A Functional Model for the Cysteinate–Ligated Non–Heme Iron Enzyme Superoxide Reductase (SOR)

Superoxide reductases (SORs) are cysteinate-ligated non–hemeiron enzymes¹ that reduce superoxide (O₂⁻) to H₂O₂ in anaerobicmicrobes.² The cysteinate of SOR is trans to the O₂⁻ binding site, and is proposed to play an important role in promoting the catalytic reaction. Herein, we report a rare example of a functional metalloenzyme active site model, that reduces O₂⁻ via atrans thiolate–ligated Fe(III)–peroxo intermediate. The trans thiolate is shown to lower the redox potential, change the spin-state, and dramatically weaken the Fe-O bond, favoring O₂⁻ reduction and H₂O₂ release.

Superoxide is a toxic byproduct of dioxygen chemistry that has been linked to a number of disease states.³ The proposed SOR mechanism involves the oxidative addition of O₂⁻ to the open site of the square pyramidal Fe^IIN₄ ^HisS^Cys active site^2c to afford a trans S^cys-ligated Fe^III-peroxo intermediate.^2d,e This intermediate displays an intense S-to-Fe(III) charge transfer band at ~600(~3500) nm, but has yet to be characterized by vibrational spectroscopy. Iron-peroxo species are extremely difficult to characterize since they are thermally unstable, and photolabile. Vibrational data have been reported for mutant SOR (E47A) peroxos generated via the addition of H₂O₂.2b,f,g Whether these are identical to the catalytic SOR intermediate remains to be determined. Although a few well-characterized synthetic nitrogen-ligated iron–peroxos have been reported,^4a,c there is a paucity of thiolate-ligated analogues.^4b Since a thiolate is likely to influence the correlation between peroxide binding mode, vibrational parameters, and spin–state, synthetic thiolate–ligated peroxos are needed to provide benchmark parameters. Prior to the work reported herein, cis thiolate-ligated [Fe^III(S^Me2N₄(tren))(OOH)]⁺ (1),^4b was the only reported example of a synthetic thiolate–ligated Fe^III-peroxo.

In situ deprotection and deprotonation of the new macrocyclic ligand cyclam-PrS–Ac·4HCl, afforded [Fe^II(cyclam–PrS)](BPh₄) (2) upon the addition of FeCl₂ and NaBPh₄. Single crystals were grown from pentane/THF at −30 °C. As shown in the ORTEP (Figure 1), the Fe²⁺ ion of 2 is ligated by three secondary amines, one tertiary amine, and a tethered apical thiolate in a square pyramidal geometry (τ= 0.13)⁵ resembling that of SOR. A related tertiary amine cyclam complex [Fe^II(Me₃-cyclam-EtS)]⁺ (3) was recently reported⁶ that reacts with H₂O₂ to afford an Fe(IV)=O.⁷ Like the SOR active site, 2 is high spin (S= 2; μ_eff=5.03 BM (MeCN); 4.91 BM (solid)). The Fe–S bond length in 2 (2.286(1) Å) falls in the usual range for synthetic Fe(II)–thiolates,^6,8 but is slightly shorter than that of SOR (Fe–S= 2.4 Å), the cysteinate sulfur of which is H-bonded to the protein backbone.^2a,c

Figure 1.

ORTEP of [Fe^II(cyclam–PrS)]⁺ (2). Selected bond lengths (Å): Fe–S(1), 2.286(1); Fe–N(1), 2.181(4); Fe–N(2,3,4)_avg, 2.16(2).

Thiolate–ligated 2 reacts rapidly with O₂ ^−• (18-crown-6-K⁺ salt) in CH₂Cl₂ at −78 °C to afford a metastable burgundy intermediate, as soon as a proton donor (MeOH; 82 equiv) is added. This intermediate is high-spin (g= 7.72, 5.40, 4.15), and displays an absorption band at λ_max= 530(1350) nm. Resonance Raman shows ν_O–O, ν_Fe–O, and ν_Fe–S stretches at 891 cm⁻¹ (Fermi doublet), 419 cm⁻¹, and 352 cm⁻¹, respectively (Figure 2). K¹⁸O₂ (50% enriched; ICON) causes the ν_O–O and ν_Fe–O to shift to 856 cm⁻¹ and 400 cm⁻¹, respectively, and addition of D⁺ (ie, MeOD) causes the Fermi doublet to collapse. These data are consistent with the formation of an Fe–hydroperoxo species, [Fe^III(cyclam– PrS)(OOH)]⁺ (4), via the proton-dependent oxidative addition of superoxide to 2. No reaction occurs in the absence of a proton donor, and O₂⁻ does not convert to H₂O₂ in the absence of 2, under the conditions examined (−78 °C, 82 equiv MeOH). Intermediate 4 represents the first example of a synthetic trans thiolate–ligated Fe^III–peroxo SOR intermediate analogue, the characterization of which provides important benchmark parameters.

Figure 2.

rRaman spectra of 4 generated from ¹⁶O₂ ⁻ (blue), ¹⁸O₂ ⁻ (red), and “decayed” product (dashed black) (571 nm excitation @ 183 K in THF/MeOH (upper panel); @ 77K in CH₂Cl₂/THF/MeOH (lower panel).

