The effects of chelation on the intratubular penetration depth of mineral trioxide aggregate.

The penetration depth and extent of mineral trioxide aggregate (MTA) crystallisation into dentinal tubules at 2, 4 and 6 weeks after chelation and MTA obturation were investigated. Standardised 12 mm human root specimens (45) were prepared with NiTi rotary files using 4% NaOCl irrigation. They were randomly allocated to three irrigants (n = 15: 4% NaOCl, 15% ethylenediaminetetraacetic acid or Edgemix) and obturated with sodium fluorescein tagged ProRoot MTA. One millimetre thick apical, middle and coronal sections were examined using confocal laser scanning microscopy to determine MTA penetration depth and area. Depths varied from 352 to 1821 µm at 6 weeks depending on section level and were unaffected by chelation. No differences (p > 0.05) were found in mean maximum penetration depth or dentine area (%) between the 3 irrigants at all time intervals. MTA mineralisation penetrated up to 90% of dentinal tubules and can extend to the cementum in roots with patent, non-infected tubules.

Nuclear Resonance Vibrational Spectroscopic Definition of the Facial Triad FeIV═O Intermediate in Taurine Dioxygenase: Evaluation of Structural Contributions to Hydrogen Atom Abstraction.

The α-ketoglutarate (αKG)-dependent oxygenases catalyze a diverse range of chemical reactions using a common high-spin FeIV═O intermediate that, in most reactions, abstract a hydrogen atom from the substrate. Previously, the FeIV═O intermediate in the αKG-dependent halogenase SyrB2 was characterized by nuclear resonance vibrational spectroscopy (NRVS) and density functional theory (DFT) calculations, which demonstrated that it has a trigonal-pyramidal geometry with the scissile C-H bond of the substrate calculated to be perpendicular to the Fe-O bond. Here, we have used NRVS and DFT calculations to show that the FeIV═O complex in taurine dioxygenase (TauD), the αKG-dependent hydroxylase in which this intermediate was first characterized, also has a trigonal bipyramidal geometry but with an aspartate residue replacing the equatorial halide of the SyrB2 intermediate. Computational analysis of hydrogen atom abstraction by square pyramidal, trigonal bipyramidal, and six-coordinate FeIV═O complexes in two different substrate orientations (one more along [σ channel] and another more perpendicular [π channel] to the Fe-O bond) reveals similar activation barriers. Thus, both substrate approaches to all three geometries are competent in hydrogen atom abstraction. The equivalence in reactivity between the two substrate orientations arises from compensation of the promotion energy (electronic excitation within the d manifold) required to access the π channel by the significantly larger oxyl character present in the pπ orbital oriented toward the substrate, which leads to an earlier transition state along the C-H coordinate.

Transport of the Thyroid Hormone Carrier Protein Transthyretin into Human Epidermoid Cells.

Purpose: Transthyretin (TTR) is a protein with a growing number of biological functions in addition to its well-established binding and circulatory transport of thyroxine, and indirect retinoid transport through interaction with retinol-binding protein. Misfolded and aggregated wild-type and mutant TTRs are involved in amyloid diseases. Several aspects of TTR pathology and physiology remain poorly understood. Receptor-mediated cellular transport of TTR has been described in a few cell types; and such studies suggest the possibility of different TTR receptors and endocytic pathways. Our main objective was to further understand the endocytic pathways for TTR.Methods: In the current study, analyses of TTR endocytic transport were performed in the human A431 cell line. The results of TTR uptake were compared with those of the iron-carrier protein transferrin (Tf, a common stardard for endocytosis studies) in the same cell types.Results: A comparison of TTR and Tf endocytosis suggested similar early, 5-10 min, accumulation kinetics. But at a later time, 30 min, TTR accumulation was 20-30% lower than that of Tf (p < .05), a result that suggests different post-endocytic fates for these two ligands. Through the use of multiple endocytosis inhibitors, biochemical evidence is provided for an internalization pathway that differs from the clathrin-mediated endocytosis of Tf.Conclusions: These results for A431 cells are compared with others reported for different cell types; and it is suggested that this same hormone carrier protein can transit into cells through multiple endocytic pathways.

Endocytic Transport as a Drug Target in Infectious Diseases.

Components of the extracellular environment can be transported into cells by molecular mechanisms collectively termed endocytosis. Cells typically use a multitude of such internalization pathways. These endocytic transport pathways have a wide range of implications for physiological regulation as well as pathological processes. Many infectious diseases, for example, involve internalization of the pathogen into the cell as part of the infection process. Selective interference with the endocytic transport of a microbe, thus, represents a therapeutic strategy that may prevent infections or decrease the rate of their progression. Herein, we provide a brief review of strategies for discovery of novel anti-infection drugs and their pharmacological implications.

Peroxide Activation for Electrophilic Reactivity by the Binuclear Non-heme Iron Enzyme AurF.

