Spectroscopic and DFT Characterization of a Highly Reactive Nonheme FeV-Oxo Intermediate.

The reaction of [(PyNMe3)FeII(CF3SO3)2], 1, with excess peracetic acid at -40 °C generates a highly reactive intermediate, 2b(PAA), that has the fastest rate to date for oxidizing cyclohexane by a nonheme iron species. It exhibits an intense 490 nm chromophore associated with an S = 1/2 EPR signal having g-values at 2.07, 2.01, and 1.94. This species was shown to be in a fast equilibrium with a second S = 1/2 species, 2a(PAA), assigned to a low-spin acylperoxoiron(III) center. Unfortunately, contaminants accompanying the 2(PAA) samples prevented determination of the iron oxidation state by Mössbauer spectroscopy. Use of MeO-PyNMe3 (an electron-enriched version of PyNMe3) and cyclohexyl peroxycarboxylic acid as oxidant affords intermediate 3b(CPCA) with a Mössbauer isomer shift δ = -0.08 mm/s that indicates an iron(V) oxidation state. Analysis of the Mössbauer and EPR spectra, combined with DFT studies, demonstrates that the electronic ground state of 3b(CPCA) is best described as a quantum mechanical mixture of [(MeO-PyNMe3)FeV(O)(OC(O)R)]2+ (∼75%) with some FeIV(O)(•OC(O)R) and FeIII(OOC(O)R) character. DFT studies of 3b(CPCA) reveal that the unbound oxygen of the carboxylate ligand, O2, is only 2.04 Å away from the oxo group, O1, corresponding to a Wiberg bond order for the O1-O2 bond of 0.35. This unusual geometry facilitates reversible O1-O2 bond formation and cleavage and accounts for the high reactivity of the intermediate when compared to the rates of hydrogen atom transfer and oxygen atom transfer reactions of FeIII(OC(O)R) ferric acyl peroxides and FeIV(O) complexes. The interaction of O2 with O1 leads to a significant downshift of the Fe-O1 Raman frequency (815 cm-1) relative to the 903 cm-1 value predicted for the hypothetical [(MeO-PyNMe3)FeV(O)(NCMe)]3+ complex.

Oxoiron(IV) Tetramethylcyclam Complexes with Axial Carboxylate Ligands: Effect of Tethering the Carboxylate on Reactivity.

Oxoiron(IV) species are implicated as reactive intermediates in nonheme monoiron oxygenases, often acting as the agent for hydrogen-atom transfer from substrate. A histidine is the most likely ligand trans to the oxo unit in most enzymes characterized thus far but is replaced by a carboxylate in the case of isopenicillin N synthase. As the effect of a trans carboxylate ligand on the properties of the oxoiron(IV) unit has not been systematically studied, we have synthesized and characterized four oxoiron(IV) complexes supported by the tetramethylcyclam (TMC) macrocycle and having a carboxylate ligand trans to the oxo unit. Two complexes have acetate or propionate axial ligands, while the other two have the carboxylate functionality tethered to the macrocyclic ligand framework by one or two methylene units. Interestingly, these four complexes exhibit substrate oxidation rates that differ by more than 100-fold, despite having Ep,c values for the reduction of the Fe═O unit that span a range of only 130 mV. Eyring parameters for 1,4-cyclohexadiene oxidation show that reactivity differences originate from differences in activation enthalpy between complexes with tethered carboxylates and those with untethered carboxylates, in agreement with computational results. As noted previously for the initial subset of four complexes, the logarithms of the oxygen atom transfer rates of 11 complexes of the FeIV(O)TMC(X) series increase linearly with the observed Ep,c values, reflecting the electrophilicity of the Fe═O unit. In contrast, no correlation with Ep,c values is observed for the corresponding hydrogen atom transfer (HAT) reaction rates; instead, the HAT rates increase as the computed triplet-quintet spin state gap narrows, consistent with Shaik's two-state-reactivity model. In fact, the two complexes with untethered carboxylates are among the most reactive HAT agents in this series, demonstrating that the axial ligand can play a key role in tuning the HAT reactivity in a nonheme iron enzyme active site.

