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

Abstract

Superoxide reductases (SORs) are cysteinate-ligated non-hemeiron enzymes 1 that reduce superoxide (O 2 −) to H 2 O 2 in anaerobicmicrobes. 2 The cysteinate of SOR is trans to the O 2 − 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 O 2 − 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 O 2 − reduction and H 2 O 2 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 O 2 − to the open site of the square pyramidal Fe II N 4 His S 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 H 2 O 2. 2b,f,g Whether these are identical to the catalytic SOR intermediate remains to be determined. Although a few well-characterized synthetic nitrogenligated 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 Me2 N 4 (tren))(OOH)] + (1), 4b was the only reported example of a synthetic thiolateligated Fe III-peroxo. In situ deprotection and deprotonation of the new macrocyclic ligand cyclam-PrS-Ac•4HCl, afforded [Fe II (cyclam-PrS)](BPh 4) (2) upon the addition of FeCl 2 and NaBPh 4. Single crystals were grown from pentane/THF at −30 °C. As shown in the ORTEP (Figure 1), the Fe 2+ 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 (Me 3-cyclam-EtS)] + (3) was recently reported 6 that reacts with H 2 O 2 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 Thiolate-ligated 2 reacts rapidly with O 2 −• (18-crown-6-K + salt) in CH 2 Cl 2 at −78 °C to afford a metastable burgundy intermediate, as soon as a proton donor (MeOH; 82 equiv) is added.

Preparation and Characterization of a Microcrystalline Non‐Heme FeIII(OOH) Complex Powder: EPR Reinvestigation of FeIII(OOH) Complexes—Improvement of the Perturbation Equations for the g Tensor of Low‐Spin FeIII

“…[11][12][13] Herein, we report the isolation of LS [Fe III 6 ) 2 , which is the first example of a microcrystalline powder for such a thermally unstable intermediate. The temperature-dependent magnetic susceptibility and the EPR properties of this complex were studied and are reported herein.…”

“…In general, these intermediates are obtained by the addition of H 2 O 2 to Fe II complexes prepared with hexa-, penta-, and tetradendate aminopyridine ligands (see Scheme 1) [3,4] and have been characterized by several techniques, in particular, EPR and resonance Raman spectroscopy. Apart from two cases, [5,6] Fe III A C H T U N G T R E N N U N G (OOH) species exhibit LS Fe III EPR features and their vibrational characteristics indicate an end-on coordination mode for the hydroperoxo ligand. [7,8] These complexes have always been prepared and studied in solution and no crystal structures have been reported to date.…”

Sulfur K-Edge X-ray Absorption Spectroscopy and Density Functional Theory Calculations on Superoxide Reductase: Role of the Axial Thiolate in Reactivity

“…The decay of this intermediate involved a diffusion controlled protonation from its pH dependence and a deuterium isotope effect of 2. Lys 14 has been identified as a participating 2 nd sphere residue (mutation of this residue decreases the turnover rate by a factor of [20][21][22][23][24][25][26][27][28][29][30] and has been proposed to assist superoxide binding to the active site. 19,20 Though no intermediate has been trapped with the physiological substrate superoxide, Olivier et.…”

“…A subsequent protonation driven H 2 O 2 release from this low-spin Fe III -OOH was proposed, though the energetic feasibility of such steps were not evaluated. 17 24 The trans effect of the thiolate has also been proposed to affect NO binding to this active site. 27 The thiolate may also play a major role in controlling the reduction potential of the active site.…”

Photooxidation of metal-bound thiolates: reactivity of sulfur containing peroxidic intermediates

“…Kovacs et al have published some elegant studies on how modified (i.e. oxidized) Co-bound cysteine affects the function of Nitrile hydratase (17)(18)(19). Unanswered questions in all of the work on oxidized Co-bound cysteine in Nitrile Hydratase are -How does nature control the oxidation of the cysteine such that one cysteine ligand is oxidized to a sulfenate while another one is oxidized to a sulfinate?…”