Superoxide reductase (SOR) catalyzes the reduction of O-2(center dot-) to H2O2. Its active site consists of a non-heme Fe2+ center in an unusual square-pyramidal [HiS(4) Cys] coordination. Like many SORs, the electronic absorption band corresponding to the oxidized active site of the SOR from Desulfoarculus baarsii exhibits a pH-dependent alkaline transition changing from ca. 644 to 560 nm as the pH increases and with an apparent pK(a) of 9.0. Variants in which the conserved amino acids glutamate 47 and lysine 48 were replaced by the neutral residues alanine (E47A) and isoleucine (K481), respectively, exhibited the same alkaline transition but at lower apparent pK(a) values of 6.7 and 7.6, respectively. Previous work [Niviere, V.; Asso, M.; Weill, C. O.; Lombard, M.; Guigliarelli, B.; Favaudon, V.; Houee-Levin, C. Biochemistry 2004, 43, 808-818] has shown that this alkaline transition is associated with the protonation/deprotonation of an unidentified base, B-, which is neither E47 nor K48. In this work, we show by resonance Raman spectroscopy that at basic pH a high-spin Fe3+-OH species is formed at the active site. The presence of the HO- ligand was directly associated with an absorption band maximum at 560 nm, whereas upon protonation, the band shifts to 644 rim. With respect to our previous work, B- can be identified with this high-spin Fe3+-OH species, which upon protonation results in a water molecule at the active site. Implications for the SOR catalytic cycle are proposed.