Enzyme Substrate Complex of the H200C Variant of Homoprotocatechuate 2,3-Dioxygenase: Mössbauer and Computational Studies.

The extradiol, aromatic ring-cleaving enzyme homoprotocatechuate 2,3-dioxygenase (HPCD) catalyzes a complex chain of reactions that involve second sphere residues of the active site. The importance of the second-sphere residue His200 was demonstrated in studies of HPCD variants, such as His200Cys (H200C), which revealed significant retardations of certain steps in the catalytic process as a result of the substitution, allowing novel reaction cycle intermediates to be trapped for spectroscopic characterization. As the H200C variant largely retains the wild-type active site structure and produces the correct ring-cleaved product, this variant presents a valuable target for mechanistic HPCD studies. Here, the high-spin Fe(II) states of resting H200C and the H200C-homoprotocatechuate enzyme-substrate (ES) complex have been characterized with Mössbauer spectroscopy to assess the electronic structures of the active site in these states. The analysis reveals a high-spin Fe(II) center in a low symmetry environment that is reflected in the values of the zero-field splitting (ZFS) (D ≈ - 8 cm(-1), E/D ≈ 1/3 in ES), as well as the relative orientations of the principal axes of the (57)Fe magnetic hyperfine (A) and electric field gradient (EFG) tensors relative to the ZFS tensor axes. A spin Hamiltonian analysis of the spectra for the ES complex indicates that the magnetization axis of the integer-spin S = 2 Fe(II) system is nearly parallel to the symmetry axis, z, of the doubly occupied dxy ground orbital deduced from the EFG and A-values, an observation, which cannot be rationalized by DFT assisted crystal-field theory. In contrast, ORCA/CASSCF calculations for the ZFS tensor in combination with DFT calculations for the EFG- and A-tensors describe the experimental data remarkably well.

Spectroscopic and Theoretical Study of Spin-Dependent Electron Transfer in an Iron(III) Superoxo Complex.

It was shown previously (J. Am. Chem. Soc. 2014, 136, 10846) that bubbling of O2 into a solution of Fe(II)(BDPP) (H2BDPP = 2,6-bis[[(S)-2-(diphenylhydroxymethyl)-1-pyrrolidinyl]methyl]pyridine) in tetrahydrofuran at -80 °C generates a high-spin (SFe = (5)/2) iron(III) superoxo adduct, 1. Mössbauer studies revealed that 1 is an exchange-coupled system, [Formula: see text], where SR = (1)/2 is the spin of the superoxo radical, of which the spectra were not well enough resolved to determine whether the coupling was ferromagnetic (S = 3 ground state) or antiferromagnetic (S = 2). The glass-forming 2-methyltetrahydrofuran solvent yields highly resolved Mössbauer spectra from which the following data have been extracted: (i) the ground state of 1 has S = 3 (J < 0); (ii) |J| > 15 cm(-1); (iii) the zero-field-splitting parameters are D = -1.1 cm(-1) and E/D = 0.02; (iv) the major component of the electric-field-gradient tensor is tilted ≈7° relative to the easy axis of magnetization determined by the MS = ±3 and ±2 doublets. The excited-state MS = ±2 doublet yields a narrow parallel-mode electron paramagnetic resonance signal at g = 8.03, which was used to probe the magnetic hyperfine splitting of (17)O-enriched O2. A theoretical model that considers spin-dependent electron transfer for the cases where the doubly occupied π* orbital of the superoxo ligand is either "in" or "out" of the plane defined by the bent Fe-OO moiety correctly predicts that 1 has an S = 3 ground state, in contrast to the density functional theory calculations for 1, which give a ground state with both the wrong spin and orbital configuration. This failure has been traced to a basis set superposition error in the interactions between the superoxo moiety and the adjacent five-membered rings of the BDPP ligand and signals a fundamental problem in the quantum chemistry of O2 activation.

