Synthetic Models for the Cysteinate-Ligated Non-Heme Iron Enzyme Superoxide Reductase: Observation and Structural Characterization by XAS of an Fe-OOH Intermediate

Superoxide reductases (SORs) belong to a new class of metalloenzymes that degrade superoxide by reducing it to hydrogen peroxide. These enzymes contain a catalytic iron site that cycles between the FeII and FeIII states during catalysis. A key step in the reduction of superoxide has been suggested to involve HO2 binding to FeII, followed by innersphere electron transfer to afford an FeIII-OO(H) intermediate. In this paper, the mechanism of the superoxide-induced oxidation of a synthetic ferrous SOR model ([FeII(SN4(tren))] (1)) to afford [Fe(SN4(tren)(solv))] (2-solv) is reported. The XANES spectrum shows that 1 remains five-coordinate in methanolic solution. Upon reaction of 1 with KO2 in MeOH at -90 °C, an intermediate (3) is formed, which is characterized by a LMCT band centered at 452(2780) nm, and a lowspin state (S ) 1/2), based on its axial EPR spectrum (g! ) 2.14; g| ) 1.97). Hydrogen peroxide is detected in this reaction, using both 1H NMR spectroscopy and a catalase assay. Intermediate 3 is photolabile, so, in lieu of a Raman spectrum, IR was used to obtain vibrational data for 3. At low temperatures, a νO-O Fermi doublet is observed in the IR at 788(2) and 781(2) cm-1, which collapses into a single peak at 784 cm-1 upon the addition of D2O. This vibrational peak diminishes in intensity over time and essentially disappears after 140 s. When 3 is generated using an 18O-labeled isotopic mixture of KO2/KO2 (23.28%), the vibration centered at 784 cm-1 shifts to 753 cm-1. This new vibrational peak is close to that predicted (740 cm-1) for a diatomic 18O-18O stretch. In addition, a νO-O vibrational peak assigned to free hydrogen peroxide is also observed (νO-O ) 854 cm-1) throughout the course of the reaction between FeII-1 and superoxide and is strongest after 100 s. XAS studies indicate that 3 possesses one sulfur scatterer at 2.33(2) Å and four nitrogen scatterers at 2.01(1) Å. Addition of two Fe-O shells, each containing one oxygen, one at 1.86(3) Å and one at 2.78(3) Å, improved the EXAFS fits, suggesting that 3 is an end-on peroxo or hydroperoxo complex, [Fe(SN4(tren))(OO(H))]. Upon warming above-50 °C, 3 is converted to 2-MeOH. In methanol and methanol:THF (THF ) tetrahydrofuran) solvent mixtures, 2-MeOH is characterized by a LMCT band at λmax ) 511(1765) nm, an intermediate spin-state (S ) 3/2), and, on the basis of EXAFS, a relatively short Fe-O bond (assigned to a coordinated methanol or methoxide) at 1.94(10) Å. Kinetic measurements in 9:1 THF:MeOH at 25 °C indicate that 3 is formed near the diffusion limit upon addition of HO2 to 1 and converts to 2-MeOH at a rate of 65(1) s-1, which is consistent with kinetic studies involving superoxide oxidation of the SOR iron site. Superoxide reductases (SORs) are metalloenzymes that belong to a newly emerging class of enzymes, which contain a non-heme, cysteinate-ligated iron center.1 This class of metalloenzymes performs at least two distinct functions and also includes the enzyme nitrile hydratase.2 SORs are used as a cellular defense mechanism against oxidative stress, destroying superoxide before it can cause extensive cellular damage. Unlike superoxide dismutases, which disproportionate superoxide to afford both hydrogen peroxide and dioxygen, SORs reduce superoxide to afford