Identification of a Catalytic Iron-Hydride at the H‑Cluster of [FeFe]-Hydrogenase

Hydrogenases couple electrochemical potential to the reversible chemical transformation of H₂ and protons, yet the reaction mechanism and composition of intermediates are not fully understood. In this Communication we describe the biophysical properties of a hydride-bound state (H_hyd) of the [FeFe]-hydrogenase from <i>Chlamydomonas reinhardtii</i>. The catalytic H-cluster of [FeFe]-hydrogenase consists of a [4Fe-4S] subcluster ([4Fe-4S]_H) linked by a cysteine thiol to an azadithiolate-bridged 2Fe subcluster ([2Fe]_H) with CO and CN^– ligands. Mössbauer analysis and density functional theory (DFT) calculations show that H_hyd consists of a reduced [4Fe-4S]_H⁺ coupled to a diferrous [2Fe]_H with a terminally bound Fe-hydride. The existence of the Fe-hydride in H_hyd was demonstrated by an unusually low Mössbauer isomer shift of the distal Fe of the [2Fe]_H subcluster. A DFT model of H_hyd shows that the Fe-hydride is part of a H-bonding network with the nearby bridging azadithiolate to facilitate fast proton exchange and catalytic turnover

Modeling Non-Heme Iron Halogenases: High-Spin Oxoiron(IV)–Halide Complexes That Halogenate C–H Bonds

The non-heme iron halogenases CytC3 and SyrB2 catalyze C–H bond halogenation in the biosynthesis of some natural products via <i>S</i> = 2 oxoiron­(IV)–halide intermediates. These oxidants abstract a hydrogen atom from a substrate C–H bond to generate an alkyl radical that reacts with the bound halide to form a C–X bond chemoselectively. The origin of this selectivity has been explored in biological systems but has not yet been investigated with synthetic models. Here we report the characterization of <i>S</i> = 2 [Fe^IV(O)­(TQA)­(Cl/Br)]⁺ (TQA = tris­(quinolyl-2-methyl)­amine) complexes that can preferentially halogenate cyclohexane. These are the first synthetic oxoiron­(IV)–halide complexes that serve as spectroscopic and functional models for the halogenase intermediates. Interestingly, the nascent substrate radicals generated by these synthetic complexes are not as short-lived as those obtained from heme-based oxidants and can be intercepted by O₂ to prevent halogenation, supporting an emerging notion that rapid rebound may not necessarily occur in non-heme oxoiron­(IV) oxidations

Mechanistic Investigation of a Non-Heme Iron Enzyme Catalyzed Epoxidation in (−)-4′-Methoxycyclopenin Biosynthesis

Mechanisms have been proposed for α-KG-dependent non-heme iron enzyme catalyzed oxygen atom insertion into an olefinic moiety in various natural products, but they have not been examined in detail. Using a combination of methods including transient kinetics, Mössbauer spectroscopy, and mass spectrometry, we demonstrate that AsqJ-catalyzed (−)-4′-methoxy­cyclopenin formation uses a high-spin Fe­(IV)-oxo intermediate to carry out epoxidation. Furthermore, product analysis on ¹⁶O/¹⁸O isotope incorporation from the reactions using the native substrate, 4′-methoxy­dehydro­cyclopeptin, and a mechanistic probe, dehydro­cyclopeptin, reveals evidence supporting oxo↔hydroxo tautomerism of the Fe­(IV)-oxo species in the non-heme iron enzyme catalysis

CmlI <i>N</i>‑Oxygenase Catalyzes the Final Three Steps in Chloramphenicol Biosynthesis without Dissociation of Intermediates