Upside Down! Crystallographic and Spectroscopic Characterization of an [Fe IV(O syn)(TMC)]2+ Complex.

Fe(II)(TMC)(OTf)2 reacts with 2-(t)BuSO2-C6H4IO to afford an oxoiron(IV) product, 2, distinct from the previously reported [Fe(IV)(Oanti)(TMC)(NCMe)](2+). In MeCN, 2 has a blue-shifted near-IR band, a higher ν(Fe═O), a larger Mössbauer quadrupole splitting, and quite a distinct (1)H NMR spectrum. Structural analysis of crystals grown from CH2Cl2 reveals a complex with the formulation of [Fe(IV)(Osyn)(TMC)(OTf)](OTf) and the shortest Fe(IV)═O bond [1.625(4) Å] found to date.

Trapping a Highly Reactive Nonheme Iron Intermediate That Oxygenates Strong C-H Bonds with Stereoretention.

An unprecedentedly reactive iron species (2) has been generated by reaction of excess peracetic acid with a mononuclear iron complex [Fe(II)(CF3SO3)2(PyNMe3)] (1) at cryogenic temperatures, and characterized spectroscopically. Compound 2 is kinetically competent for breaking strong C-H bonds of alkanes (BDE ≈ 100 kcal·mol(-1)) through a hydrogen-atom transfer mechanism, and the transformations proceed with stereoretention and regioselectively, responding to bond strength, as well as to steric and polar effects. Bimolecular reaction rates are at least an order of magnitude faster than those of the most reactive synthetic high-valent nonheme oxoiron species described to date. EPR studies in tandem with kinetic analysis show that the 490 nm chromophore of 2 is associated with two S = 1/2 species in rapid equilibrium. The minor component 2a (∼5% iron) has g-values at 2.20, 2.19, and 1.99 characteristic of a low-spin iron(III) center, and it is assigned as [Fe(III)(OOAc)(PyNMe3)](2+), also by comparison with the EPR parameters of the structurally characterized hydroxamate analogue [Fe(III)(tBuCON(H)O)(PyNMe3)](2+) (4). The major component 2b (∼40% iron, g-values = 2.07, 2.01, 1.95) has unusual EPR parameters, and it is proposed to be [Fe(V)(O)(OAc)(PyNMe3)](2+), where the O-O bond in 2a has been broken. Consistent with this assignment, 2b undergoes exchange of its acetate ligand with CD3CO2D and very rapidly reacts with olefins to produce the corresponding cis-1,2-hydroxoacetate product. Therefore, this work constitutes the first example where a synthetic nonheme iron species responsible for stereospecific and site selective C-H hydroxylation is spectroscopically trapped, and its catalytic reactivity against C-H bonds can be directly interrogated by kinetic methods. The accumulated evidence indicates that 2 consists mainly of an extraordinarily reactive [Fe(V)(O)(OAc)(PyNMe3)](2+) (2b) species capable of hydroxylating unactivated alkyl C-H bonds with stereoretention in a rapid and site-selective manner, and that exists in fast equilibrium with its [Fe(III)(OOAc)(PyNMe3)](2+) precursor.

A Long-Lived Fe(III)-(Hydroperoxo) Intermediate in the Active H200C Variant of Homoprotocatechuate 2,3-Dioxygenase: Characterization by Mössbauer, Electron Paramagnetic Resonance, and Density Functional Theory Methods.

