Modeling the reactivity of superoxide reducing metalloenzymes with a nitrogen and sulfur coordinated iron complex.

Superoxide is a potent cellular poison.1 As such, organisms have evolved a number of pathways to degrade superoxide before it can cause cellular damage. The best understood are the superoxide dismutases (SOD), which catalyze the disproportionation of superoxide.2 In some anaerobic organisms a different protection scheme has evolved. These organisms utilize superoxide reductases (SORs), which catalyze the reduction of superoxide to peroxide, thus eliminating the production of dioxygen.3 Two different SORs have recently been structurally characterized; neelaredoxin (from the hyperthermophilic archeon, Pyrococcus furiou) and rubreodoxin oxidoreductase (Rbo, also known as desulfoferredoxin, from DesulfoVibrio desulfuricans).3,4 The active sites of both of these enzymes contain a cysteinate-ligated iron center that cycles between the +2 (active form) and +3 (resting form) oxidation states. These lie near the surface of the protein exposed to solvent. In both enzymes, the metal center is fivecoordinate in the reduced form and is ligated by four equatorial histidines and one axial cysteinate (Scheme 1). Upon oxidation by a protonated superoxide anion, a nearby glutamate residue binds to the metal center in the site trans to the cysteinate (Scheme 1). Presented below is a five-coordinate Fe(II) complex which models the reactive properties of SORs. X-ray quality crystals of [FeSN4(tren)](PF6) (1) were grown from acetonitrile/diethyl ether (1:10) at -30 °C.5,6 The iron center of 1 is in a distorted triganol bypyramidal environment with the sulfur trans to an amine nitrogen in the apical position (Figure 1). All of the bond lengths are typical for an Fe(II) complex in this ligand environment and compare well with those found in the reduced iron center of Rbo.4a,f Complex 1 is five-coordinate, suggesting that small molecules such as HO2 could possibly bind to, and potentially oxidize the metal center. Upon exposure to a wet acetonitrile solution of potassium superoxide and sodium hexaflurophosphate, the pale yellow-green solution of 1 changes to deep blue (Figure 2).7 Furthermore, the resulting solution’s electronic absorption spectrum is reminiscent of the oxidized high-pH form of SOR with a λmax at 582 nm ( ) 1975 M-1 cm-1), suggesting that the solution contained the oxidized form of 1, possibly with a sixth ligand in the open site.4 When this reaction is performed at low temperatures, an intermediate is observed. We are currently in the process of characterizing this intermediate by EXAFS and resonance Raman. This method of generating oxidized 1 was not suitable on a preparative scale, because complete decomposition of the product occurs after a few minutes. This occurs presumably as a result of degradative ligand oxidation by hydrogen peroxide produced in the reaction.8 The oxidized form of 1 was rationally synthesized starting from an Fe(III) source. Addition of FeCl3 in methanol to a methanolic solution of 3-methyl-3-mercaptobutanone and tris(2-aminoethyl)amine instantly produces a deep blue solution. Exchange of chloride for tetraphenyl borate followed by crystallization from MeCN/Et2O afforded blue needles. These crystals produced a dark blue solution with an electronic absorption spectrum identical to that of the solution containing 1 and superoxide, and a spectrum similar to that of neelaredoxin and Rbo. The IR spectrum of the * Author to whom correspondence should be addressed. E-mail: kovacs@ chem.washington.edu. (1) (a) Totter, J. R. Proc. Natl. Acad. Sci. U.S.A. 1980, 77, 1763-1767. (b) Burdon, R. H. Free Radical Biol. Med. 1995, 18, 775-794. (2) (a) Lyons, T. J.; Gralla, E. B.; Valentine, J. S. Met. Ions Biol. Syst. 1999, 36, 125-177. (b) Riley, D. P. Chem. ReV. 1999, 99, 2573-2587. (c) Culotta, V. C. Curr. Top. Cell Regul. 2000, 36, 117-132. (3) Jenney, F. E., Jr.; Verhagen, M. F.; Cui, X.; Adams, M. W. W. Science 1999, 286, 306-309. (4) (a) Coulter, E. D.; Emerson, J. P.; Kurtz, D. M. Jr.; Cabelli, D. E. J. Am. Chem. Soc. 2000, 122, 11555-11556. (b) Lombard, M.; Fontecave, M.; Touati, D.; Niviere, V. J. J. Biol. Chem. 2000, 275, 115-121. (c) Liochev, S. I.; Fridovich, I. J. Biol. Chem. 1997, 272, 25573-2557. (d) Yeh, A. P.; Hu, Y.; Jenney, F. E., Jr.; Adams, M. W.; Rees, D. C. Biochemistry 2000, 39, 2499-2508. (e) Chem, L.; Sharma, P.; Le Gall, J.; Mariano, A. M.; Teixeira, M.; Xavier, A. V. Eur. J. Biochem. 1994, 39, 2499-2508. (f) Coehlo, A. V.; Matias, P.; Fülöp, U.; Thompson, A.; Gonzalez, A.; Carrondo, M. A. J. Bioinorg. Chem. 1997, 2, 680689. (5) Compound 1 was prepared by combining 2 equiv of 3-methyl-3mercapto-2-butanone with ferrous chloride in methanol, adding 1 equiv of tris(2-aminoethyl)amine (tren) followed by stirring for 24 h. (6) Crystal data for 1 (FeC11H25N4SPF6): brown plates, 0.28 × 0.16 × 0.16 mm, orthorhombic, space group P21212, a ) 8.209(2) Å, b ) 12.742(1) Å, c ) 18.090(2) Å, R ) â ) γ ) 90°, V ) 1892(1) Å3, Z ) 4, Mo radiation λ ) 0.7107 Å. For 5636 unique reflections collected at 161 K, the current discrepancy indices are R ) 0.046 and Rw ) 0.097 (direct methods using SIR92 software). (7) Complex 1 readily reacts with dioxygen to afford a μ-oxo dimer which has been characterized by X-ray crystallography: Shearer, J.; Kaminsky, W.; Kovacs, J. A. Unpublished results. (8) The reaction of 1 with superoxide in wet DMSO shows high catalase activity, suggesting that hydrogen peroxide is produced during the reaction. Such high activity is not observed in the absence of 1 and in the presence of 2. Addition of H2O2 to solutions of 2 results in its rapid and complete decomposition. Figure 1. ORTEP of [FeSN4(tren)] (1) showing 50% probability ellipsoids and atom-labeling scheme. All H atoms have been omitted for clarity. Selected bond lengths for 1: Fe-N(1), 2.091(3) Å; Fe-N(2), 2.268(3) Å; Fe-N(3), 2.131(3) Å; Fe-N(4), 2.117(3) Å; Fe-S, 2.329(1) Å.