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.

Mechanistic pathways of metalloenzymes are controlled by the metal ion's electronic and magnetic properties, which are tuned by the coordinated ligands. The functional advantage gained by incorporating cysteinates into the active site of non-heme iron enzymes such as superoxide reductase (SOR) is not entirely understood. Herein we compare the structural and redox properties of a series of structurally-related thiolate, alkoxide, and amine-ligated Fe(II) complexes in order to determine how the thiolate influences properties critical to function. Thiolates are shown to reduce metal ion Lewis acidity relative to alkoxides and amines, and have a strong trans influence thereby helping to maintain an open coordination site. Comparison of the redox potentials of the structurally analogous compounds described herein indicates that alkoxide ligands favor the higher-valent Fe 3+ oxidation state, amine ligands favor the reduced Fe 2+ oxidation state, and thiolates fall somewhere in between. These properties provide a functional advantange for substrate reducing enzymes in that they provide a site at the metal ion for substrate to bind, and a moderate potential that facilitates both substrate reduction, and regeneration of the catalytically active reduced state. Redox potentials for structurally-related Co(II) complexes are shown to be cathodically-shifted relative to their Fe(II) analogues, making them ineffective reducing agents for substrates such as superoxide.