only hydrogen peroxide.3 This is especially advantageous for anaerobic organisms, which do not have the cellular machinery to deal with dioxygen. All SORs reported to date (including the enzymes rubredoxin oxidoreductase (Rbo, also known as desulfoferrodoxin) and neelaredoxin) contain, in their reduced state, a redox-active Fe2+ ion ligated by four equatorial histidines and a cysteinate trans to an open site.4 As * Corresponding author. E-mail address: [email protected]. † University of Washington. ‡ Haverford College. (1) Kurtz, D. M., Jr.; Coulter, E. D. Chemtracts 2001, 14, 407-435. (2) (a) Shearer, J.; Kovacs. J. A. Nitrile Hydratase: An Unusual FeIII CysteineLigated Metalloenzyme. Encyclopedia of Catalysis; John Wiley & Sons: New York, in press. (b) Kobyashi, M. Nat. Biotechnol. 1998, 16, 733736. (3) (a) Jovanoviæ, T.; Ascenso, C.; Hazlett, K. R. O.; Sikkink, R.; Kerbs, C.; Litwiller, R.; Benson, L. M.; Moura, I.; Moura, J. J. G.; Radolf, J. D.; Huynh, B. H.; Naylor, S.; Rusnak, F. J. Biol. Chem. 2000, 275, 2843928448. (b) Jenney, F. E., Jr.; Verhagen, M. F. J. M.; Cui, X.; Adams, M. W. W. Science 1999, 286, 306-309. (4) (a) Coelho, A. V.; Matias, P.; Fülöp, V.; Thompson, A.; Gonzalez, A.; Coronado, M. A. J. Biol. Inorg. Chem. 1997, 2, 680-689. (b) Andrew, P. Y.; Hu, Y.; Jenney, F. E.; Adams, M. W. W.; Rees, D. C. Biochemistry 2000, 39, 2499-2508. Published on Web 09/10/2002 10.1021/ja012722b CCC: $22.00 © 2002 American Chemical Society J. AM. CHEM. SOC. 2002, 124, 11709-11717 9 11709 such, they resemble the heme iron enzyme cytochrome P450.5 The hydroperoxyl radical (HO2) oxidizes the Fe2+ of SOR to afford Fe3+ and hydrogen peroxide. Upon release of hydrogen peroxide, a glutamate coordinates to the iron center, to afford a six-coordinate ferric species (the oxidized resting state).4b Recently, a number of flash photolysis studies aimed at elucidating the mechanism of SOR catalysis were reported.6 These studies show that upon exposure of reduced SOR to HO2 an intermediate species forms, near the diffusion limit, which has been proposed to be an FeIII-OO(H) species.1 This is supported by its transient nature and electronic absorption spectrum, which contains an intense low-energy charge-transfer band characteristic of an Fe3+ complex.6 This intermediate peroxide species rapidly converts to the oxidized resting state via the displacement of hydrogen peroxide by Glu47 (Scheme 1). It has been suggested that proton transfer in this final step of the mechanism involves a catalytically essential conserved Lys48.38 A recent study demonstrated that by mutating Glu47 to alanine, the transient intermediate is stabilized, making it possible to characterize by resonance Raman. Under these conditions, an νO-O stretch is observed at 850 cm-1 which was attributed to a deprotonated side-on ferric peroxide species.7 Que and Girerd have shown that a side-on ferric peroxide (Fe(η-O2)) will convert to an end-on ferric hydroperoxide (FeIII(η1-OOH)) upon protonation.8 This observation, along with the proposed involvement of a proton-transfer step in SOR catalysis, suggests that an end-on ferric hydroperoxide may be involved in the mechanism of superoxide reduction by SOR. Non-heme FeIII-hydroperoxo species have been implicated as important intermediates in several biological systems.9 As such, a number of synthetic non-heme alkyland hydroperoxo-FeIII model complexes have been prepared, including those reported