Trapping a Highly Reactive Nonheme Iron Intermediate That Oxygenates Strong CH Bonds with Stereoretention

An unprecedentedly reactive iron species (2) has been generated by reaction of excess peracetic acid with a mononuclear iron complex [FeII(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 [FeIII(OOAc)(PyNMe3)]2+, also by comparison with the EPR parameters of the structurally characterized hydroxamate analogue [FeIII(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 [FeV(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 [FeV(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 [FeIII(OOAc)(PyNMe3)]2+ precursor

Mössbauer EPR, and DFT Studies of Oxygen Activation in Enzymes and Biologically Relevant Synthetic Complexes

Long before Joseph Priestley’s observation that heating lead and mercury oxides generates oxygen, Leonardo da Vinci proposed that one of the two main gases that makeup air must be capable of supporting both flames and life. Today, nearly five hundred years after da Vinci’s death, researchers are busy trying to understand the evolution andactivation of oxygen in biochemical reactions, as well as the maladies that result when oxygen activation is not tightly regulated. At the forefront of the field of ‘oxygen activation’ are questions regarding how O2 can be made to react with high specificity at ambient temperature and how we can understand nature’s oxygen activation strategies

Defining Requirements for Heme Binding in PGRMC1 and Identifying Key Elements that Influence Protein Dimerization

Progesterone receptor membrane component 1 (PGRMC1) binds heme via a surface-exposed site and displays some structural resemblance to cytochrome b5 despite their different functions. In the case of PGRMC1, it is the protein interaction with drug-metabolizing cytochrome P450s and the epidermal growth factor receptor that has garnered the most attention. These interactions are thought to result in a compromised ability to metabolize common chemotherapy agents and to enhance cancer cell proliferation. X-ray crystallography and immunoprecipitation data have suggested that heme-mediated PGRMC1 dimers are important for facilitating these interactions. However, more recent studies have called into question the requirement of heme binding for PGRMC1 dimerization. Our study employs spectroscopic and computational methods to probe and define heme binding and its impact on PGRMC1 dimerization. Fluorescence, electron paramagnetic resonance and circular dichroism spectroscopies confirm heme binding to apo-PGRMC1 and were used to demonstrate the stabilizing effect of heme on the wild-type protein. We also utilized variants (C129S and Y113F) to precisely define the contributions of disulfide bonds and direct heme coordination to PGRMC1 dimerization. Understanding the key factors involved in these processes has important implications for downstream protein–protein interactions that may influence the metabolism of chemotherapeutic agents. This work opens avenues for deeper exploration into the physiological significance of the truncated-PGRMC1 model and developing design principles for potential therapeutics to target PGRMC1 dimerization and downstream interactions

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

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

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(star)) (TPA(star) = 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, Mossbauer 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

Kinetic analysis of amino acid radicals formed in H2O2-driven Cu-I LPMO reoxidation implicates dominant homolytic reactivity

Lytic polysaccharide monooxygenases (LPMOs) have been proposed to react with both O-2 and H2O2 as cosubstrates. In this study, the H2O2 reaction with reduced Hypocrea jecorina LPMO9A (Cu-I-HjLPMO9A) is demonstrated to be 1,000-fold faster than the O-2 reaction while producing the same oxidized oligosaccharide products. Analysis of the reactivity in the absence of polysaccharide substrate by stopped-flow absorption and rapid freeze-quench (RFQ) electron paramagnetic resonance (EPR) and magnetic circular dichroism (MCD) yields two intermediates corresponding to neutral tyrosyl and tryptophanyl radicals that are formed along minor reaction pathways. The dominant reaction pathway is characterized by RFQ EPR and kinetic modeling to directly produce Cu-II-HjLPMO9A and indicates homolytic O-O cleavage. Both optical intermediates exhibit magnetic exchange coupling with the Cu-II sites reflecting facile electron transfer (ET) pathways, which may be protective against uncoupled turnover or provide an ET pathway to the active site with substrate bound. The reactivities of nonnative organic peroxide cosubstrates effectively exclude the possibility of a ping-pong mechanism

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

Characterization of a High-Spin Non-Heme Fe-III-O Intermediate and Its Quantitative Conversion to an Fe-IV = O Complex

We have generated a high-spin Fe-III-OOH complex supported by tetramethylcyclam via protonation of its conjugate base and characterized it in detail using various spectroscopic methods. This Fe-III-OOH species can be converted quantitatively to an Fe-IV=O complex via O-O bond cleavage; this is the first example of such a conversion. This conversion is promoted by two factors: the strong Fe-III-OOH bond, which inhibits Fe-O bond lysis, and the addition of protons, which facilitates O-O bond cleavage. This example provides a synthetic precedent for how O-O bond cleavage of high-spin Fe-III-peroxo intermediates of non-heme iron enzymes may be promoted

Protonation of a Peroxodiiron(III) Complex and Conversion to a Diiron(III/IV) Intermediate: Implications for Proton-Assisted O-O Bond Cleavage in Nonheme Diiron Enzymes

Oxygenation of a diiron(II) complex, [Fe-2(II)(mu-OH)(2)(BnBQA)(2)(NCMe)(2)](2+) [2, where BnBQA is N-benzyl-N,N-bis(2-quinolinylmethyl)amine], results in the formation of a metastable peroxocliferric intermediate, 3. The treatment of 3 with strong acid affords its conjugate acid, 4, in which the (mu-oxo)(mu-1,2-peroxo)diiron(III) core of 3 is protonated at the oxo bridge. The core structures of 3 and 4 are characterized in detail by UV-vis, Mossbauer, resonance Raman, and X-ray absorption spectroscopies. Complex 4 is shorter-lived than 3 and decays to generate in similar to 20% yield of a diiron(III/IV) species 5, which can be identified by electron paramagnetic resonance and Mossbauer spectroscopies. This reaction sequence demonstrates for the first time that protonation of the oxo bridge of a (mu-oxo)(mu-1,2-peroxo)diiron(III) complex leads to cleavage of the peroxo O-O bond and formation of a high-valent diiron complex, thereby mimicking the steps involved in the formation of intermediate X in the activation cycle of ribonucleotide reductase

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