A functional model for the cysteinate-ligated non-heme iron enzyme superoxide reductase (SOR)

Abstract

Superoxide reductases (SORs) are cysteine-ligated, non-heme iron enzymes that reduce toxic superoxide radicals (O2-). The functional role of the trans cysteinate, as well as the mechanism by which SOR reduces O2-, is unknown. Herein is described a rare example of a functional metalloenzyme analogue, which catalytically reduces superoxide in a proton-dependent mechanism, via a trans thiolate-ligated iron-peroxo intermediate, the first example of its type. Acetic-acid-promoted H2O2 release, followed by Cp2Co reduction, regenerates the active Fe(II) catalyst. The thiolate ligand and its trans positioning relative to the substrate are shown to contribute significantly to the catalyst's function, by lowering the redox potential, changing the spin state, and dramatically lowering the nuFe-O stretching frequency well-below that of any other reported iron-peroxo, while leaving nuO-O high, so as to favor superoxide reduction and Fe-O, as opposed to O-O, bond cleavage. Thus we provide critical insight into the relationship between the SOR structure and its function, as well as important benchmark parameters for characterizing highly unstable thiolate-ligated iron-peroxo intermediates.

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).

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).

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.

Figure 4

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