The extradiol-cleaving dioxygenase homoprotocatechuate 2,3-dioxygenase (HPCD) binds substrate homoprotocatechuate (HPCA) and O2 sequentially in adjacent ligand sites of the active site Fe(II). Kinetic and spectroscopic studies of HPCD have elucidated catalytic roles of several active site residues, including the crucial acid-base chemistry of His200. In the present study, reaction of the His200Cys (H200C) variant with native substrate HPCA resulted in a decrease in both kcat and the rate constants for the activation steps following O2 binding by >400 fold. The reaction proceeds to form the correct extradiol product. This slow reaction allowed a long-lived (t1/2 = 1.5 min) intermediate, H200C-HPCAInt1 (Int1), to be trapped. Mössbauer and parallel mode electron paramagnetic resonance (EPR) studies show that Int1 contains an S1 = 5/2 Fe(III) center coupled to an SR = 1/2 radical to give a ground state with total spin S = 2 (J > 40 cm(-1)) in Hexch = JS1·SR. Density functional theory (DFT) property calculations for structural models suggest that Int1 is a (HPCA semiquinone(•))Fe(III)(OOH) complex, in which OOH is protonated at the distal O and the substrate hydroxyls are deprotonated. By combining Mössbauer and EPR data of Int1 with DFT calculations, the orientations of the principal axes of the (57)Fe electric field gradient and the zero-field splitting tensors (D = 1.6 cm(-1), E/D = 0.05) were determined. This information was used to predict hyperfine splittings from bound (17)OOH. DFT reactivity analysis suggests that Int1 can evolve from a ferromagnetically coupled Fe(III)-superoxo precursor by an inner-sphere proton-coupled-electron-transfer process. Our spectroscopic and DFT results suggest that a ferric hydroperoxo species is capable of extradiol catalysis.

Oxoiron(IV) Complex of the Ethylene-Bridged Dialkylcyclam Ligand Me2EBC.

We report herein the first example of an oxoiron(IV) complex of an ethylene-bridged dialkylcyclam ligand, [Fe(IV)(O)(Me2EBC)(NCMe)](2+) (2; Me2EBC = 4,11-dimethyl-1,4,8,11-tetraazabicyclo[6.6.2]hexadecane). Complex 2 has been characterized by UV-vis, (1)H NMR, resonance Raman, Mössbauer, and X-ray absorption spectroscopy as well as electrospray ionization mass spectrometry, and its properties have been compared with those of the closely related [Fe(IV)(O)(TMC)(NCMe)](2+) (3; TMC = 1,4,8,11-tetramethyl-1,4,8,11-tetraazacyclotetradecane), the intensively studied prototypical oxoiron(IV) complex of the macrocyclic tetramethylcyclam ligand. Me2EBC has an N4 donor set nearly identical with that of TMC but possesses an ethylene bridge in place of the 1- and 8-methyl groups of TMC. As a consequence, Me2EBC is forced to deviate from the trans-I configuration typically found for Fe(IV)(O)(TMC) complexes and instead adopts a folded cis-V stereochemistry that requires the MeCN ligand to coordinate cis to the Fe(IV)═O unit in 2 rather than in the trans arrangement found in 3. However, switching from the trans geometry of 3 to the cis geometry of 2 did not significantly affect their ground-state electronic structures, although a decrease in ν(Fe═O) was observed for 2. Remarkably, despite having comparable Fe(IV/III) reduction potentials, 2 was found to be significantly more reactive than 3 in both oxygen-atom-transfer (OAT) and hydrogen-atom-transfer (HAT) reactions. A careful analysis of density functional theory calculations on the HAT reactivity of 2 and 3 revealed the root cause to be the higher oxyl character of 2, leading to a stronger O---H bond specifically in the quintet transition state.

Spectroscopic identification of an Fe(III) center, not Fe(IV), in the crystalline Sc-O-Fe adduct derived from [Fe(IV)(O)(TMC)]²âº.

The apparent Sc(3+) adduct of [Fe(IV)(O)(TMC)](2+) (1, TMC = 1,4,8,11-tetramethyl-1,4,8,11-tetraazacyclotetradecane) has been synthesized in amounts sufficient to allow its characterization by various spectroscopic techniques. Contrary to the earlier assignment of a +4 oxidation state for the iron center of 1, we establish that 1 has a high-spin iron(III) center based on its Mössbauer and EPR spectra and its quantitative reduction by 1 equiv of ferrocene to [Fe(II)(TMC)](2+). Thus, 1 is best described as a Sc(III)-O-Fe(III) complex, in agreement with previous DFT calculations (Swart, M. Chem. Commun. 2013, 49, 6650.). These results shed light on the interaction of Lewis acids with high-valent metal-oxo species.

