Marlène Martinho

A Synthetic High‐Spin Oxoiron(IV) Complex: Generation, Spectroscopic Characterization, and Reactivity

High versus low: The high-yield generation of a synthetic high-spin oxoiron(IV) complex, [Fe(IV)(O)(TMG(3)tren)](2+) (see picture, TMG(3)tren = 1,1,1-tris{2-[N2-(1,1,3,3-tetramethylguanidino)]ethyl}amine), has been achieved by using the very bulky tetradentate TMG(3)tren ligand, in order to both sterically protect the oxoiron(IV) moiety and enforce a trigonal bipyramidal geometry at the iron center, for which an S = 2 ground state is favored.

Proton- and Reductant-Assisted Dioxygen Activation by a Nonheme Iron(II) Complex to Form an Oxoiron(IV) Intermediate

Dioxygen activation by mononuclear iron oxygenases in general requires two electrons and protons to facilitate the reductive cleavage of the O-O bond and formation of a high-valent iron oxidant.[1,2] For enzymes with an iron(III) resting state, the oxidant is postulated to have a formally FeV oxidation state, e.g. FeIV(O)(porphyrin radical) for cytochrome P450[i] and FeV(O)(OH) for the Rieske dioxygenases.[ii] On the other hand, enzymes with an iron(II) resting state often require a tetrahydropterin or an α-keto acid cofactor to form an FeIV(O) intermediate.[2] Such intermediates have recently been trapped and characterized for several enzymes.[iii]

Human deoxyhypusine hydroxylase, an enzyme involved in regulating cell growth, activates O2 with a nonheme diiron center

Deoxyhypusine hydroxylase is the key enzyme in the biosynthesis of hypusine containing eukaryotic translation initiation factor 5A (eIF5A), which plays an essential role in the regulation of cell proliferation. Recombinant human deoxyhypusine hydroxylase (hDOHH) has been reported to have oxygen- and iron-dependent activity, an estimated iron/holoprotein stoichiometry of 2, and a visible band at 630 nm responsible for the blue color of the as-isolated protein. EPR, Mössbauer, and XAS spectroscopic results presented herein provide direct spectroscopic evidence that hDOHH has an antiferromagnetically coupled diiron center with histidines and carboxylates as likely ligands, as suggested by mutagenesis experiments. Resonance Raman experiments show that its blue chromophore arises from a (μ-1,2-peroxo)diiron(III) center that forms in the reaction of the reduced enzyme with O2, so the peroxo form of hDOHH is unusually stable. Nevertheless we demonstrate that it can carry out the hydroxylation of the deoxyhypusine residue present in the elF5A substrate. Despite a lack of sequence similarity, hDOHH has a nonheme diiron active site that resembles both in structure and function those found in methane and toluene monooxygenases, bacterial and mammalian ribonucleotide reductases, and stearoyl acyl carrier protein Δ9-desaturase from plants, suggesting that the oxygen-activating diiron motif is a solution arrived at by convergent evolution. Notably, hDOHH is the only example thus far of a human hydroxylase with such a diiron active site.

Shape‐Selective Interception by Hydrocarbons of the O2‐Derived Oxidant of a Biomimetic Nonheme Iron Complex

Picky ferryl: The complex [Fe(Tp(Ph(2)))(BF)] (Tp(Ph(2)) = hydrotris(3,5-diphenylpyrazolyl)borate; BF = benzoylformate) reacts with O(2) to generate an oxidant (see picture; O red, pink; Fe yellow; N blue; C gray; H white) that oxidizes added hydrocarbons shape-selectively. Discrimination derives from a cleft formed by two phenyl groups of the Tp(Ph(2)) ligand, favoring oblate spheroidal substrates.

Insights into the P-to-Q conversion in the catalytic cycle of methane monooxygenase from a synthetic model system

For the catalytic cycle of soluble methane monooxygenase (sMMO), it has been proposed that cleavage of the O–O bond in the (μ-peroxo)diiron(III) intermediate P gives rise to the diiron(IV) intermediate Q with an Fe2(μ–O)2 diamond core, which oxidizes methane to methanol. As a model for this conversion, (μ–oxo) diiron(III) complex 1 ([FeIII2(μ–O)(μ–O2H3)(L)2]3+, L = tris(3,5-dimethyl-4-methoxypyridyl-2-methyl)amine) has been treated consecutively with one eq of H2O2 and one eq of HClO4 to form 3 ([FeIV2(μ–O)2(L)2]4+). In the course of this reaction a new species, 2, can be observed before the protonation step; 2 gives rise to a cationic peak cluster by ESI-MS at m/z 1,399, corresponding to the {[Fe2O3L2H](OTf)2}+ ion in which 1 oxygen atom derives from 1 and the other two originate from H2O2. Mössbauer studies of 2 reveal the presence of two distinct, exchange coupled iron(IV) centers, and EXAFS fits indicate a short Fe–O bond at 1.66 Å and an Fe–Fe distance of 3.32 Å. Taken together, the spectroscopic data point to an HO-FeIV-O-FeIV = O core for 2. Protonation of 2 results in the loss of H2O and the formation of 3. Isotope labeling experiments show that the [FeIV2(μ–O)2] core of 3 can incorporate both oxygen atoms from H2O2. The reactions described here serve as the only biomimetic precedent for the conversion of intermediates P to Q in the sMMO reaction cycle and shed light on how a peroxodiiron(III) unit can transform into an [FeIV2(μ–O)2] core.

