Trapping and spectroscopic characterization of an FeIII-superoxo intermediate from a nonheme mononuclear iron-containing enzyme Michael M. Mbughunia, Mrinmoy Chakrabartib, Joshua A. Haydenb, Emile L. Bominaarb, Michael P. Hendrichb, Eckard Münckb, and John D. Lipscomba,1 aDepartment of Biochemistry, Molecular Biology and Biophysics, and Center for Metals in Biocatalysis, University of Minnesota, Minneapolis, MN 55455, and bDepartment of Chemistry, Carnegie Mellon University, Pittsburgh, PA 15213 Edited by Edward I. Solomon, Stanford University, Stanford, CA, and approved August 4, 2010 (received for review July 9, 2010) III •− Fe -O2 intermediates are well known in heme enzymes, but none have been characterized in the nonheme mononuclear FeII enzyme family. Many steps in the O2 activation and reaction cycle of FeII-containing homoprotocatechuate 2,3-dioxygenase are made detectable by using the alternative substrate 4-nitrocatechol (4NC) and mutation of the active site His200 to Asn (H200N). Here, the first intermediate (Int-1) observed after adding O2 to the H200N- 4NC complex is trapped and characterized using EPR and Möss- ∕ III bauer (MB) spectroscopies. Int-1 is a high-spin (S1 ¼ 5 2) Fe anti- ∕ ≈ −1 ferromagnetically (AF) coupled to an S2 ¼ 1 2 radical (J 6cm in H • ¼ JS1 S2). It exhibits parallel-mode EPR signals at g ¼ 8.17 from the S ¼ 2 multiplet, and g ¼ 8.8 and 11.6 from the S ¼ 3 multiplet. 17 These signals are broadened significantly by O2 hyperfine inter- ≈ III •− actions (A17O 180 MHz). Thus, Int-1 is an AF-coupled Fe -O2 spe- cies. The experimental observations are supported by density functional theory calculations that show nearly complete transfer of spin density to the bound O2. Int-1 decays to form a second intermediate (Int-2). MB spectra show that it is also an AF-coupled FeIII-radical complex. Int-2 exhibits an EPR signal at g ¼ 8.05 arising 17 from an S ¼ 2 state. The signal is only slightly broadened by O2 (<3% spin delocalization), suggesting that Int-2 is a peroxo- FeIII-4NC semiquinone radical species. Our results demonstrate II facile electron transfer between Fe ,O2, and the organic ligand, thereby supporting the proposed wild-type enzyme mechanism. oxygen activation ∣ oxygenase ∣ spectroscopy ∣ superoxide Scheme 1. Proposed mechanism for extradiol dioxygenases. In the case of − 2,3-HPCD, R is −CH2COO and B is His200. When R is −NO2 and His200 is chan- ost mononuclear nonheme iron-containing oxidases and ged to Asn, the reaction stalls before reaching intermediate C. Peroxide is Moxygenases are proposed to initiate their oxygen activation slowly released and the product is 4NC quinone. II cycles by binding O2 to an active site Fe (1–8). Internal electron transfer to form an FeIII-superoxo species converts the kinetically to O2 forms adjacent substrate and oxygen radicals (Scheme 1B). inert triplet ground state of O2 to a doublet that can participate in Recombination of the radicals would begin the ring cleavage and the many types of chemistry characteristic of this mechanistically oxygen insertion reactions of this enzyme that eventually yield a diverse group of enzymes. The same strategy is usually employed muconic semialdehyde adduct as the product. A localized radical by heme-containing oxygenases and oxidases, leading in some on the 4NC semiquinone at the incipient position of oxygen at- cases to comparatively stable FeIII-superoxo intermediates that tack would account for the lack of ring planarity. Although this is have been structurally and spectroscopically characterized (9– the only structurally characterized nonheme Fe-superoxo species, 12). Instability of the putative superoxo intermediate in all the iron oxidation state differs from all of the other postulated mononuclear nonheme iron-containing enzymes has prevented Fe-superoxo intermediates. similar characterization, although a superoxide level species The mechanism that emerges from the structural and kinetic has been reported for the dinuclear iron site of myo-inositol oxy- studies does not require a change in metal oxidation state to form genase (13). II a reactive intermediate (22). However, our studies of 2,3-HPCD In recent studies of the nonheme Fe -containing homoproto- II II catechuate 2,3-dioxygenase (2,3-HPCD), we have shown that in which Fe is replaced with Mn suggest that transient forma- three intermediates of the catalytic cycle can be trapped in one crystal for structural analysis (14). One of these intermediates Author contributions: M.P.H., E.M., and J.D.L. designed research; M.M.M., M.C., J.A.H., and II has been proposed to be an Fe -superoxo species based on the E.L.B. performed research; M.M.M., M.C., J.A.H., E.L.B., M.P.H., E.M., and