The redox-induced structural changes at the active site of the superoxide reductase (SOR) from Desulfoarculus baarsii and Treponema pallidum have been monitored by means of FTIR difference spectroscopy coupled to electrochemistry. With this technique, the structure and interactions formed by individual amino acids at a redox site can be detected. The infrared data on wild-type, Glu47Ala, and Lys48Ile mutants of the SOR from D. baarsii provide experimental support for the conclusion that the two different coordination motifs observed in the three-dimensional structure of the SOR from Pyrococcus furiosus [Yeh, A. P., Hu, Y., Jenney, F. E., Adams, M. W. W., and Rees, D. (2000) Biochemistry 39, 2499-2508] correspond to the two redox forms of the SOR iron center. We extend this result to the center II iron of SOR of the desulfoferrodoxin type. Similar structural changes are also observed upon iron oxidation in the SOR of T. pallidum. In D. baarsii, the IR modes of the Glu47 side chain support that it provides a monodentate ligand to the oxidized iron, while it does not interact with Fe(2+). Structural changes at the level of peptide bond(s) observed upon iron oxidation in wild-type are suppressed in the Glu47Ala mutant. We propose that the presence of the Glu side chain plays an important role for the structural reorganization accompanying iron oxidation. We identified the infrared modes of the Lys48 side chain and found that a change in its environment occurs upon iron oxidation. The lack of other structural changes upon the Lys48Ile mutation shows that the catalytic role of Lys, as evidenced by pulse radiolysis experiments [Lombard, M., Houée-Levin, C., Touati, D., Fontecave, M., and Nivière, V. (2001) Biochemistry 40, 5032-5040], is purely electrostatic, guiding superoxide toward the reduced iron.