Modeling TauD-J: a high-spin nonheme oxoiron(IV) complex with high reactivity toward C-H bonds.

High-spin oxoiron(IV) species are often implicated in the mechanisms of nonheme iron oxygenases, their C-H bond cleaving properties being attributed to the quintet spin state. However, the few available synthetic S = 2 Fe(IV)═O complexes supported by polydentate ligands do not cleave strong C-H bonds. Herein we report the characterization of a highly reactive S = 2 complex, [Fe(IV)(O)(TQA)(NCMe)](2+) (2) (TQA = tris(2-quinolylmethyl)amine), which oxidizes both C-H and C═C bonds at -40 °C. The oxidation of cyclohexane by 2 occurs at a rate comparable to that of the oxidation of taurine by the TauD-J enzyme intermediate after adjustment for the different temperatures of measurement. Moreover, compared with other S = 2 complexes characterized to date, the spectroscopic properties of 2 most closely resemble those of TauD-J. Together these features make 2 the best electronic and functional model for TauD-J to date.

An unusual peroxo intermediate of the arylamine oxygenase of the chloramphenicol biosynthetic pathway.

Streptomyces venezuelae CmlI catalyzes the six-electron oxygenation of the arylamine precursor of chloramphenicol in a nonribosomal peptide synthetase (NRPS)-based pathway to yield the nitroaryl group of the antibiotic. Optical, EPR, and Mössbauer studies show that the enzyme contains a nonheme dinuclear iron cluster. Addition of O(2) to the diferrous state of the cluster results in an exceptionally long-lived intermediate (t(1/2) = 3 h at 4 °C) that is assigned as a peroxodiferric species (CmlI-peroxo) based upon the observation of an (18)O(2)-sensitive resonance Raman (rR) vibration. CmlI-peroxo is spectroscopically distinct from the well characterized and commonly observed cis-µ-1,2-peroxo (µ-η(1):η(1)) intermediates of nonheme diiron enzymes. Specifically, it exhibits a blue-shifted broad absorption band around 500 nm and a rR spectrum with a ν(O-O) that is at least 60 cm(-1) lower in energy. Mössbauer studies of the peroxo state reveal a diferric cluster having iron sites with small quadrupole splittings and distinct isomer shifts (0.54 and 0.62 mm/s). Taken together, the spectroscopic comparisons clearly indicate that CmlI-peroxo does not have a µ-η(1):η(1)-peroxo ligand; we propose that a µ-η(1):η(2)-peroxo ligand accounts for its distinct spectroscopic properties. CmlI-peroxo reacts with a range of arylamine substrates by an apparent second-order process, indicating that CmlI-peroxo is the reactive species of the catalytic cycle. Efficient production of chloramphenicol from the free arylamine precursor suggests that CmlI catalyzes the ultimate step in the biosynthetic pathway and that the precursor is not bound to the NRPS during this step.

Redesigning the blue copper azurin into a redox-active mononuclear nonheme iron protein: preparation and study of Fe(II)-M121E azurin.

Much progress has been made in designing heme and dinuclear nonheme iron enzymes. In contrast, engineering mononuclear nonheme iron enzymes is lagging, even though these enzymes belong to a large class that catalyzes quite diverse reactions. Herein we report spectroscopic and X-ray crystallographic studies of Fe(II)-M121E azurin (Az), by replacing the axial Met121 and Cu(II) in wild-type azurin (wtAz) with Glu and Fe(II), respectively. In contrast to the redox inactive Fe(II)-wtAz, the Fe(II)-M121EAz mutant can be readily oxidized by Na2IrCl6, and interestingly, the protein exhibits superoxide scavenging activity. Mössbauer and EPR spectroscopies, along with X-ray structural comparisons, revealed similarities and differences between Fe(II)-M121EAz, Fe(II)-wtAz, and superoxide reductase (SOR) and allowed design of the second generation mutant, Fe(II)-M121EM44KAz, that exhibits increased superoxide scavenging activity by 2 orders of magnitude. This finding demonstrates the importance of noncovalent secondary coordination sphere interactions in fine-tuning enzymatic activity.

