Superoxide reductase (SOR) is a non-heme iron enzyme that reduces superoxide to peroxide at a diffusion-controlled rate. Sulfur K-edge X-ray absorption spectroscopy (XAS) is used to investigate the ground-state electronic structure of the resting high-spin and CN-bound low-spin Fe-III forms of the I Fe SOR from Pyrococcus furiosus. A computational model with constrained imidazole rings (necessary for reproducing spin states), H-bonding interaction to the thiolate (necessary for reproducing Fe-S bond covalency of the high-spin and low-spin forms), and H-bonding to the exchangeable axial ligand (necessary to reproduce the ground state of the low-spin form) was developed and then used to investigate the enzymatic reaction mechanism. Reaction of the resting ferrous site with superoxide and protonation leading to a high-spin Fe-III-OOH species and its subsequent protonation resulting in H2O2 release is calculated to be the most energetically favorable reaction pathway. Our results suggest that the thiolate acts as a covalent anionic ligand. Replacing the thiolate with a neutral noncovalent ligand makes protonation very endothermic and greatly raises the reduction potential. The covalent nature of the thiolate weakens the Fe-III bond to the proximal oxygen of this hydroperoxo species, which raises its pK(a), by an additional 5 log units relative to the pKa of a primarily anionic ligand, facilitating its protonation. A comparison with cytochrome P450 indicates that the stronger equatorial ligand field from the porphyrin results in a low-spin Fe-III-OOH species that would not be capable of efficient H2O2 release due to a spin-crossing barrier associated with formation of a high-spin 5C Fe-III product. Additionally, the presence of the dianionic porphyrin pi ring in cytochrome P450 allows O-O heterolysis, forming an Fe-IV-oxo porphyrin radical species, which is calculated to be extremely unfavorable for the non-heme SOR ligand environment. Finally, the 5C Fe-III site that results from the product release at the end of the O-2(-) reduction cycle is calculated to be capable of reacting with a second O-2, resulting in superoxide dismutase (SOD) activity. However, in contrast to FeSOD, the 5C Fe-III site of SOR, which is more positively charged, is calculated to have a high affinity for binding a sixth anionic ligand, which would inhibit its SOD activity.