Substrate-Mediated Oxygen Activation by Homoprotocatechuate 2,3-Dioxygenase: Intermediates Formed by a Tyrosine 257 Variant

Homoprotocatechuate (HPCA; 3,4-dihydroxyphenylacetate or 4-carboxymethyl catechol) and O₂ bind in adjacent ligand sites of the active site Fe^II of homoprotocatechuate 2,3-dioxygenase (FeHPCD). We have proposed that electron transfer from the chelated aromatic substrate through the Fe^II to O₂ gives both substrates radical character. This would promote reaction between the substrates to form an alkylperoxo intermediate as the first step in aromatic ring cleavage. Several active site amino acids are thought to promote these reactions through acid/base chemistry, hydrogen bonding, and electrostatic interactions. Here the role of Tyr257 is explored by using the Tyr257Phe (Y257F) variant, which decreases <i>k</i>_cat by about 75%. The crystal structure of the FeHPCD-HPCA complex has shown that Tyr257 hydrogen bonds to the deprotonated C2-hydroxyl of HPCA. Stopped-flow studies show that at least two reaction intermediates, termed Y257F_Int1^HPCA and Y257F_Int2^HPCA, accumulate during the Y257F-HPCA + O₂ reaction prior to formation of the ring-cleaved product. Y257F_Int1^HPCA is colorless and is formed as O₂ binds reversibly to the HPCA–enzyme complex. Y257F_Int2^HPCA forms spontaneously from Y257F_Int1^HPCA and displays a chromophore at 425 nm (ε₄₂₅ = 10 500 M^–1 cm^–1). Mössbauer spectra of the intermediates trapped by rapid freeze quench show that both intermediates contain Fe^II. The lack of a chromophore characteristic of a quinone or semiquinone form of HPCA, the presence of Fe^II, and the low O₂ affinity suggest that Y257F_Int1^HPCA is an HPCA-Fe^II-O₂ complex with little electron delocalization onto the O₂. In contrast, the intense spectrum of Y257F_Int2^HPCA suggests the intermediate is most likely an HPCA quinone-Fe^II-(hydro)­peroxo species. Steady-state and transient kinetic analyses show that steps of the catalytic cycle are slowed by as much as 100-fold by the mutation. These effects can be rationalized by a failure of Y257F to facilitate the observed distortion of the bound HPCA that is proposed to promote transfer of one electron to O₂

Spectroscopic and Theoretical Investigation of a Complex with an [OFe^IV–O–Fe^IVO] Core Related to Methane Monooxygenase Intermediate <b>Q</b>

Previous efforts to model the diiron­(IV) intermediate <b>Q</b> of soluble methane monooxygenase have led to the synthesis of a diiron­(IV) TPA complex, <b>2</b>, with an O=Fe^IV–O–Fe^IV–OH core that has two ferromagnetically coupled S_loc = 1 sites. Addition of base to <b>2</b> at −85 °C elicits its conjugate base <b>6</b> with a novel OFe^IV–O–Fe^IVO core. In frozen solution, <b>6</b> exists in two forms, <b>6a</b> and <b>6b</b>, that we have characterized extensively using Mössbauer and parallel mode EPR spectroscopy. The conversion between <b>2</b> and <b>6</b> is quantitative, but the relative proportions of <b>6a</b> and <b>6b</b> are solvent dependent. <b>6a</b> has two equivalent high-spin (<i>S</i>_loc = 2) sites, which are antiferromagnetically coupled; its quadrupole splitting (0.52 mm/s) and isomer shift (0.14 mm/s) match those of intermediate <b>Q</b>. DFT calculations suggest that <b>6a</b> assumes an anti conformation with a dihedral OFe–FeO angle of 180°. Mössbauer and EPR analyses show that <b>6b</b> is a diiron­(IV) complex with ferromagnetically coupled <i>S</i>_loc = 1 and <i>S</i>_loc = 2 sites to give total spin <i>S</i>_t = 3. Analysis of the zero-field splittings and magnetic hyperfine tensors suggests that the dihedral OFe–FeO angle of <b>6b</b> 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 <b>6b</b>, facilitating protonation of one oxo group to regenerate <b>2</b>, a protonation step difficult to achieve for mononuclear Fe^IVO complexes. Complex <b>6</b> 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 Sc³⁺ 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 Sc³⁺ adduct of [Fe^III(η²-O₂)­(TMC)]⁺ by an alternative route, which was found to have an Fe³⁺–(μ-η²:η²-peroxo)–Sc³⁺ 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

Upside Down! Crystallographic and Spectroscopic Characterization of an [Fe^IV(O_syn)(TMC)]²⁺ Complex

Fe^II(TMC)­(OTf)₂ reacts with 2-^tBuSO₂–C₆H₄IO to afford an oxoiron­(IV) product, <b>2</b>, distinct from the previously reported [Fe^IV(O_anti)­(TMC)­(NCMe)]²⁺. In MeCN, <b>2</b> has a blue-shifted near-IR band, a higher ν­(FeO), a larger Mössbauer quadrupole splitting, and quite a distinct ¹H NMR spectrum. Structural analysis of crystals grown from CH₂Cl₂ reveals a complex with the formulation of [Fe^IV(O_syn)­(TMC)­(OTf)]­(OTf) and the shortest Fe^IVO bond [1.625(4) Å] found to date