by Que,8a,d,10a,f Lippard,10b and Girerd,8b in order to gain more information about these species.8,10 The spectroscopic characterization of FeIII-peroxo complexes39 is extremely difficult due to their high reactivity and photolability.9d,11 There are even fewer structurally characterized FeIII-peroxo complexes, with the majority of those reported being iron dimers.10b,12 For example, Lippard12a recently reported the X-ray absorption spectrum of a synthetic diiron complex containing a terminal hydroperoxo, and Que and co-workers12b reported the crystal structure of a (μ-1,2-peroxo)diiron complex. In a previous paper we communicated preliminary results involving the oxidation of the five-coordinate ferrous complex, [Fe(SN4(tren))] (1), by KO2 in MeCN to afford the sixcoordinate ferric complex [Fe(SN4(tren))(MeCN)] (2MeCN).13 Hydrogen peroxide was shown to form in this reaction using a catalase assay. We now describe the mechanism of HO2 oxidation of 1 and the observation and characterization of an FeIII-OO(H) intermediate. Experimental Section General Methods. All reactions were carried out under an atmosphere of dinitrogen or argon using a glovebox or standard Schlenk techniques. Chemical reagents purchased from commercial vendors were of the highest purity available and used without further purification. O2 (23.28% isotopic enrichment) was purchased from ICON isotopes. All solvents, with the exception of tetrahydrofuran (THF) and “superdry” MeOH, were rigorously degassed and purified according to standard procedures.14 THF was purified by refluxing it over sodium/ benzophenone ketal and distilling it onto KO2. This was then vacuum transferred immediately prior to use. Superdry MeOH was distilled from Na onto Mg/I2, where it was refluxed for several hours and was then distilled onto 4 Å molecular sieves immediately prior to use. “WetMeCN” refers to MeCN that was not dried or distilled prior to use but which was rigorously degassed. D2O was incorporated into acetone by adding 1 drop of D2O to acetone (previously dried over 4 Å molecular sieves), followed by repeated drying and D2O additions. IR spectra were recorded on a Perkin-Elmer 1720 FTIR either as KBr pellets or in a Beckman solution IR cell equipped with KBr windows. Electronic absorption spectra were recorded using a Hewlett-Packard 8453 diode array spectrometer. Low-temperature electronic absorption spectra were (5) Loew, G. H.; Harris, D. L. Chem. ReV. 2000, 100, 407-420. (6) (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.; Houee-Levin, C.; Touati, D.; Fontecave, M.; Niviere, V. Biochemistry 2001, 40, 5032-5040. (c) Niviere, V.; Lombard, M.; Fontecave, M.; Houee-Levin, C. FEBS Lett. 2001, 497, 171-173. (7) Mathe, C.; Mattioli, T. A.; Horner, O.; Lombard, M.; Latour, J.-M.; Fontecave, M.; Niviere, V. J. Am. Chem. Soc. 2002, 124, 4966-4967. (8) (a) Ho, R. Y. N.; Roelfes, G.; Hermant, R.; Hage, R.; Feringa, B. L.; Que, L., Jr. Chem. Commun. 1999, 2161-2162. (b) Simaan, A. J.; Banse, F.; Mialane, P.; Boussac, A.; Un, S.; Kargar-Grisel, T.; Bouchoux, G.; Girerd, J.-J. Eur. J. Inorg. Chem. 1999, 993-996. (c) Simaan, A. J.; Dopner, S.; Banse, F.; Bourcier, S.; Bouchoux, G.; Boussac, A.; Hildebrandt, P.; Girerd, J.-J. Eur. J. Inorg. Chem 2000, 1627-1633. (d) Ho, R. Y. N.; Roelfes, G.; Feringa, B. L.; Que, L., Jr. J. Am. Chem. Soc. 1999, 121, 264-265. (9) (a) Que, L., Jr.; Watanabe, Y. Science 2001, 292, 651-653. (b) Girerd, J.