Characterization of a paramagnetic mononuclear nonheme iron-superoxo complex.

O2 bubbling into a THF solution of Fe(II)(BDPP) (1) at -80 °C generates a reversible bright yellow adduct 2. Characterization by resonance Raman and Mössbauer spectroscopy provides complementary insights into the nature of 2. The former shows a resonance-enhanced vibration at 1125 cm(-1), which can be assigned to the ν(O-O) of a bound superoxide, while the latter reveals the presence of a high-spin iron(III) center that is exchange-coupled to the superoxo ligand, like the Fe(III)-O2(-) pair found for the O2 adduct of 4-nitrocatechol-bound homoprotocatechuate 2,3-dioxygenase. Lastly, 2 oxidizes dihydroanthracene to anthracene, supporting the notion that Fe(III)-O2(-) species can carry out H atom abstraction from a C-H bond to initiate the 4-electron oxidation of substrates proposed for some nonheme iron enzymes.

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.

Identification of a low-spin acylperoxoiron(III) intermediate in bio-inspired non-heme iron-catalysed oxidations.

Synthetically useful hydrocarbon oxidations are catalysed by bio-inspired non-heme iron complexes using hydrogen peroxide as oxidant, and carboxylic acid addition enhances their selectivity and catalytic efficiency. Talsi has identified a low-intensity g=2.7 electron paramagnetic resonance signal in such catalytic systems and attributed it to an oxoiron(V)-carboxylate oxidant. Herein we report the use of Fe(II)(TPA*) (TPA*=tris(3,5-dimethyl-4-methoxypyridyl-2-methyl)amine) to generate this intermediate in 50% yield, and have characterized it by ultraviolet-visible, resonance Raman, Mössbauer and electrospray ionization mass spectrometric methods as a low-spin acylperoxoiron(III) species. Kinetic studies show that this intermediate is not itself the oxidant but decays via a unimolecular rate-determining step to unmask a powerful oxidant. The latter is shown by density functional theory calculations to be an oxoiron(V) species that oxidises substrate without a barrier. This study provides a mechanistic scenario for understanding catalyst reactivity and selectivity as well as a basis for improving catalyst design.

Spectroscopic and theoretical investigation of a complex with an [O═Fe(IV)-O-Fe(IV)═O] core related to methane monooxygenase intermediate Q.

Previous efforts to model the diiron(IV) intermediate Q of soluble methane monooxygenase have led to the synthesis of a diiron(IV) TPA complex, 2, with an O=Fe(IV)-O-Fe(IV)-OH core that has two ferromagnetically coupled Sloc = 1 sites. Addition of base to 2 at -85 °C elicits its conjugate base 6 with a novel O═Fe(IV)-O-Fe(IV)═O core. In frozen solution, 6 exists in two forms, 6a and 6b, that we have characterized extensively using Mössbauer and parallel mode EPR spectroscopy. The conversion between 2 and 6 is quantitative, but the relative proportions of 6a and 6b are solvent dependent. 6a has two equivalent high-spin (Sloc = 2) sites, which are antiferromagnetically coupled; its quadrupole splitting (0.52 mm/s) and isomer shift (0.14 mm/s) match those of intermediate Q. DFT calculations suggest that 6a assumes an anti conformation with a dihedral O═Fe-Fe═O angle of 180°. Mössbauer and EPR analyses show that 6b is a diiron(IV) complex with ferromagnetically coupled Sloc = 1 and Sloc = 2 sites to give total spin St = 3. Analysis of the zero-field splittings and magnetic hyperfine tensors suggests that the dihedral O═Fe-Fe═O angle of 6b is ∼90°. DFT calculations indicate that this angle is enforced by hydrogen bonding to both terminal oxo groups from a shared water molecule. The water molecule preorganizes 6b, facilitating protonation of one oxo group to regenerate 2, a protonation step difficult to achieve for mononuclear Fe(IV)═O complexes. Complex 6 represents an intriguing addition to the handful of diiron(IV) complexes that have been characterized.