Structural, EPR, and Mössbauer Characterization of (μ-Alkoxo)(μ-Carboxylato)Diiron(II,III) Model Complexes for the Active Sites of Mixed-Valent Diiron Enzymes

To obtain structural and spectroscopic models for the diiron­(II,III) centers in the active sites of diiron enzymes, the (μ-alkoxo)­(μ-carboxylato)­diiron­(II,III) complexes [Fe^IIFe^III(<i>N</i>-Et-HPTB)­(O₂CPh)­(NCCH₃)₂]­(ClO₄)₃ (<b>1</b>) and [Fe^IIFe^III(<i>N</i>-Et-HPTB)­(O₂CPh)­(Cl)­(HOCH₃)]­(ClO₄)₂ (<b>2</b>) (<i>N</i>-Et-HPTB = <i>N,N,N</i>′<i>,N</i>′-tetrakis­(2-(1-ethyl-benzimidazolylmethyl))-2-hydroxy-1,3-diaminopropane) have been prepared and characterized by X-ray crystallography, UV–visible absorption, EPR, and Mössbauer spectroscopies. Fe1–Fe2 separations are 3.60 and 3.63 Å, and Fe1–O1–Fe2 bond angles are 128.0° and 129.4° for <b>1</b> and <b>2</b>, respectively. Mössbauer and EPR studies of <b>1</b> show that the Fe^III (<i>S</i>_A = 5/2) and Fe^II (<i>S</i>_B = 2) sites are antiferromagnetically coupled to yield a ground state with <i>S</i> = 1/2 (<i>g</i> <b>=</b> 1.75, 1.88, 1.96); Mössbauer analysis of solid <b>1</b> yields <i>J</i> = 22.5 ± 2 cm^–1 for the exchange coupling constant (H = <i>J</i><b>S</b>_A·<b>S</b>_B convention). In addition to the <i>S</i> = 1/2 ground-state spectrum of <b>1</b>, the EPR signal for the <i>S</i> = 3/2 excited state of the spin ladder can also be observed, the first time such a signal has been detected for an antiferromagnetically coupled diiron­(II,III) complex. The anisotropy of the ⁵⁷Fe magnetic hyperfine interactions at the Fe^III site is larger than normally observed in mononuclear complexes and arises from admixing <i>S</i> > 1/2 excited states into the <i>S</i> = 1/2 ground state by zero-field splittings at the two Fe sites. Analysis of the “<i>D</i>/<i>J</i>” mixing has allowed us to extract the zero-field splitting parameters, local <i>g</i> values, and magnetic hyperfine structural parameters for the individual Fe sites. The methodology developed and followed in this analysis is presented in detail. The spin Hamiltonian parameters of <b>1</b> are related to the molecular structure with the help of DFT calculations. Contrary to what was assumed in previous studies, our analysis demonstrates that the deviations of the <i>g</i> values from the free electron value (<i>g</i> = 2) for the antiferromagnetically coupled diiron­(II,III) core in complex <b>1</b> are predominantly determined by the anisotropy of the effective <i>g</i> values of the ferrous ion and only to a lesser extent by the admixture of excited states into ground-state ZFS terms (<i>D</i>/<i>J</i> mixing). The results for <b>1</b> are discussed in the context of the data available for diiron­(II,III) clusters in proteins and synthetic diiron­(II,III) complexes

Structural, EPR, and Mössbauer Characterization of (μ-Alkoxo)(μ-Carboxylato)Diiron(II,III) Model Complexes for the Active Sites of Mixed-Valent Diiron Enzymes

