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

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 Fe(II) and Fe(III) states during catalysis. A key step in the reduction of superoxide has been suggested to involve HO(2) binding to Fe(II), followed by innersphere electron transfer to afford an Fe(III)-OO(H) intermediate. In this paper, the mechanism of the superoxide-induced oxidation of a synthetic ferrous SOR model ([Fe(II)(S(Me2)N(4)(tren))](+) (1)) to afford [Fe(III)(S(Me2)N(4)(tren)(solv))](2+) (2-solv) is reported. The XANES spectrum shows that 1 remains five-coordinate in methanolic solution. Upon reaction of 1 with KO(2) in MeOH at -90 degrees 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(perpendicular) = 2.14; g(parallel) = 1.97). Hydrogen peroxide is detected in this reaction, using both (1)H 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 nu(O-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 D(2)O. This vibrational peak diminishes in intensity over time and essentially disappears after 140 s. When 3 is generated using an (18)O-labeled isotopic mixture of K(18)O(2)/K(16)O(2) (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 (18)O-(18)O stretch. In addition, a nu(O-O) vibrational peak assigned to free hydrogen peroxide is also observed (nu(O-O) = 854 cm(-1)) throughout the course of the reaction between Fe(II)-1 and superoxide and is strongest after 100 s. XAS studies indicate that 3 possesses one sulfur scatterer at 2.33(2) A and four nitrogen scatterers at 2.01(1) A. Addition of two Fe-O shells, each containing one oxygen, one at 1.86(3) A and one at 2.78(3) A, improved the EXAFS fits, suggesting that 3 is an end-on peroxo or hydroperoxo complex, [Fe(III)(S(Me2)N(4)(tren))(OO(H))](+). Upon warming above -50 degrees 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 lambda(max) = 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) A. Kinetic measurements in 9:1 THF:MeOH at 25 degrees C indicate that 3 is formed near the diffusion limit upon addition of HO(2) 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.