Nitric oxide (NO) is frequently used to probe the substrate–binding site of “spectroscopically silent” non-heme Fe2+ sites of metalloenzymes, such as superoxide reductase (SOR). Herein we use NO to probe the superoxide binding site of our thiolate–ligated biomimetic SOR model [FeII(SMe2N4(tren))]+ (1). Like NO–bound trans cysteinate-ligated SOR (SOR-NO), the rhombic S= 3/2 EPR signal of NO–bound cis thiolate-ligated [Fe(SMe2N4(tren)(NO)]+ (2; g = 4.44, 3.54, 1.97), isotopically sensitive νNO(ν15NO) stretching frequency (1685(1640) cm−1), and 0.05 Å decrease in Fe–S bond length are shown to be consistent with the oxidative addition of NO to Fe(II) to afford an Fe(III)–NO− {FeNO}7 species containing high–spin (S= 5/2) Fe(III) antiferromagnetically coupled to NO− (S= 1). The cis versus trans positioning of the thiolate does not appear to influence these properties. Although it has yet to be crystallographically characterized, SOR-NO is presumed to possess a bent Fe-NO similar to that of 2 (Fe–N–O= 151.7(4)°). The N–O bond is shown to be more activated in 2 relative to N– and O–ligated {FeNO}7 complexes, and this is attributed to the electron donating properties of the thiolate ligand. Hydrogen bonding to the cysteinate sulfur attenuates N–O bond activation in SOR as shown by its higher νNO frequency (1721 cm−1). In contrast, the νO–O frequency of SOR peroxo intermediate and its analogues is not affected by H-bonds to the cysteinate sulfur, or other factors influencing the Fe–SR bond strength. These only influence the νFe–O frequency. Reactions between 1 and NO2− are shown to result in the proton–dependent heterolytic cleavage of an N–O bond. The mechanism of this reaction is proposed to involve both FeII–NO2− and {FeNO}6 intermediates similar to those implicated in the mechanism of NiR–promoted NO2− reduction.

Kinetic studies aimed at determining the most probable mechanism for the proton-dependent [FeII(SMe2N4(tren))]+ (1) promoted reduction of superoxide via a thiolate-ligated hydroperoxo intermediate [FeIII(SMe2N4(tren))(OOH)]+ (2) are described. Rate laws are derived for three proposed mechanisms, and it is shown that they should conceivably be distinguishable by kinetics. For weak proton donors with pKa(HA) >pKa(HO2) rates are shown to correlate with proton donor pKa, and display first-order dependence on iron, and half-order dependence on superoxide and proton donor HA. Proton donors acidic enough to convert O2− to HO2 (in tetrahydrofuran, THF), that is, those with pKa(HA) < pKa(HO2), are shown to display first-order dependence on both superoxide and iron, and rates which are independent of proton donor concentration. Relative pKa values were determined in THF by measuring equilibrium ion pair acidity constants using established methods. Rates of hydroperoxo 2 formation displays no apparent deuterium isotope effect, and bases, such as methoxide, are shown to inhibit the formation of 2. Rate constants for p-substituted phenols are shown to correlate linearly with the Hammett substituent constants σ−. Activation parameters ((ΔH‡ = 2.8 kcal/mol, ΔS‡ = −31 eu) are shown to be consistent with a low-barrier associative mechanism that does not involve extensive bond cleavage. Together, these data are shown to be most consistent with a mechanism involving the addition of HO2 to 1 with concomitant oxidation of the metal ion, and reduction of superoxide (an “oxidative addition” of sorts), in the rate-determining step. Activation parameters for MeOH- (ΔH‡ = 13.2 kcal/mol and ΔS‡ = −24.3 eu), and acetic acid- (ΔH‡ = 8.3 kcal/mol and ΔS‡ = −34 eu) promoted release of H2O2 to afford solvent-bound [FeIII(SMe2N4(tren))(OMe)]+ (3) and [FeIII(SMe2N4(tren))(O(H)Me)]+ (4), respectively, are shown to be more consistent with a reaction involving rate-limiting protonation of an Fe(III)–OOH, than with one involving rate-limiting O–O bond cleavage. The observed deuterium isotope effect (kH/kD = 3.1) is also consistent with this mechanism.