EPR and Mössbauer Spectroscopy of Intact Mitochondria Isolated from Yah1p-Depleted Saccharomyces cerevisiae†

Yah1p, an [Fe 2S 2]-containing ferredoxin located in the matrix of Saccharomyces cerevisiae mitochondria, functions in the synthesis of Fe/S clusters and heme a prosthetic groups. EPR, Mossbauer spectroscopy, and electron microscopy were used to characterize the Fe that accumulates in Yah1p-depleted isolated intact mitochondria. Gal- YAH1 cells were grown in standard rich media (YPD and YPGal) under O 2 or argon atmospheres. Mitochondria were isolated anaerobically, then prepared in the as-isolated redox state, the dithionite-treated state, and the O 2-treated state. The absence of strong EPR signals from Fe/S clusters when Yah1p was depleted confirms that Yah1p is required in Fe/S cluster assembly. Yah1p-depleted mitochondria, grown with O 2 bubbling through the media, accumulated excess Fe (up to 10 mM) that was present as 2-4 nm diameter ferric nanoparticles, similar to those observed in mitochondria from yfh1Delta cells. These particles yielded a broad isotropic EPR signal centered around g = 2, characteristic of superparamagnetic relaxation. Treatment with dithionite caused Fe (3+) ions of the nanoparticles to become reduced and largely exported from the mitochondria. Fe did not accumulate in mitochondria isolated from cells grown under Ar; a significant portion of the Fe in these organelles was in the high-spin Fe (2+) state. This suggests that the O 2 used during growth of Gal- YAH1 cells is responsible, either directly or indirectly, for Fe accumulation and for oxidizing Fe (2+) --> Fe (3+) prior to aggregation. Models are proposed in which the accumulation of ferric nanoparticles is caused either by the absence of a ligand that prevents such precipitation in wild-type mitochondria or by a more oxidizing environment within the mitochondria of Yah1p-depleted cells exposed to O 2. The efficacy of reducing accumulated Fe along with chelating it should be considered as a strategy for its removal in diseases involving such accumulations.

An Inverted and More Oxidizing Isomer of [FeIV(O)(tmc)(NCCH3)]2+†

High-valent oxoiron species are often invoked as the oxidants in the catalytic cycles of dioxygen activating mononuclear nonheme iron enzymes.[1] To date, such iron(IV) intermediates have been characterized for four enzymes, lending strong support for this notion.[2] Within the same time frame, synthetic nonheme complexes containing oxoiron(IV) units have also been described that serve as models for such reactive intermediates.[3] The first crystallographically characterized and most extensively studied member of this family of synthetic oxoiron(IV) complexes is [FeIV(O)(TMC)(NCCH3)](OTf)2 (1-NCCH3)[4] (TMC = 1,4,8,11-tetramethyl-1,4,8,11-tetraazacyclotetradecane). Its structure features a short Fe=O bond (rFe=O = 1.646 A) with an acetonitrile bound trans to the oxo atom.[4] The macrocyclic TMC ligand adopts a trans-I (R,S,R,S) configuration, such that all four methyl groups are oriented in the same direction with respect to the FeN4 plane,[5] and anti to the oxo atom. On the other hand, monoanionic X ligands coordinate syn to the methyl groups in crystal structures of five-coordinate [FeII(TMC)(X)]+ complexes.[6] Herein, we report the unexpected preparation of an inverted isomer of 1-NCCH3 in which the oxo group binds to the site syn to the four methyl groups (Scheme 1). The conversion of 1-NCCH3 to its inverted isomer is effected by treatment with PhIO in the presence of tetrafluroborate, an otherwise inert anion. The switch in binding site of the oxo group engenders changes in the spectroscopic properties of the oxoiron(IV) complex and, more importantly, a significantly enhanced reactivity in hydrogen-atom abstraction and oxo-transfer reactions.

Mössbauer and DFT Study of the Ferromagnetically Coupled Diiron(IV) Precursor to a Complex with an FeIV2O2 Diamond Core