J.D.L. analyzed long Fe-O bond distances and an unexpected lack of planarity of data; and E.L.B., M.P.H., E.M., and J.D.L. wrote the paper. the aromatic ring of the alternative substrate 4-nitrocatechol The authors declare no conflict of interest. (4NC), which chelates the iron in ligand sites adjacent to that This article is a PNAS Direct Submission. of the O2. In accord with the mechanism postulated for this 1To whom correspondence should be addressed. E-mail: [email protected]. – enzyme class as illustrated in Scheme 1 (1, 8, 15 21), we have This article contains supporting information online at www.pnas.org/lookup/suppl/ II proposed that net electron transfer from 4NC through the Fe doi:10.1073/pnas.1010015107/-/DCSupplemental. 16788–16793 ∣ PNAS ∣ September 28, 2010 ∣ vol. 107 ∣ no. 39 www.pnas.org/cgi/doi/10.1073/pnas.1010015107 Downloaded by guest on September 27, 2021 tion of an oxidized metal center may occur (23). The MnII-re- placed enzyme is fully active and has no detectable change in structure. Our studies showed that, upon addition of O2 to the enzyme complexed with the normal substrate [homoprotocatech- uate or 3,4 dihydroxyphenylacetate (HPCA)], an intermediate is formed with a lifetime of a few milliseconds. The EPR character- istics of this intermediate are consistent with a MnIII-superoxo formulation. This observation suggests that a similar intermediate might exist in the native enzyme as a transient FeIII-superoxo species (Scheme 1A). Ongoing rapid freeze quench (RFQ) studies of the WT 2,3- HPCD with HPCA as a substrate and the native FeII in the active site have not revealed accumulation of an FeIII-superoxo species that can be detected by EPR or Mössbauer experiments. These results suggest that if such a species is formed, it is very short- lived, so a strategy to slow the reaction is required to detect the putative ferric-superoxo intermediate. Two methods have been described to slow the oxygen activation and substrate attack portions of the reaction cycle. The first is to use an alternative substrate with electron withdrawing substituents, such as 4NC (17). The second is to change the key active site acid-base catalyst and hydrogen bonding residue His200 to an Asn residue (2,3- HPCD variant H200N) (19). Using either one of these strategies results in a slow reaction that gives the expected ring-cleaved product at the end of the cycle. When both strategies are used simultaneously, the reaction slows further and the 4NC aromatic Fig. 1. Parallel-mode EPR spectra (colored lines) and simulations (thin black ring is not opened; rather, it is converted to 4NC quinone and lines). (A) 2 K spectrum 10 s after mixing H200N-4NC complex with O2 satu- rated buffer at 4 °C. (B) Sample of A at 9 K. (C) Spectrum at 10 K for a sample H2O2 is released (19). Independent of whether ring cleavage prepared as in A but with 70% enriched 17O .(D) Sample from A at 9 K after (with HPCA) or ring oxidation (with 4NC) is catalyzed by 2 λ 610 10 min incubation at 4 °C. Conditions prior to mixing: 1.64 mM H200N-4NC, H200N, a common blue intermediate ( max ¼ nm) is formed. 200 mM MOPS buffer pH 7.5. Simulation parameters: B, S1 ¼ 5∕2, S2 ¼ 1∕2, 6 −1 −0 48 −1 ∕ 0 20 2 01 2 02 1 98 2 04 The rate constant for the formation of this intermediate depends J ¼þ cm , D1 ¼ . cm , E D1 ¼ . , g1 ¼ . , g2 ¼ð . ; . ; . Þ, 0 29 90 55 17 ≈ 180 linearly on the O2 concentration (19). We speculate that this r ¼ . nm, rθ ¼ °, and rφ ¼ °; C, same as B but with A O MHz −1 −1 species is the initial oxy intermediate, potentially the elusive (I ¼ 5∕2); D, S1 ¼ 5∕2, S2 ¼ 1∕2, J ¼þ40 cm , D1 ¼þ0.5 cm , III ∕ 0 13 2 01 2 00 Fe -superoxo species of the mononuclear nonheme iron oxi- E D1 ¼ . , g1 ¼ . , and g2 ¼ . EPR conditions: frequency, dase/oxygenase family. The transient kinetic, EPR, and Möss- 9.24 GHz; power, 20 mW; and modulation amplitude, 10 G. bauer studies reported here show this to be the case. exhibits, also in parallel mode, a new EPR signal at g ¼ 8.05 Results (Figs. 1D and 2). Absorption Spectra Show That an Intermediate Precedes Substrate The EPR spectra of Int-1 over a range of temperatures (spec- Oxidation. The stoichiometric H200N-4NC complex exhibits an tra at 2 and 9 K are shown in Fig. 1 A and B) show that the absorption spectrum characteristic of the dianionic form of g ¼ 8.17 resonance originates from a ground doublet, and the 4NC at 518 nm. Mixing this complex with O2 causes a shift to g ¼ 8.8 and 11.6 resonances from excited states. The Mössbauer CHEMISTRY 506 nm within 25 ms at 4 °C showing that an intermediate (Int-1) spectra, shown below, reveal that the iron in both Int-1 and Int-2 III is formed (Fig.