CmlI catalyzes the six-electron oxidation of an aryl-amine precursor (NH₂-CAM) to the aryl-nitro group of chloramphenicol (CAM). The active site of CmlI contains a (hydr)­oxo- and carboxylate-bridged dinuclear iron cluster. During catalysis, a novel diferric-peroxo intermediate <b>P</b> is formed and is thought to directly effect oxygenase chemistry. Peroxo intermediates can facilitate at most two-electron oxidations, so the biosynthetic pathway of CmlI must involve at least three steps. Here, kinetic techniques are used to characterize the rate and/or dissociation constants for each step by taking advantage of the remarkable stability of <b>P</b> in the absence of substrates (decay <i>t</i>_1/2 = 3 h at 4 °C) and the visible chromophore of the diiron cluster. It is found that diferrous CmlI (CmlI^red) can react with NH₂-CAM and O₂ in either order to form a <b>P</b>-NH₂-CAM intermediate. <b>P</b>-NH₂-CAM undergoes rapid oxygen transfer to form a diferric CmlI (CmlI^ox) complex with the aryl-hydroxylamine [NH­(OH)-CAM] pathway intermediate. CmlI^ox-NH­(OH)-CAM undergoes a rapid internal redox reaction to form a CmlI^red-nitroso-CAM (NO-CAM) complex. O₂ binding results in formation of <b>P</b>-NO-CAM that converts to CmlI^ox-CAM by enzyme-mediated oxygen atom transfer. The kinetic analysis indicates that there is little dissociation of pathway intermediates as the reaction progresses. Reactions initiated by adding pathway intermediates from solution occur much more slowly than those in which the intermediate is generated in the active site as part of the catalytic process. Thus, CmlI is able to preserve efficiency and specificity while avoiding adventitious chemistry by performing the entire six-electron oxidation in one active site

Mechanism for Six-Electron Aryl-N-Oxygenation by the Non-Heme Diiron Enzyme CmlI

The ultimate step in chloramphenicol (CAM) biosynthesis is a six-electron oxidation of an aryl-amine precursor (NH₂-CAM) to the aryl-nitro group of CAM catalyzed by the non-heme diiron cluster-containing oxygenase CmlI. Upon exposure of the diferrous cluster to O₂, CmlI forms a long-lived peroxo intermediate, <b>P</b>, which reacts with NH₂-CAM to form CAM. Since <b>P</b> is capable of at most a two-electron oxidation, the overall reaction must occur in several steps. It is unknown whether <b>P</b> is the oxidant in each step or whether another oxidizing species participates in the reaction. Mass spectrometry product analysis of reactions under ¹⁸O₂ show that both oxygen atoms in the nitro function of CAM derive from O₂. However, when the single-turnover reaction between ¹⁸O₂-<b>P</b> and NH₂-CAM is carried out in an ¹⁶O₂ atmosphere, CAM nitro groups contain both ¹⁸O and ¹⁶O, suggesting that <b>P</b> can be reformed during the reaction sequence. Such reformation would require reduction by a pathway intermediate, shown here to be NH­(OH)-CAM. Accordingly, the aerobic reaction of NH­(OH)-CAM with diferric CmlI yields <b>P</b> and then CAM without an external reductant. A catalytic cycle is proposed in which NH₂-CAM reacts with <b>P</b> to form NH­(OH)-CAM and diferric CmlI. Then the NH­(OH)-CAM rereduces the enzyme diiron cluster, allowing <b>P</b> to reform upon O₂ binding, while itself being oxidized to NO-CAM. Finally, the reformed <b>P</b> oxidizes NO-CAM to CAM with incorporation of a second O₂-derived oxygen atom. The complete six-electron oxidation requires only two exogenous electrons and could occur in one active site

Mechanistic Investigation of Oxidative Decarboxylation Catalyzed by Two Iron(II)- and 2‑Oxoglutarate-Dependent Enzymes

Two non-heme iron enzymes, IsnB and AmbI3, catalyze a novel decarboxylation-assisted olefination to produce indole vinyl isonitrile, an important building block for many natural products. Compared to other reactions catalyzed by this enzyme family, decarboxylation-assisted olefination represents an attractive biosynthetic route and a mechanistically unexplored pathway in constructing a CC bond. Using mechanistic probes, transient state kinetics, reactive intermediate trapping, spectroscopic characterizations, and product analysis, we propose that both IsnB and AmbI3 initiate stereoselective olefination via a benzylic C–H bond activation by an Fe­(IV)–oxo intermediate, and the reaction likely proceeds through a radical- or carbocation-induced decarboxylation to complete CC bond installation