The ν_Fe–O stretch of 4 is significantly lower than all other reported synthetic iron peroxides (range: 450–639 cm⁻¹),^4a but compares well with that of the only reported SOR peroxo (438 cm⁻¹).2a,e The ν_O–O stretch (891 cm⁻¹) is unusually high (reported range: 820–860 cm⁻¹).^4a The DFT optimized structure of 4 minimizes with Fe-S and Fe-O distances of 2.36 Å and 1.95 Å, respectively, and a protonated peroxo O-O distance of 1.44 Å. This Fe-O(peroxo) distance is significantly longer than the few reported Fe–(η¹-OOH) structures(1.76–1.86 Å)^4a reflecting the trans influence of the thiolate sulfur. The calculated ν_Fe–S (345 cm⁻¹), ν_Fe–O (400 cm⁻¹) and ν_O–O (933 cm⁻¹) stretches are in reasonable agreement with the experimental data. When the thiolate is replaced with an amine, or alkoxide, trans to the peroxo,⁹ then the calculated ν_Fe–O (495 cm⁻¹ and 420 cm⁻¹, respectively) is considerably higher. These vibrational data, along with the calculated force constant (k_Fe–O = 1.20 mdynes/cm² for 4 vs reported range= 2.2-2.1 mdynes/cm²),^4a indicate that the Fe– O(peroxide) bond is significantly weakened upon the introduction of a trans thiolate into the coordination sphere.

Addition of HOAc to metastable 4 at −78 °C releases H₂O₂ (as detected using an amplex red assay), and cleanly affords a new aqua blue species λ_max = 604(1350) nm (Figure 3). When this reaction is monitored by EPR, the high–spin signal associated with 4 is replaced with a new low–spin signal at g= 2.37, 2.30, 1.89. The ν_O–O and ν_Fe–O stretches disappear in the rRaman spectrum, and new stretches are observed at 339, 409, and 421 cm⁻¹. Although this aqua blue species proved too unstable to isolate, it was unambiguously identified by ESI-mass spectrometry as acetate–bound [Fe^III(cyclam–PrS)(OAc)]⁺ (5), a model for Glu–bound SOR.

Figure 3.

Conversion of peroxo–bound 4 (0.5 mM in CH₂Cl₂) to acetate–bound [Fe^III(cyclam–PrS)(OAc)]⁺ (5) via the addition of HOAc (0.1 equivaliquots every two minutes) at −78 °C.

Addition of a sacrificial reductant (Cp₂Co) to 5 at low temperatures (−78 °C) regenerates 2, which then reacts with a second equiv of O₂ ^−• to re-afford peroxo 4. Addition of a second equiv of HOAc releases H₂O₂ (Figure 4), thereby mimicking the proposed SOR catalytic cycle involving glutamic acid,2d,e,i and demonstrating that reduction of O₂ ^−• by 2 is catalytic. Thus far, five turnovers have been achieved.

Figure 4.

The catalytic cycle involving [Fe^II(cyclam–PrS)]⁺ (2) induced superoxide (O₂⁻) reduction

The thiolate ligand and its trans positioning relative to the substrate appear to contribute significantly to the function of our biomimetic catalyst. First, the pendent thiolate arm of 2 causes the redox potential to shift anodically by +480 mV relative to [Fe^II(cyclam)(MeCN)₂] (from +700 mV to +220 mV vs SCE), making it better suited to promote superoxide reduction. Second, the trans thiolate changes the spin–state from S= 1/2^4b to S= 5/2: the majority of nitrogen-ligated Fe(III)-OOH’s are S= 1/2,^4a as is cis-thiolate ligated 1.^4b Third, the thiolate dramatically shifts the ν_Fe–O stretch and decreases the k_Fe–O force constant well-below all other reported iron peroxides.^4a Peroxo 4 partially converts to methoxide-bound [Fe^III(cyclam–PrS)(OMe)]⁺ (6; g= 2.34, 2.26, 1.95; ν_Fe–S= 357 cm⁻¹) within minutes at −78 °C, whereas cisligated peroxo 1 takes hours (t_1/2= 63.9 hrs) to convert to [Fe^III(S^Me2N₄(tren))(OMe)]⁺ under the same conditions. Methoxide–bound 6 was identified via its independent synthesis involving Cp₂Fe⁺ oxidation of 2 in MeOH, in the presence of ⁱPr₂EtN.

In conclusion, the data described herein indicates that like the enzyme, SOR intermediate–analogue 4 is better suited to promote Fe–O, as opposed to O–O, bond cleavage. This is in contrast to P450 and its analogue 3. Kinetics studies, and studies aimed at determining the pK_a of the proximal and distal peroxo oxygens of 4 are currently underway.

Supplementary Material

si20060708_060. Supporting Information Available.

Detailed ligand syntheses and description of UV/vis monitored catalytic turnover, ¹H NMR, ESI mass spec (ligand, 5), EPR, 1/χ vs. T plot and CV of 2, UV/vis of 4, Amplex Red assay, and X-ray tables. This information is available free of charge via the Internet at http://pubs.act.org.