Sc3+-triggered oxoiron(IV) formation from O2 and its non-heme iron(II) precursor via a Sc3+-peroxo-Fe3+ intermediate.

We report that redox-inactive Sc(3+) can trigger O2 activation by the Fe(II)(TMC) center (TMC = tetramethylcyclam) to generate the corresponding oxoiron(IV) complex in the presence of BPh4(-) as an electron donor. To model a possible intermediate in the above reaction, we generated an unprecedented Sc(3+) adduct of [Fe(III)(η(2)-O2)(TMC)](+) by an alternative route, which was found to have an Fe(3+)-(µ-η(2):η(2)-peroxo)-Sc(3+) core and to convert to the oxoiron(IV) complex. These results have important implications for the role a Lewis acid can play in facilitating O-O bond cleavage during the course of O2 activation at non-heme iron centers.

Intermediate P* from soluble methane monooxygenase contains a diferrous cluster.

During a single turnover of the hydroxylase component (MMOH) of soluble methane monooxygenase from Methylosinus trichosporium OB3b, several discrete intermediates are formed. The diiron cluster of MMOH is first reduced to the Fe(II)Fe(II) state (H(red)). O2 binds rapidly at a site away from the cluster to form the Fe(II)Fe(II) intermediate O, which converts to an Fe(III)Fe(III)-peroxo intermediate P and finally to the Fe(IV)Fe(IV) intermediate Q. Q binds and reacts with methane to yield methanol and water. The rate constants for these steps are increased by a regulatory protein, MMOB. Previously reported transient kinetic studies have suggested that an intermediate P* forms between O and P in which the g = 16 EPR signal characteristic of the reduced diiron cluster of H(red) and O is lost. This was interpreted as signaling oxidation of the cluster, but a low level of accumulation of P* prevented further characterization. In this study, three methods for directly detecting and trapping P* are applied together to allow its spectroscopic and kinetic characterization. First, the MMOB mutant His33Ala is used to specifically slow the decay of P* without affecting its formation rate, leading to its nearly quantitative accumulation. Second, spectra-kinetic data collection is used to provide a sensitive measure of the formation and decay rate constants of intermediates as well as their optical spectra. Finally, the substrate furan is included to react with Q and quench its strong chromophore. The optical spectrum of P* closely mimics those of H(red) and O, but it is distinctly different from that of P. The reaction cycle rate constants allowed prediction of the times for maximal accumulation of the intermediates. Mössbauer spectra of rapid freeze-quench samples at these times show that the intermediates are formed at almost exactly the predicted levels. The Mössbauer spectra show that the diiron cluster of P*, quite unexpectedly, is in the Fe(II)Fe(II) state. Thus, the loss of the g = 16 EPR signal results from a change in the electronic structure of the Fe(II)Fe(II) center rather than oxidation. The similarity of the optical and Mössbauer spectra of H(red), O, and P* suggests that only subtle changes occur in the electronic and physical structure of the diiron cluster as P* forms. Nevertheless, the changes that do occur are necessary for O2 to be activated for hydrocarbon oxidation.

Cyanobacterial aldehyde deformylase oxygenation of aldehydes yields n-1 aldehydes and alcohols in addition to alkanes.