Characterization of the Fleeting Hydroxoiron(III) Complex of the Pentadentate TMC-py Ligand

Nonheme mononuclear hydroxoiron­(III) species are important intermediates in biological oxidations, but well-characterized examples of synthetic complexes are scarce due to their instability or tendency to form μ-oxodiiron­(III) complexes, which are the thermodynamic sink for such chemistry. Herein, we report the successful stabilization and characterization of a mononuclear hydroxoiron­(III) complex, [Fe^III(OH)­(TMC-py)]²⁺ (<b>3</b>; TMC-py = 1<i>-</i>(pyridyl-2′-methyl)-4,8,11-trimethyl-1,4,8,11-tetrazacyclotetradecane), which is directly generated from the reaction of [Fe^IV(O)­(TMC-py)]²⁺ (<b>2</b>) with 1,4-cyclohexadiene at −40 °C by H-atom abstraction. Complex <b>3</b> exhibits a UV spectrum with a λ_max at 335 nm (ε ≈ 3500 M^–1 cm^–1) and a molecular ion in its electrospray ionization mass spectrum at <i>m</i>/<i>z</i> 555 with an isotope distribution pattern consistent with its formulation. Electron paramagnetic resonance and Mössbauer spectroscopy show <b>3</b> to be a high-spin Fe­(III) center that is formed in 85% yield. Extended X-ray absorption fine structure analysis reveals an Fe–OH bond distance of 1.84 Å, which is also found in [(TMC-py)­Fe^III–O–Cr^III(OTf)₃]⁺ (<b>4</b>) obtained from the reaction of <b>2</b> with Cr­(OTf)₂. The <i>S</i> = 5/2 spin ground state and the 1.84 Å Fe–OH bond distance are supported computationally. Complex <b>3</b> reacts with 1-hydroxy-2,2,6,6-tetramethylpiperidine (TEMPOH) at −40 °C with a second-order rate constant of 7.1 M^–1 s^–1 and an OH/OD kinetic isotope effect value of 6. On the basis of density functional theory calculations, the reaction between <b>3</b> and TEMPOH is classified as a proton-coupled electron transfer as opposed to a hydrogen-atom transfer

Characterization of [4Fe-4S] Cluster Vibrations and Structure in Nitrogenase Fe Protein at Three Oxidation Levels via Combined NRVS, EXAFS, and DFT Analyses

Azotobacter vinelandii nitrogenase Fe protein (<i>Av2</i>) provides a rare opportunity to investigate a [4Fe-4S] cluster at three oxidation levels in the same protein environment. Here, we report the structural and vibrational changes of this cluster upon reduction using a combination of NRVS and EXAFS spectroscopies and DFT calculations. Key to this work is the synergy between these three techniques as each generates highly complementary information and their analytical methodologies are interdependent. Importantly, the spectroscopic samples contained no glassing agents. NRVS and DFT reveal a systematic 10–30 cm^–1 decrease in Fe–S stretching frequencies with each added electron. The “oxidized” [4Fe-4S]²⁺ state spectrum is consistent with and extends previous resonance Raman spectra. For the “reduced” [4Fe-4S]¹⁺ state in Fe protein, and for any “all-ferrous” [4Fe-4S]⁰ cluster, these NRVS spectra are the first available vibrational data. NRVS simulations also allow estimation of the vibrational disorder for Fe–S and Fe–Fe distances, constraining the EXAFS analysis and allowing structural disorder to be estimated. For oxidized <i>Av2</i>, EXAFS and DFT indicate nearly equal Fe–Fe distances, while addition of one electron decreases the cluster symmetry. However, addition of the second electron to form the all-ferrous state induces significant structural change. EXAFS data recorded to <i>k</i> = 21 Å^–1 indicates a 1:1 ratio of Fe–Fe interactions at 2.56 Å and 2.75 Å, a result consistent with DFT. Broken symmetry (BS) DFT rationalizes the interplay between redox state and the Fe–S and Fe–Fe distances as predominantly spin-dependent behavior inherent to the [4Fe-4S] cluster and perturbed by the <i>Av2</i> protein environment

