Like many non-heme iron enzymes, reduced iron superoxide dismutase (Fe2+SOD) reacts with nitric oxide (NO) to yield an {Fe-NO}(7) system. Electron paramagnetic resonance (EPR) data obtained for this Fe-NO adduct of FeSOD (NO-FeSOD) exhibit two rhombic S = 3/2 signals of comparable population; E/D = 0.128 (42%) and 0.154 (58%). While similar results were previously reported for NO-FeSOD [Niederhoffer, E. C.; Fee, J. A.; Papaefthymiou, V.; Munck, E. Magnetic Resonance Studies Involving Iron Superoxide Dismutase from Escherichia coli. Isotope and Nuclear Chemistry Division Annual Report, Los Alamos National Laboratory: Los Alamos, NM, 1987], detailed geometric and electronic structure descriptions of these {Fe-NO}(7) systems had not yet been developed. Therefore, in addition to EPR spectroscopy, we have used electronic absorption, magnetic circular dichroism (MCD), variable-temperature, variable-field MCD, and resonance Raman spectroscopies to determine ground-state spin Hamiltonian parameters, electronic transition energies, oscillator strengths, and transition polarizations for NO-FeSOD. These spectroscopic parameters have been used in conjunction with density functional theory (DFT) and semiempirical INDO/S-Cl calculations to generate an experimentally calibrated active site model for NO-FeSOD. Our studies indicate that NO binds to the active site of Fe2+SOD to form a six-coordinate {Fe-NO}(7) system with an Fe-N-O angle of similar to145degrees. DFT computations performed on this model of NO-FeSOD reveal that the NO ligand is formally reduced by the ferrous center to yield NO- and an Fe3+ center that are strongly antiferromagnetically coupled. DFT calculations reveal that NO binding to Fe2+SOD also lowers the pK of the coordinated water ligand by at least 3.3 pH units, suggesting that this process is associated with increased acidity and probable ionization of the axial solvent ligand. To explore the origin of the two {Fe-NO}(7) systems observed by EPR spectroscopy, additional calculations have been performed on slightly perturbed NO-FeSOD models. Significantly, semiempirical INDO/S-Cl computations reveal that the rhombicity of NO-FeSOD is altered by changes in the Fe-N-O angle or rotation about the Fe-N(O) bond, suggesting that the two species observed by EPR spectroscopy merely differ slightly with respect to the orientation of the NO ligand. Indeed, our EPR data obtained on NO-FeSOD variants indicate that the relative population of the S = 3/2 signals can be altered by perturbations in the second sphere of the protein active site. These results provide compelling evidence that the second coordination sphere is able to modulate the geometric and electronic structures of NO-FeSOD.