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Synthetic Models for the Cysteinate-Ligated Non-Heme Iron Enzyme Superoxide Reductase:  Observation and Structural Characterization by XAS of an FeIII−OOH Intermediate

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journal contribution
posted on 10.09.2002, 00:00 by Jason Shearer, Robert C. Scarrow, Julie A. Kovacs
Superoxide reductases (SORs) belong to a new class of metalloenzymes that degrade superoxide by reducing it to hydrogen peroxide. These enzymes contain a catalytic iron site that cycles between the FeII and FeIII states during catalysis. A key step in the reduction of superoxide has been suggested to involve HO2 binding to FeII, followed by innersphere electron transfer to afford an FeIII−OO(H) intermediate. In this paper, the mechanism of the superoxide-induced oxidation of a synthetic ferrous SOR model ([FeII(SMe2N4(tren))]+ (1)) to afford [FeIII(SMe2N4(tren)(solv))]2+ (2-solv) is reported. The XANES spectrum shows that 1 remains five-coordinate in methanolic solution. Upon reaction of 1 with KO2 in MeOH at −90 °C, an intermediate (3) is formed, which is characterized by a LMCT band centered at 452(2780) nm, and a low-spin state (S = 1/2), based on its axial EPR spectrum (g = 2.14; g = 1.97). Hydrogen peroxide is detected in this reaction, using both 1H NMR spectroscopy and a catalase assay. Intermediate 3 is photolabile, so, in lieu of a Raman spectrum, IR was used to obtain vibrational data for 3. At low temperatures, a νO-O Fermi doublet is observed in the IR at 788(2) and 781(2) cm-1, which collapses into a single peak at 784 cm-1 upon the addition of D2O. This vibrational peak diminishes in intensity over time and essentially disappears after 140 s. When 3 is generated using an 18O-labeled isotopic mixture of K18O2/K16O2 (23.28%), the vibration centered at 784 cm-1 shifts to 753 cm-1. This new vibrational peak is close to that predicted (740 cm-1) for a diatomic 18O−18O stretch. In addition, a νO-O vibrational peak assigned to free hydrogen peroxide is also observed (νO-O = 854 cm-1) throughout the course of the reaction between FeII1 and superoxide and is strongest after 100 s. XAS studies indicate that 3 possesses one sulfur scatterer at 2.33(2) Å and four nitrogen scatterers at 2.01(1) Å. Addition of two Fe−O shells, each containing one oxygen, one at 1.86(3) Å and one at 2.78(3) Å, improved the EXAFS fits, suggesting that 3 is an end-on peroxo or hydroperoxo complex, [FeIII(SMe2N4(tren))(OO(H))]+. Upon warming above −50 °C, 3 is converted to 2-MeOH. In methanol and methanol:THF (THF = tetrahydrofuran) solvent mixtures, 2-MeOH is characterized by a LMCT band at λmax = 511(1765) nm, an intermediate spin-state (S = 3/2), and, on the basis of EXAFS, a relatively short Fe−O bond (assigned to a coordinated methanol or methoxide) at 1.94(10) Å. Kinetic measurements in 9:1 THF:MeOH at 25 °C indicate that 3 is formed near the diffusion limit upon addition of HO2 to 1 and converts to 2-MeOH at a rate of 65(1) s-1, which is consistent with kinetic studies involving superoxide oxidation of the SOR iron site.

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