O–H Activation by an Unexpected Ferryl Intermediate during Catalysis by 2‑Hydroxyethylphosphonate Dioxygenase

Activation of O–H bonds by inorganic metal-oxo complexes has been documented, but no cognate enzymatic process is known. Our mechanistic analysis of 2-hydroxy­ethyl­phosphonate dioxygenase (HEPD), which cleaves the C1–C2 bond of its substrate to afford hydroxy­methyl­phosphonate on the biosynthetic pathway to the commercial herbicide phosphino­thricin, uncovered an example of such an O–H-bond-cleavage event. Stopped-flow UV–visible absorption and freeze-quench Mössbauer experiments identified a transient iron­(IV)-oxo (ferryl) complex. Maximal accumulation of the intermediate required both the presence of deuterium in the substrate and, importantly, the use of ²H₂O as solvent. The ferryl complex forms and decays rapidly enough to be on the catalytic pathway. To account for these unanticipated results, a new mechanism that involves activation of an O–H bond by the ferryl complex is proposed. This mechanism accommodates all available data on the HEPD reaction

The Two Faces of Tetramethylcyclam in Iron Chemistry: Distinct Fe–O–M Complexes Derived from [Fe^IV(O_{<i>anti</i>/<i>syn</i>})(TMC)]²⁺ Isomers

Tetramethylcyclam (TMC, 1,4,8,11-tetramethyl-1,4,8,11-tetraazacyclotetradecane) exhibits two faces in supporting an oxoiron­(IV) moiety, as exemplified by the prototypical [(TMC)­Fe^IV(O_<i>anti</i>)­(NCCH₃)]­(OTf)₂, where <i>anti</i> indicates that the O atom is located on the face opposite all four methyl groups, and the recently reported <i>syn</i> isomer [(TMC)­Fe^IV(O_<i>syn</i>)­(OTf)]­(OTf). The ability to access two isomers of [(TMC)­Fe^IV(O_{<i>anti</i>/<i>syn</i>})] raises the fundamental question of how ligand topology can affect the properties of the metal center. Previously, we have reported the formation of [(CH₃CN)­(TMC)­Fe^III–O_<i>anti</i>–Cr^III(OTf)₄(NCCH₃)] (<b>1</b>) by inner-sphere electron transfer between Cr­(OTf)₂ and [(TMC)­Fe^IV(O_<i>anti</i>)­(NCCH₃)]­(OTf)₂. Herein we demonstrate that a new species <b>2</b> is generated from the reaction between Cr­(OTf)₂ and [(TMC)­Fe^IV(O_<i>syn</i>)­(NCCH₃)]­(OTf)₂, which is formulated as [(TMC)­Fe^III–O_<i>syn</i>–Cr^III(OTf)₄(NCCH₃)] based on its characterization by UV–vis, resonance Raman, Mössbauer, and X-ray absorption spectroscopic methods, as well as electrospray mass spectrometry. Its pre-edge area (30 units) and Fe–O distance (1.77 Å) determined by X-ray absorption spectroscopy are distinctly different from those of <b>1</b> (11-unit pre-edge area and 1.81 Å Fe–O distance) but more closely resemble the values reported for [(TMC)­Fe^III–O_<i>syn</i>–Sc^III(OTf)₄(NCCH₃)] (<b>3</b>, 32-unit pre-edge area and 1.75 Å Fe–O distance). This comparison suggests that <b>2</b> has a square pyramidal iron center like <b>3</b>, rather than the six-coordinate center deduced for <b>1</b>. Density functional theory calculations further validate the structures for <b>1</b> and <b>2</b>. The influence of the distinct TMC topologies on the coordination geometries is further confirmed by the crystal structures of [(Cl)­(TMC)­Fe^III–O_<i>anti</i>–Fe^IIICl₃] (<b>4</b>_<b>Cl</b>) and [(TMC)­Fe^III–O_<i>syn</i>–Fe^IIICl₃]­(OTf) (<b>5</b>). Complexes <b>1</b>–<b>5</b> thus constitute a set of complexes that shed light on ligand topology effects on the coordination chemistry of the oxoiron moiety