A spectroscopic study of the Fe(III)-EDTA-peroxide complex using electron paramagnetic resonance (EPR), low-temperature absorption (LT-ABS), variable-temperature-variable-field (VTVH) magnetic circular dichroism (MCD), and resonance Raman (rR) spectroscopies is reported. Density functional (DFT) and INDO/S-CI molecular orbital (MO) calculations are used to derive an experimentally calibrated bonding scheme. The molecule is described as a B-coordinate, mononuclear, high-spin ferric-peroxide complex with a side-on eta(2)-FeO2 arrangement. EPR spectroscopy shows that the zero-field splitting (ZFS) is negative with D = -1.0 +/- 0.25 cm-l and E/D = 0.21. MCD and LT-ABS spectroscopies lead to the identification of at least six excited states below 35 000 cm(-1). The lowest two are assigned as ligand field sextet-quartet transitions, and the remaining transitions have peroxide to iron ligand-to-metal charge transfer (LMCT) character. The polarization of the LMCT bands in the principal axis system of the D-tensor are derived by VTVH-MCD spectroscopy. The Fe-O and O-O stretching vibrations are observed at 459 and 816 cm(-1), respectively. rR excitation profiles are simulated to obtain excited-state distortions. The weak CT bands in the visible region mainly enhance the O-O stretch, whereas the UV band dominantly enhances the metal-ligand stretch and only weakly the O-O stretch. A normal coordinate analysis shows little mechanical coupling between the two stretches and gives force constants of 3.02 mdyn/Angstrom for the O-O stretch and 1.56 mdyn/Angstrom for the Fe-O stretch. MO calculations show that the bonding is dominated by a strong, covalent a-bond formed between the Fe-d(xy) and peroxide-pi*(sigma) orbitals with little contributions from pi- or sigma-symmetry iron-peroxide interactions. A back-bonding interaction between Fe-3d and the peroxide-o*-orbital that contributes to the weak O-O bond in oxyhemocyanin is not present in the Fe(III)-EDTA-peroxide complex. A calculation of the D-tensor from the INDO/S-CIS(D) wave functions gives good agreement with the experimental values and defines the orientation of the D-tensor in the molecular coordinate system. This allows the polarizations obtained from VTVH-MCD spectroscopy to be related to a molecular axis system and assists in band assignments. The negative D-value and the observed excited distortions are explained by the extensive anisotropic covalency of the FeO2 sigma-bond. Possible contributions of the electronic structure to the reactivity of non-heme iron enzymes are considered, and protonation of the peroxide ligand is proposed to lead to highly reactive species.