Mössbauer Characterization of an Unusual High-Spin Side-On Peroxo−Fe<sup>3+</sup> Species in the Active Site of Superoxide Reductase from <i>Desulfoarculus baarsii</i>. Density Functional Calculations on Related Models<sup>†</sup>

Superoxide reductase (SOR) is an Fe protein that catalyzes the reduction of superoxide to give H<sub>2</sub>O<sub>2</sub>. Recently, the mutation of the Glu47 residue into alanine (E47A) in the active site of SOR from <i>Desulfoarculus baarsii</i> has allowed the stabilization of an iron−peroxo species when quickly reacted with H<sub>2</sub>O<sub>2</sub> [Mathé et al. (2002) <i>J. Am. Chem. Soc.</i> <i>124</i>, 4966−4967]. To further investigate this non-heme peroxo−iron species, we have carried out a Mössbauer study of the <sup>57</sup>Fe-enriched E47A SOR from <i>D. baarsii</i> reacted quickly with H<sub>2</sub>O<sub>2</sub>. Considering the Mössbauer data, we conclude, in conjunction with the other spectroscopic data available and with the results of density functional calculations on related models, that this species corresponds to a high-spin side-on peroxo−Fe<sup>3+</sup> complex. This is one of the first examples of such a species in a biological system for which Mössbauer parameters are now available: δ<sub>/Fe</sub> = 0.54 (1) mm/s, Δ<i>E</i><sub>Q</sub> = −0.80 (5) mm/s, and the asymmetry parameter η = 0.60 (5) mm/s. The Mössbauer and spin Hamiltonian parameters have been evaluated on a model from the side-on peroxo complex (model <b>2</b>) issued from the oxidized iron center in SOR from <i>Pyrococcus furiosus</i>, for which structural data are available in the literature [Yeh et al. (2000) <i>Biochemistry 39</i>, 2499−2508]. For comparison, similar calculations have been carried out on a model derived from <b>2</b> (model <b>3</b>), where the [CH<sub>3</sub>−S]<sup>1-</sup> group has been replaced by the neutral [NH<sub>3</sub>]<sup>0</sup> group [Neese and Solomon (1998) <i>J. Am. Chem. Soc.</i> <i>120</i>, 12829−12848]. Both models <b>2</b> and <b>3</b> contain a formally high-spin Fe<sup>3+</sup> ion (i.e., with empty minority spin orbitals). We found, however, a significant fraction (∼0.6 for <b>2</b>, ∼0.8 for <b>3</b>) of spin (equivalently charge) spread over two occupied (minority spin) orbitals. The quadrupole splitting value for <b>2</b> is found to be negative and matches quite well the experimental value. The computed quadrupole tensors are rhombic in the case of <b>2</b> and axial in the case of <b>3</b>. This difference originates directly from the presence of the thiolate ligand in <b>2</b>. A correlation between experimental isomer shifts for Fe<sup>3+</sup> mononuclear complexes with computed electron densities at the iron nucleus has been built and used to evaluate the isomer shift values for <b>2</b> and <b>3</b> (0.56 and 0.63 mm/s, respectively). A significant increase of isomer shift value is found upon going from a methylthiolate to a nitrogen ligand for the Fe<sup>3+</sup> ion, consistent with covalency effects due to the presence of the axial thiolate ligand. Considering that the isomer shift value for <b>3</b> is likely to be in the 0.61−0.65 mm/s range [Horner et al. (2002) <i>Eur. J. Inorg. Chem.</i>, 3278−3283], the isomer shift value for a high-spin η<sup>2</sup>-O<sub>2</sub> Fe<sup>3+</sup> complex with an axial thiolate group can be estimated to be in the 0.54−0.58 mm/s range. The occurrence of a side-on peroxo intermediate in SOR is discussed in relation to the recent data published for a side-on peroxo−Fe<sup>3+</sup> species in another biological system [Karlsson et al. (2003) <i>Science</i> <i>299</i>, 1039−1042].