Aldehyde-deformylating oxygenase (ADO) catalyzes O2-dependent release of the terminal carbon of a biological substrate, octadecanal, to yield formate and heptadecane in a reaction that requires external reducing equivalents. We show here that ADO also catalyzes incorporation of an oxygen atom from O2 into the alkane product to yield alcohol and aldehyde products. Oxygenation of the alkane product is much more pronounced with C9-10 aldehyde substrates, so that use of nonanal as the substrate yields similar amounts of octane, octanal, and octanol products. When using doubly-labeled [1,2-13C]-octanal as the substrate, the heptane, heptanal and heptanol products each contained a single 13C-label in the C-1 carbons atoms. The only one-carbon product identified was formate. [18O]-O2 incorporation studies demonstrated formation of [18O]-alcohol product, but rapid solvent exchange prevented similar determination for the aldehyde product. Addition of [1-13C]-nonanol with decanal as the substrate at the outset of the reaction resulted in formation of [1-13C]-nonanal. No 13C-product was formed in the absence of decanal. ADO contains an oxygen-bridged dinuclear iron cluster. The observation of alcohol and aldehyde products derived from the initially formed alkane product suggests a reactive species similar to that formed by methane monooxygenase (MMO) and other members of the bacterial multicomponent monooxygenase family. Accordingly, characterization by EPR and Mössbauer spectroscopies shows that the electronic structure of the ADO cluster is similar, but not identical, to that of MMO hydroxylase component. In particular, the two irons of ADO reside in nearly identical environments in both the oxidized and fully reduced states, whereas those of MMOH show distinct differences. These favorable characteristics of the iron sites allow a comprehensive determination of the spin Hamiltonian parameters describing the electronic state of the diferrous cluster for the first time for any biological system. The nature of the diiron cluster and the newly recognized products from ADO catalysis hold implications for the mechanism of C-C bond cleavage.

A Mononuclear Carboxylate-Rich Oxoiron(IV) Complex: a Structural and Functional Mimic of TauD Intermediate 'J'

The pentadentate ligand (n)Bu-P2DA (2(b), (n)Bu-P2DA = N-(1',1'-bis(2-pyridyl)pentyl)iminodiacetate) was designed to bind an iron center in a carboxylate-rich environment similar to that found in the active sites of TauD and other α-ketoglutarate-dependent mononuclear non-heme iron enzymes. The iron(II) complex (n)Bu(4)N[Fe(II)(Cl)((n)Bu-P2DA)] (3(b)-Cl) was synthesized and crystallographically characterized to have a 2-pyridine-2-carboxylate donor set in the plane perpendicular to the Fe-Cl bond. Reaction of 3(b)-Cl with N-heterocyclic amines such as pyridine or imidazole yielded the N-heterocyclic amine adducts [Fe(II)(N)((n)Bu-P2DA)]. These adducts in turn reacted with oxo-transfer reagents at -95 °C to afford a short-lived oxoiron(IV) complex [Fe(IV)(O)((n)Bu-P2DA)] (5(b)) in yields as high as 90% depending on the heterocycle used. Complex 5(b) exhibits near-IR absorption features (λ(max) = 770 nm) and Mossbauer parameters (δ = 0.04 mm/s; ΔE(Q) = 1.13 mm/s; D = 27±2 cm(-1)) characteristic of an S = 1 oxoiron(IV) species. Direct evidence for an Fe=O bond of 1.66 Å was found from EXAFS analysis. DFT calculations on 5(b) in its S =1 spin state afforded a geometry-optimized structure consistent with the EXAFS data. They further demonstrated that the replacement of two pyridine donors in [Fe(IV)(O)(N4Py)](2+) (N4Py = N,N-(bis(2-pyridyl)methyl)N-bis(2-pyridylmethyl)amine) with carboxylate donors in 5(b) decreased the energy gap between the ground S = 1 and the excited S = 2 states, reflecting the weaker equatorial ligand field of 5(b) and accounting for its larger D value. Complex 5(b) reacted readily with dihydrotoluene, methyldiphenylphosphine and ferrocene at -60 °C, and in all cases was approximately a 5-fold more reactive oxidant than [Fe(IV)(O)(N4Py)](2+). The reactivity differences between these two complexes may arise from a combination of electronic and steric factors. Carboxylate-rich 5(b) represents the closest structural mimic reported thus far of the oxoiron(IV) intermediate ('J') found in TauD and provides us with vital insights into the role carboxylate ligands play in modulating the spectroscopic and reactivity properties of the non-heme oxoiron(IV) moiety.