Recently, we reported the reaction of the (mu-oxo)diiron(III) complex 1 ([Fe(III)(2)(mu-O)(mu-O(2)H(3))(L)(2)](3+), L = tris(3,5-dimethyl-4-methoxypyridyl-2-methyl)amine) with 1 equiv of H(2)O(2) to yield a diiron(IV) intermediate, 2 (Xue, G.; Fiedler, A. T.; Martinho, M.; Munck, E.; Que, L., Jr. Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 20615-20). Upon treatment with HClO(4), complex 2 converted to a species with an Fe(IV)(2)(mu-O)(2) diamond core that serves as the only synthetic model to date for the diiron(IV) core proposed for intermediate Q of soluble methane monooxygenase. Here we report detailed Mossbauer and density functional theory (DFT) studies of 2. The Mossbauer studies reveal that 2 has distinct Fe(IV) sites, a and b. Studies in applied magnetic fields show that the spins of sites a and b (S(a) = S(b) = 1) are ferromagnetically coupled to yield a ground multiplet with S = 2. Analysis of the applied field spectra of the exchange-coupled system yields for site b a set of parameters that matches those obtained for the mononuclear [LFe(IV)(O)(NCMe)](2+) complex, showing that site b (labeled Fe(O)) has a terminal oxo group. Using the zero-field splitting parameters of [LFe(IV)(O)(NCMe)](2+) for our analysis of 2, we obtained parameters for site a that closely resemble those reported for the nonoxo Fe(IV) complex [(beta-BPMCN)Fe(IV)(OH)(OO(t)Bu)](2+), suggesting that a (labeled Fe(OH)) coordinates a hydroxo group. A DFT optimization performed on 2 yielded an Fe-Fe distance of 3.39 A and an Fe-(mu-O)-Fe angle of 131 degrees , in good agreement with the results of our previous EXAFS study. The DFT calculations reproduce the Mossbauer parameters (A-tensors, electric field gradient, and isomer shift) of 2 quite well, including the observation that the largest components of the electric field gradients of Fe(O) and Fe(OH) are perpendicular. The ferromagnetic behavior of 2 seems puzzling given that the Fe-(mu-O)-Fe angle is large but can be explained by noting that the orbital structures of Fe(O) and Fe(OH) are such that the unpaired electrons at the two sites delocalize into orthogonal orbitals at the bridging oxygen, rationalizing the ferromagnetic behavior of 2. Thus, inequivalent coordinations at Fe(O) and Fe(OH) define magnetic orbitals favorable for ferromagnetic ineractions.

An ultra-stable oxoiron(IV) complex and its blue conjugate base

Treatment of [FeII(L)](OTf)2 (4), (where L = 1,4,8-Me3cyclam-11-CH2C(O)NMe2) with iodosylbenzene yielded the corresponding S = 1 oxoiron(IV) complex [FeIV(O(L)](OTf)2 (5) in nearly quantitative yield. The remarkably high stability of 5 (t1/2 ≈ 5 days at 25 °C) facilitated its characterization by X-ray crystallography and a raft of spectroscopic techniques. Treatment of 5 with strong base was found to generate a distinct, significantly less stable S = 1 oxoiron(IV) complex, 6 (t1/2 ~ 1.5 hrs. at 0 °C), which could be converted back to 5 by addition of a strong acid; these observations indicate that 5 and 6 represent a conjugate acid-base pair. That 6 can be formulated as [FeIV(O)(L-H)](OTf) was further supported by ESI mass spectrometry, spectroscopic and electrochemical studies, and DFT calculations. The close structural similarity of 5 and 6 provided a unique opportunity to probe the influence of the donor trans to the FeIV=O unit upon its reactivity in H-atom transfer (HAT) and O-atom transfer (OAT), and 5 was found to display greater reactivity than 6 in both OAT and HAT. While the greater OAT reactivity of 5 is expected on the basis of its higher redox potential, its higher HAT reactivity does not follow the anti-electrophilic trend reported for a series of [FeIV(O)(TMC)(X)] complexes (TMC = tetramethylcyclam) and thus appears to be inconsistent with the Two-State Reactivity rationale that is the prevailing explanation for the relative facility of oxoiron(IV) complexes to undergo HAT.

Mössbauer Evidence for an Exchange-Coupled {[Fe4S4]1+ Nip1+} A-Cluster in Isolated α Subunits of Acetyl-Coenzyme A Synthase/Carbon Monoxide Dehydrogenase

The active site A-cluster in the alpha subunit of the title enzyme consists of an Fe4S4 cluster coordinated to a [Nip Nid] subcomponent. The cluster must be activated for catalysis using low-potential reductants such as Ti(III) citrate. Relative to the inactive {[Fe4S4]2+ Nip2+ Nid2+} state, the activated state appears to be 2-electrons more reduced, but the location of these electrons within the A-cluster is uncertain, with {[Fe4S4]2+ Nip0 Nid2+} and {[Fe4S4]1+ Nip1+ Nid2+} configurations proposed. Recombinant apo-alpha subunits oligomerize after activation with NiCl2. The dimer fraction, upon reduction with excess Ti(III)citrate, exhibited Mössbauer spectra consisting of two quadrupole doublets representing 51% and 21% of the Fe, with parameters indicating [Fe4S4]1+ states. Spectra recorded in strong magnetic fields were typical of diamagnetic systems, indicating an exchange-coupled S = 0 {[Fe4S4]1+ Nip1+} state. Additional treatment with CO altered the doublet Mössbauer parameters, suggesting an interaction with CO, but maintaining the cluster in the {[Fe4S4]1+ Nip1+} state. Reduction with substoichiometric equivalents of Ti(III) citrate afforded an EPR signal typical of Ni1+ ions, with g parallel = 2.10 and g perpendicular = 2.02. Addition of more Ti caused the signal intensity to decline, suggesting that it arises from the semireduced {[Fe4S4]2+ Nip1+} state.