To obtain structural and spectroscopic models for the diiron­(II,III) centers in the active sites of diiron enzymes, the (μ-alkoxo)­(μ-carboxylato)­diiron­(II,III) complexes [Fe^IIFe^III(<i>N</i>-Et-HPTB)­(O₂CPh)­(NCCH₃)₂]­(ClO₄)₃ (<b>1</b>) and [Fe^IIFe^III(<i>N</i>-Et-HPTB)­(O₂CPh)­(Cl)­(HOCH₃)]­(ClO₄)₂ (<b>2</b>) (<i>N</i>-Et-HPTB = <i>N,N,N</i>′<i>,N</i>′-tetrakis­(2-(1-ethyl-benzimidazolylmethyl))-2-hydroxy-1,3-diaminopropane) have been prepared and characterized by X-ray crystallography, UV–visible absorption, EPR, and Mössbauer spectroscopies. Fe1–Fe2 separations are 3.60 and 3.63 Å, and Fe1–O1–Fe2 bond angles are 128.0° and 129.4° for <b>1</b> and <b>2</b>, respectively. Mössbauer and EPR studies of <b>1</b> show that the Fe^III (<i>S</i>_A = 5/2) and Fe^II (<i>S</i>_B = 2) sites are antiferromagnetically coupled to yield a ground state with <i>S</i> = 1/2 (<i>g</i> <b>=</b> 1.75, 1.88, 1.96); Mössbauer analysis of solid <b>1</b> yields <i>J</i> = 22.5 ± 2 cm^–1 for the exchange coupling constant (H = <i>J</i><b>S</b>_A·<b>S</b>_B convention). In addition to the <i>S</i> = 1/2 ground-state spectrum of <b>1</b>, the EPR signal for the <i>S</i> = 3/2 excited state of the spin ladder can also be observed, the first time such a signal has been detected for an antiferromagnetically coupled diiron­(II,III) complex. The anisotropy of the ⁵⁷Fe magnetic hyperfine interactions at the Fe^III site is larger than normally observed in mononuclear complexes and arises from admixing <i>S</i> > 1/2 excited states into the <i>S</i> = 1/2 ground state by zero-field splittings at the two Fe sites. Analysis of the “<i>D</i>/<i>J</i>” mixing has allowed us to extract the zero-field splitting parameters, local <i>g</i> values, and magnetic hyperfine structural parameters for the individual Fe sites. The methodology developed and followed in this analysis is presented in detail. The spin Hamiltonian parameters of <b>1</b> are related to the molecular structure with the help of DFT calculations. Contrary to what was assumed in previous studies, our analysis demonstrates that the deviations of the <i>g</i> values from the free electron value (<i>g</i> = 2) for the antiferromagnetically coupled diiron­(II,III) core in complex <b>1</b> are predominantly determined by the anisotropy of the effective <i>g</i> values of the ferrous ion and only to a lesser extent by the admixture of excited states into ground-state ZFS terms (<i>D</i>/<i>J</i> mixing). The results for <b>1</b> are discussed in the context of the data available for diiron­(II,III) clusters in proteins and synthetic diiron­(II,III) complexes

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) (<i>D</i> ≈ – 8 cm^–1, <i>E</i>/<i>D</i> ≈ 1/3 in ES), as well as the relative orientations of the principal axes of the ⁵⁷Fe magnetic hyperfine (<b>A</b>) 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 <i>S</i> = 2 Fe^II system is nearly parallel to the symmetry axis, <i>z</i>, of the doubly occupied d_<i>xy</i> ground orbital deduced from the EFG and <b>A</b>-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 <b>A</b>-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 (<i>J. Am. Chem. Soc.</i> <b>2014</b>, <i>136</i>, 10846) that bubbling of O₂ into a solution of Fe^II(BDPP) (H₂BDPP = 2,6-bis­[[(<i>S</i>)-2-(diphenylhydroxymethyl)-1-pyrrolidinyl]­methyl]­pyridine) in tetrahydrofuran at −80 °C generates a high-spin (<i>S</i>_Fe = ⁵/₂) iron­(III) superoxo adduct, <b>1</b>. Mössbauer studies revealed that <b>1</b> is an exchange-coupled system, Ĥex=JŜFe·ŜR, where <i>S</i>_R = 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 (<i>S</i> = 3 ground state) or antiferromagnetic (<i>S</i> = 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 <b>1</b> has <i>S</i> = 3 (<i>J</i> < 0); (ii) |<i>J</i>| > 15 cm^–1; (iii) the zero-field-splitting parameters are <i>D</i> = −1.1 cm^–1 and <i>E</i>/<i>D</i> = 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 <i>M</i>_<i>S</i> = ±3 and ±2 doublets. The excited-state <i>M</i>_<i>S</i> = ±2 doublet yields a narrow parallel-mode electron paramagnetic resonance signal at <i>g</i> = 8.03, which was used to probe the magnetic hyperfine splitting of ¹⁷O-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 <b>1</b> has an <i>S</i> = 3 ground state, in contrast to the density functional theory calculations for <b>1</b>, 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

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 <i>k</i>_cat 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 (<i>t</i>_1/2 = 1.5 min) intermediate, H200C-HPCA_Int1 (<i>Int1</i>), to be trapped. Mössbauer and parallel mode electron paramagnetic resonance (EPR) studies show that <i>Int1</i> contains an <i>S</i>₁ = 5/2 Fe^III center coupled to an <i>S</i>_R = 1/2 radical to give a ground state with total spin <i>S</i> = 2 (<i>J</i> > 40 cm^–1) in Hexch=JŜ1·ŜR. Density functional theory (DFT) property calculations for structural models suggest that <i>Int1</i> 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 <i>Int1</i> with DFT calculations, the orientations of the principal axes of the ⁵⁷Fe electric field gradient and the zero-field splitting tensors (<i>D</i> = 1.6 cm^–1, <i>E</i>/<i>D</i> = 0.05) were determined. This information was used to predict hyperfine splittings from bound ¹⁷OOH. DFT reactivity analysis suggests that <i>Int1</i> 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

Spectroscopic Identification of an Fe^III Center, not Fe^IV, in the Crystalline Sc–O–Fe Adduct Derived from [Fe^IV(O)(TMC)]²⁺

The apparent Sc³⁺ adduct of [Fe^IV(O)­(TMC)]²⁺ (<b>1</b>, 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 <b>1</b>, we establish that <b>1</b> 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, <b>1</b> 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