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 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 FeIII 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 FeIII-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 FeIII bond to the proximal oxygen of this hydroperoxo species, which raises its pKa 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 FeIII-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 FeIII product. Additionally, the presence of the dianionic porphyrin pi ring in cytochrome P450 allows O-O heterolysis, forming an FeIV-oxo porphyrin radical species, which is calculated to be extremely unfavorable for the non-heme SOR ligand environment. Finally, the 5C FeIII site that results from the product release at the end of the O2- reduction cycle is calculated to be capable of reacting with a second O2-, resulting in superoxide dismutase (SOD) activity. However, in contrast to FeSOD, the 5C FeIII 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.

Fig. 1

Active site of SOR from P. furiosus at 1.7 Å resolution (pdb:1DQI). Residues Ile113 and Asn112 are in the vicinity of the active site and proposed to H-bond to the Cys111 thiolate.

Figure 2

S K-edge XAS of superoxide reductase in its reduced (black), resting oxidized (blue) and CN⁻ bound oxidized (red) forms. The 2^nd derivatives of the data are shown in dotted lines. The Fe^II pre-edge feature is indicated by the black arrow.

Figure 3

The crystal structure of the active site of resting oxidized SOR (A), the unconstrained model (B), H-bonded only model (C), α carbon constrained model (D), H-bonded and α-carbon constrained model (E) and overlay between the optimized model E (in blue) and X-tal structure (atom colored) from 1DQI (F). The constrained atoms are circled in the respective structures. Bond lengths of the protein crystal structure are given in Å and are color coded as Fe-S, Fe-N and Fe-O.

Figure 4

The MO diagrams of the resting six-coordinate high-spin Fe^III active site without, (left) and with (right) H-bonding interactions. The S_3p co-efficients are indicated in text. Only the β contours are shown. The dyz and dxy contours of the model without H-bonding are not shown to avoid crowding.

Figure 5

β contours of the E (left) and E +H₂O (right) models of the linear Fe-CN bound LS forms. The two α unoccupied contours are not shown for clarity. The designations of the orbitals are written next to them and the %Fe_3d, CN_2p, S_3p are given as well.

Figure 6

Reaction mechanism of SOR with H-bonding to the CysS⁻ included. The numbers represent ΔE of individual steps (ΔE calculated as E_products - E_reactants) and were calculated using a PCM model (ε = 4.0) and are given in Kcal/mol. Solvation energy of 260 Kcal/mole was used for a proton (H⁺_solv) in this scheme. The computational model used for this calculation is described in Figure 3E. The H-bonds to thiolate and the axial ligand are not shown for clarity. ± In the E47A mutant where the Glu is replaced by Ala, the axial ligand has been shown to be OH⁻ using rR. The binding of a OH⁻ from water is calculated to be exothermic by −17 Kcal/mole.

Figure 7

Comparison of energies of superoxide oxidation by SOR (upper panel) and SOD (lower panel) active sites (energies (ΔE) were calculated using a PCM model (ε = 4.0) and are given in Kcal/mol); Solvation energy of 260 Kcal/mol was used for an H⁺.