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.

Iron K-edge X-ray spectroscopy (XANES and EXAFS) was used to study iron coordination in frozen solutions of soybean lipoxygenase-1 (SLO). The intensity of the 1s-->3d pre-edge transition of native iron(II) lipoxygenase is greater than what was found for six-coordinate high-spin iron(II) model complexes, but comparable to that of a five-coordinate model. This and a relatively short average bond length determined by EXAFS (2.13 A) indicate that the native lipoxygenase in our frozen samples is five-coordinate, excluding possible bonds longer than 2.5 A. The coordination of the iron(II) in native lipoxygenase changes when methanol (as low as 0.1%) or glycerol (20%) is added to the buffer prior to freezing. The addition of methanol diminishes the pre-edge transition and increases EXAFS-derived bond lengths by 0.04 A, indicating a change to six-coordination. The small pre-edge feature in active iron(III) lipoxygenase suggests six-coordination. EXAFS indicates a short, 1.88 A Fe-O bond, which, given other spectroscopic and crystallographic evidence, is assigned to coordinated hydroxide. The average of the remaining bond lengths is 2.11 A. The iron coordination in iron(III) lipoxygenase is less affected by the presence of alcohols than is the site in the iron(II) enzyme. Bond valence sums indicate that the bond lengths for lipoxygenase derived from our EXAFS analyses are comparable to those of crystallographically characterized model complexes. The flexibility of the coordination number in SLON (native SLO) and the presence of an [FeIIIOH]2+ unit in SLOA (active SLO) are of possible mechanistic importance.

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Nitrile hydratase (NHase) is an iron-containing metalloenzyme that converts nitriles to amides. The mechanism by which this biochemical reaction occurs is unknown. One mechanism that has been proposed involves nucleophilic attack of an Fe-bound nitrile by water (or hydroxide). Reported herein is a five-coordinate model compound ([Fe III (S 2 Me2 N 3 (Et,Pr))] + ) containing Fe(III) in an environment resembling that of NHase, which reversibly binds a variety of nitriles, alcohols, amines, and thiocyanate. XAS shows that five-coordinate [Fe III (S 2 Me2 N 3 (Et,Pr))] + reacts with both methanol and acetonitrile to afford a six-coordinate solvent-bound complex.Competitive binding studies demonstrate that MeCN preferentially binds over ROH, suggesting that nitriles would be capable of displacing the H 2 O coordinated to the iron site of NHase. Thermodynamic parameters were determined for acetonitrile (ΔH = −6.2(±0.2) kcal/mol, ΔS = −29.4(±0.8) eu), benzonitrile (−4.2(±0.6) kcal/mol, ΔS = −18(±3) eu), and pyridine (ΔH = −8(±1) kcal/mol, ΔS = −41(±6) eu) binding to [Fe III (S 2 Me2 N 3 (Et,Pr))] + using variable-temperature electronic absorption spectroscopy. Ligand exchange kinetics were examined for acetonitrile, isopropylnitrile, benzonitrile, and 4-tert-butylpyridine using 13 C NMR line-broadening analysis, at a variety of temperatures. Activation parameters for ligand exchange were determined to be ΔH ‡ = 7.1(±0.8) kcal/mol, ΔS ‡ = −10(±1) eu (acetonitrile), ΔH ‡ = 5.4(±0.6) kcal/mol, ΔS ‡ = −17(±2) eu (iso-propionitrile), ΔH ‡ = 4.9(±0.8) kcal/mol, ΔS ‡ = −20(±3) eu (benzonitrile), and ΔH ‡ = 4.7(±1.4) kcal/mol ΔS ‡ = −18(±2) eu (4-tert-butylpyridine). The thermodynamic parameters for pyridine binding to a related complex, [Fe III (S 2 Me2 N 3 (Pr,Pr))] + (ΔH = −5.9(±0.8) kcal/mol, ΔS = −24(±3) eu), are also reported, as well as kinetic parameters for 4-tert-butylpyridine exchange (ΔH ‡ = 3.1(±0.8) kcal/mol, ΔS ‡ )−25(±3) eu). These data show for the first time that, when it is contained in a ligand environment similar to that of NHase, Fe(III) is capable of forming a stable complex with nitriles. Also, the rates of ligand exchange demonstrate that low-spin Fe(III) in this ligand environment is more labile than expected. Furthermore, comparison of (Tables S-6-S-9), van't Hoff plots for ligand binding to 1 and 2, variable-temperature electronic absorption spectra (Figures S-3-S-7), and EPR spectra for "substrate"-bound 2 , and crystallographic data for 2-NCS (Tables S-10-S-14) (PDF). This material is available free of charge via the Internet at http://pubs.acs.org. HHS Public AccessAuthor manuscript (S 2 Me2 N 3 (Pr,Pr))] + demonstrates how minor distortions induced by ligand constraints can dramatically alter the reactivity of a metal complex.Performing reactions under environmentally friendly conditions has recently become a desirable goal in the search for new catalysts. 1 Enzymes are often viewed as ideal in this respect because of their ability to perform chemical transformations und...