)-SOR) with all three types of bonds forming an Fe(His)4CysGlu center. We first found that Apo-SOR without bound iron showed multiple unfolding pathways only from the β-barrel core. Then, using Holo-SOR with a ferric ion, we found that a single Fe-O(Glu) bond can tightly connect the flexible N-terminal fragment to the β-barrel and stabilize the whole protein, showing a complete protein unfolding scenario, while the single Fe-N(His) bond was weak and unable to provide such a stabilization. Moreover, when multiple Fe-N bonds are present, a similar stabilization effect can be achieved. Our results showed that the iron-ligand bond modulates protein structure and stability in different modes at the single-bond level.