-J.; B., F.; Simaan, A. J. Struct. Bonding 2000, 97, 145-177. (c) Newcomb, M.; Toy, P. H. Acc. Chem. Res. 2000, 33, 449-455. (d) Solomon, E. I.; Brunold, T. C.; Davis, M. I.; Kemsley, J. N.; Lee, S.-K.; Lehnert, N.; Neese, F.; Skulan, A. J.; Yang, Y.-S.; Zhou, J. Chem. ReV. 2000, 100, 235-349. (e) Mialane, P.; Nivorojkine, A.; Pratviel, G.; Azéma, L.; Slany, M.; Godde, F.; Simaan, A.; Banse, F.; Kargar-Grisel, T.; Bouchoux, G.; Sainton, J.; Horner, O.; Guilhem, J.; Tchertanova, L.; Meunier, B.; Girerd, J.-J. Inorg. Chem. 1999, 38, 1085-1092. (f) Que, L., Jr.; Ho, R. Y. N. Chem. ReV. 1996, 96, 2607-2624. (10) (a) Kim, J.; Larka, E.; Wilkinson, E. C.; Que, L., Jr. Angew. Chem., Int. Ed. Engl. 1995, 34, 2048-2051. (b) Kim K.; Lippard S. J. J. Am. Chem. Soc. 1996, 118, 4914-4915. (c) Rabion, A.; Chen, S.; Wang, J.; Buchanan, R. M.; Series, J.-L.; Fish, R. H. J. Am. Chem. Soc. 1995, 117, 1235612357. (d) Roelfs, G.; Lubben, M.; Chen, K.; Ho, R. Y. N.; Meetsma, A.; Genseberger, S.; Hermant, R. M.; Hage, R.; Mandal, S. K.; Young, V. G.; Zang, Y.; Kooijman, H.; Spek, A. L.; Que, L., Jr.; Feringa, B. L. Inorg. Chem. 1999, 38, 1929-1936. (e) Wada, A.; Ogo, S.; Watanabe, Y.; Mukai, M.; Kitagawa, T.; Jitsukawa, K.; Masuda, H.; Einaga, H. Inorg. Chem. 1999, 38, 3592-3593. (f) Zang, Y.; Kim, J.; Dong, Y.; Wilkinson, E. C.; Appelman, E. H.; Que, L., Jr. J. Am. Chem. Soc. 1997, 119, 4197-4205. (g) Bernal, I.; Jensen, I. M.; Jensen, K. B.; McKenzie, C. J.; Toftlund, H.; Tuchagues, J.-P. J. Chem. Soc., Dalton Trans. 1995, 3667-3669. (h) Simaan, A. J.; Banse, F.; Mialane, P.; Kargar-Grisel, T.; Bouchoux, G.; Boussac, A.; Un, S.; Girerd J.-J. Eur. J. Inorg. Chem. 1999, 993-996. (11) Lehnert, N.; Ho, R. Y. N.; Que, L., Jr.; Solomon E. I. J. Am. Chem. Soc. 2001 123, 8271-8290. (12) (a) Mizoguchi, T. J.; Kuzelka, J.; Spingler, B.; DuBois, J. L.; Davydov, R. M.; Hedman, B.; Hodgson, K. O. Lippard, S. J. Inorg. Chem. 2001, 40, 4662-4673. (b) Dong, Y.; Yan, S.; Young, V. G., Jr.; Que, L., Jr. Angew. Chem., Int. Ed. Engl. 1996, 35, 618-620. (c) Ookubo, T.; Sugimoto, H.; Nagayama, T.; Masuda, H.; Sato, T.; Tanaka, K.; Maeda, Y.; Okawa, H.; Hayashi, Y.; Uehara, A.; Suzuki, M. J. Am. Chem. Soc. 1996, 118, 701702. (d.) Friant, P.; Goulon, J.; Fischer, J.; Ricard, L.; Schappacher, M.; Weiss, R.; Momenteau, M. NouV. J. Chim. 1985, 9, 33-40. (e) Kitajima, N.; Tamura, N.; Amagai, H.; Fukui, H.; Moro-oka, Y.; Mizutani, Y.; Kitagawa, T.; Mathur, R.; Heerwegh, K.; Reed, C. A.; Randall, C. R.; Que, L., Jr.; Tatsumi, K. J. Am. Chem. Soc. 1994, 116, 9071-9085. (13) Shearer, J.; Nehring, J.; Kaminsky, W.; Kovacs, J. A. Inorg. Chem. 2001, 40, 5483-5484. (14) Perrin, D. D.; Armarego, W. L. F.; Perrin, D. R.Purification of Laboratory Chemicals, 2nd ed.; Pergamon Press: Elmsford, NY, 1980. Scheme 1 A R T I C L E S Shearer et al. 11710 J. AM. CHEM. SOC. 9 VOL. 124, NO. 39, 2002 recorded in a custom-built low-temperature copper-block sample holder inserted into a stainless steel cryostat and cooled to-90 °C by a stream of cold nitrogen gas. EPR spectra were recorded on a Bruker EPX CW-EPR spectrometer operating at X-band frequency at 130 K. Solution-state magnetic moments were calculated using the Evans’ method, corrected for superconducting solenoids, on a Bruker DPX 300 FTNMR spectrometer.15 The temperature of all NMR experiments was calculated using van Geet’s method.16 Solid-state magnetic measurements were recorded on a Quantum Design MPMS-5S SQUID