Sulfur K-Edge X-ray Absorption Spectroscopy and Density Functional Theory Calculations on Superoxide Reductase:  Role of the Axial Thiolate in Reactivity

Abstract

Superoxide reductase (SOR) is a non-heme iron enzyme which 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 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 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 H 2 O 2 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 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 which would not be capable of efficient H 2 O 2 release due to a spin-crossing barrier associated with formation of a high spin 5C Fe III product. Additionally, the presence of the di-anionic porphyrin π ring in Cytochrome P450 allows O-O heterolysis forming a 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 which 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 positive is calculated to have a high affinity for the binding a 6 th anionic ligand which would inhibit its SOD activity.

Influence of the Nitrogen Donors on Nonheme Iron Models of Superoxide Reductase: High-Spin FeIII−OOR Complexes

“…These results support the strikingly similar findings that have been obtained for (hydro)peroxo-iron(III) adducts of SOR, where a weakening of the S Cys -Fe III bond correlates with a strengthening of the Fe-O bond, but has little or no affect on the O-O bond 6,23. In fact, there is a growing body of evidence to suggest that the nature of the S-Fe interaction in SOR plays a critical role in catalysis 3,23,74,75. Our findings and those on the enzyme are in agreement, and show that the traditional “push effect” invoked for heme enzymes, where the thiolate donor helps to weaken the O-O bond and thereby favor O-O bond cleavage, is not observed in either our low-spin or high-spin nonheme iron models, or in the native enzyme itself.…”

Substrate Binding Preferences and pKaDeterminations of a Nitrile Hydratase Model Complex: Variable Solvent Coordination to [(bmmp-TASN)Fe]OTf

“…An amine nitrogen occupies the position trans to the bridging hydroxide. The iron-ligand bond distances of [(LFe) 2 OH] + are within the ranges expected for high spin Fe(III) 2,11,40…”

Valence tautomerism in synthetic models of cytochrome P450

“…Thus, the aqueous environment stabilizes the ferric thiolate state only. In these heterogeneous systems, the thermodynamic reduction potentials (E 0 Fe III/II = 0.0 V vs. NHE) are shifted toward positive potentials relative to analogous synthetic complexes in organic medium (−0.3 V vs. NHE) consistent with the presence of hydrogen bonding to the axial thiolate ligand (32,33). We propose that similar hydrogen bonding interaction of the thiolate ligand with water shifts the equilibrium to the Fe III -thiolate state.…”