Mössbauer Characterization of an Unusual High-Spin Side-On Peroxo−Fe³⁺ Species in the Active Site of Superoxide Reductase from Desulfoarculus baarsii. Density Functional Calculations on Related Models^†

Superoxide reductase (SOR) is an Fe protein that catalyzes the reduction of superoxide to give H₂O₂. Recently, the mutation of the Glu47 residue into alanine (E47A) in the active site of SOR from Desulfoarculus baarsii has allowed the stabilization of an iron−peroxo species when quickly reacted with H₂O₂ [Mathé et al. (2002) J. Am. Chem. Soc. 124, 4966−4967]. To further investigate this non-heme peroxo−iron species, we have carried out a Mössbauer study of the ⁵⁷Fe-enriched E47A SOR from D. baarsii reacted quickly with H₂O₂. 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−Fe³⁺ complex. This is one of the first examples of such a species in a biological system for which Mössbauer parameters are now available: δ_/Fe = 0.54 (1) mm/s, ΔE_Q = −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 2) issued from the oxidized iron center in SOR from Pyrococcus furiosus, for which structural data are available in the literature [Yeh et al. (2000) Biochemistry 39, 2499−2508]. For comparison, similar calculations have been carried out on a model derived from 2 (model 3), where the [CH₃−S]^1- group has been replaced by the neutral [NH₃]⁰ group [Neese and Solomon (1998) J. Am. Chem. Soc. 120, 12829−12848]. Both models 2 and 3 contain a formally high-spin Fe³⁺ ion (i.e., with empty minority spin orbitals). We found, however, a significant fraction (∼0.6 for 2, ∼0.8 for 3) of spin (equivalently charge) spread over two occupied (minority spin) orbitals. The quadrupole splitting value for 2 is found to be negative and matches quite well the experimental value. The computed quadrupole tensors are rhombic in the case of 2 and axial in the case of 3. This difference originates directly from the presence of the thiolate ligand in 2. A correlation between experimental isomer shifts for Fe³⁺ mononuclear complexes with computed electron densities at the iron nucleus has been built and used to evaluate the isomer shift values for 2 and 3 (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 Fe³⁺ ion, consistent with covalency effects due to the presence of the axial thiolate ligand. Considering that the isomer shift value for 3 is likely to be in the 0.61−0.65 mm/s range [Horner et al. (2002) Eur. J. Inorg. Chem., 3278−3283], the isomer shift value for a high-spin η²-O₂ Fe³⁺ 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−Fe³⁺ species in another biological system [Karlsson et al. (2003) Science 299, 1039−1042].