| Sumario: | 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 forms of the 1Fe 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—OOH species and its subsequent protonation resulting in HO 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 bond to the proximal oxygen of this hydroperoxo species, which raises its pK by an additional 5 log units relative to the pK 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—OOH species that would not be capable of efficient HO release due to a spin-crossing barrier associated with formation of a high-spin 5C Fe" product. Additionally, the presence of the dianionic porphyrin π ring in cytochrome P450 allows O—O heterolysis, forming an Fe—oxo porphyrin radical species, which is calculated to be extremely unfavorable for the non-heme SOR ligand environment. Finally, the 5C Fe site that results from the product release at the end of the O—reduction cycle is calculated to be capable of reacting with a second O—, resulting in superoxide dismutase (SOD) activity. However, in contrast to FeSOD, the SC Fe 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.
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