Understanding how the thiolate sulfur contributes to the function of the non-heme iron enzyme superoxide reductase

Abstract

Toxic superoxide radicals, generated via adventitious reduction of dioxygen, have been implicated in a number of disease states. The cysteinate-ligated non-heme iron enzyme superoxide reductase (SOR) degrades superoxide via reduction. Biomimetic analogues which provide insight into why nature utilizes a trans-thiolate to promote SOR function are described. Spectroscopic and/or structural characterization of the first examples of thiolate-ligated Fe (III)-peroxo complexes provides important benchmark parameters for the identification of biological intermediates. Oxidative addition of superoxide is favored by low redox potentials. The trans influence of the thiolate appears to significantly weaken the Fe-O peroxo bond, favoring proton-induced release of H 2O 2 from a high-spin Fe(III)-OOH complex.

FIGURE 1

Proposed mechanism for SOR-catalyzed reduction of superoxide via hydroperoxo (T₁) and solvent-bound (T₂) intermediates.

FIGURE 2

Comparison of the thiolate-ligated non-heme, and heme, iron active sites of SOR (left) and P450 (right).

FIGURE 3

ORTEP diagram of thiolate-ligated, five-coordinate [Fe^III(S₂^Me2N₃(Pr,Pr))]⁺ (1) synthesized by Kovacs group postdocs Steve Shoner and Jeff Ellison.

FIGURE 4

ORTEP diagram of thiolate-ligated, biomimetic superoxide reducing catalyst [Fe^II(S^Me2N₄(tren))]⁺ (2) synthesized by Jason Shearer, a graduate student in the Kovacs lab.

FIGURE 5

Low-temperature detection of the hydroperoxo intermediate [Fe^III(S^Me2N₄(tren))(OOH)]⁺ (3) using electronic absorption spectroscopy, and its conversion to solvent-bound [Fe^III(S^Me2N₄(tren))(OMe)]⁺ (4) upon warming.

FIGURE 6

X-Band EPR spectrum of tangerine orange [Fe^III(S^Me2N₄(tren))(OOH)]⁺ (3) at 7 K in an MeOH/EtOH (5:2) glass.

FIGURE 7

Low-temperature IR spectrum of ¹⁶O-labeled (· · ·) and ¹⁸O-labeled (23%; —) [Fe^III(S^Me2N₄(tren))(OOH)]⁺ (3) showing the peroxo ν_O–O Fermi doublet that shifts upon incorporation of ¹⁸O.

FIGURE 8

ORTEP diagram of solvent-bound [Fe^III(S^Me2N₄(tren))(MeCN)]²⁺ showing that MeCN binds trans to the imine nitrogen and cis to the thiolate sulfur.

FIGURE 9

ORTEP diagram of thiolate-ligated [Fe^III(S^Me2N₄(tren))(N₃)]⁺.

FIGURE 10

ORTEP diagram of acetate-bound [Fe^III(S^Me2N₄(tren))(OAc)]⁺ (5), a mimic for the glutamate-bound SOR resting state.

FIGURE 11

Proton-induced conversion of [Fe^III(S^Me2N₄(tren))(OOH)]⁺ (3) to solvent-bound [Fe^III(S^Me2N₄(tren)(MeOH))]²⁺ (6) as monitored by electronic absorption spectroscopy at low temperatures.

FIGURE 12

Catalytic cycle involving [Fe^II(S^Me2N₄(tren))]⁺ (2)-promoted reduction of superoxide to afford H₂O₂, via the sequential protonation and Cp₂Co-promoted reduction of hydroperoxo- and solvent-bound intermediates.

FIGURE 13

ORTEP diagram of trans-thiolate-ligated, biomimetic superoxide reducing catalyst [Fe^II(cyclam-PrS)]⁺ (8) synthesized by Terutaka Kitagawa, a graduate student in the Kovacs lab.

FIGURE 14

Low-temperature electronic absorption spectrum and DFT-calculated structure of the hydroperoxo intermediate [Fe^III(cyclam-PrS)(OOH)]⁺ (9) formed upon addition of superoxide to reduced [Fe^II(cyclam-PrS](BPh₄) (8) in the presence of a proton source.

FIGURE 15

Catalytic cycle involving [Fe^II(cyclam-PrS](BPh₄) (8)-promoted reduction of superoxide to afford H₂O₂, via the sequential protonation and Cp₂Co-promoted reduction of hydroperoxo- and acetate-bound intermediates.