Binuclear non-heme iron enzymes activate O2 for diverse chemistries that include oxygenation of organic substrates and hydrogen atom abstraction. This process often involves the formation of peroxo-bridged biferric intermediates, only some of which can perform electrophilic reactions. To elucidate the geometric and electronic structural requirements to activate peroxo reactivity, the active peroxo intermediate in 4-aminobenzoate N-oxygenase (AurF) has been characterized spectroscopically and computationally. A magnetic circular dichroism study of reduced AurF shows that its electronic and geometric structures are poised to react rapidly with O2. Nuclear resonance vibrational spectroscopic definition of the peroxo intermediate formed in this reaction shows that the active intermediate has a protonated peroxo bridge. Density functional theory computations on the structure established here show that the protonation activates peroxide for electrophilic/single-electron-transfer reactivity. This activation of peroxide by protonation is likely also relevant to the reactive peroxo intermediates in other binuclear non-heme iron enzymes.

Disruption of endocytic transport by transthyretin aggregates.

The cytotoxicity of amyloidogenic proteins such as transthyretin (TTR) has implications for neurodegeneration and other pathologies, but is not well understood. In the current study, potential effects of misfolded, aggregated TTRs (agTTR) upon a major cell membrane function-endocytosis-were assessed. Internalization of transferrin (Tf), a ligand whose receptor-mediated endocytosis is well characterized, was analyzed in different cell types after treatment with agTTR. The results indicate disruption of Tf endocytosis: 20-25% inhibition by agTTR relative to the same concentrations of normal soluble TTR, or relative to another control protein, albumin (p<0.05 for agTTR relative to controls). No statistically significant difference was observed for cell surface Tf binding between agTTR-treated and control cells. This is the first evidence for endocytic disruption by agTTR, and presents a novel cytotoxicity mechanism that may account for previously reported inhibitory effects of amyloidogenic TTR on neuronal growth.

Electronic Structure of the Ferryl Intermediate in the α-Ketoglutarate Dependent Non-Heme Iron Halogenase SyrB2: Contributions to H Atom Abstraction Reactivity.

Low temperature magnetic circular dichroism (LT MCD) spectroscopy in combination with quantum-chemical calculations are used to define the electronic structure associated with the geometric structure of the Fe(IV)═O intermediate in SyrB2 that was previously determined by nuclear resonance vibrational spectroscopy. These studies elucidate key frontier molecular orbitals (FMOs) and their contribution to H atom abstraction reactivity. The VT MCD spectra of the enzymatic S = 2 Fe(IV)═O intermediate with Br(-) ligation contain information-rich features that largely parallel the corresponding spectra of the S = 2 model complex (TMG3tren)Fe(IV)═O (Srnec, M.; Wong, S. D.; England, J; Que, L; Solomon, E. I. Proc. Natl. Acad. Sci. USA 2012, 109, 14326-14331). However, quantitative differences are observed that correlate with π-anisotropy and oxo donor strength that perturb FMOs and affect reactivity. Due to π-anisotropy, the Fe(IV)═O active site exhibits enhanced reactivity in the direction of the substrate cavity that proceeds through a π-channel that is controlled by perpendicular orientation of the substrate C-H bond relative to the halide-Fe(IV)═O plane. Also, the increased intrinsic reactivity of the SyrB2 intermediate relative to the ferryl model complex is correlated to a higher oxyl character of the Fe(IV)═O at the transition states resulting from the weaker ligand field of the halogenase.

Activities of apocynin in cytotoxicity assays of potential pathological relevance.

Low levels of some reactive oxygen and nitrogen species (ROS, RNS) are of physiological importance; but high levels result in oxidative stress and can perturb many cell functions including signal transduction and transport, and contribute to aging and chronic diseases. Apocynin (1-(4-hydroxy-3-methoxyphenyl)ethanone) is a phytochemical with reported antioxidant activities in some experimental models of human disease. The major objectives of the current study were to test the antioxidant capacity of apocynin in a hemin-peroxide assay, and test its capacity to moderate pro-oxidant-dependent inhibition of a cell function-endocytic transport. Apocynin, tested at concentrations up to 20µM, did not exhibit statistically significant antioxidant activity (94.3±7.8% relative to controls, p>0.05) in the oxidation assay. When tested against the inhibition of endocytic transport by hydrogen peroxide, apocynin treatment did not significantly rescue such inhibition in the cell types tested (p>0.05, relative to peroxide alone). When cells were treated with a cytotoxic protein aggregate that increased both ROS and RNS, apocynin treatment only inhibited production of the latter (30.0±3.6% inhibition relative to controls without apocynin, p<0.05). The results provide evidence that apocynin, unlike other phytochemicals such as curcumin, does not exhibit antioxidant activity in the heme-peroxide assay. The results also provide the first evidence that apocynin does not rescue hydrogen peroxide-mediated inhibition of endocytic transport, nor prevent hydrogen peroxide production by a cytotoxic protein aggregate. In the latter toxicity assay, however, apocynin could moderate oxidative stress by decreasing cellular levels of RNS.

Spectroscopic and computational insight into the activation of O2 by the mononuclear Cu center in polysaccharide monooxygenases.

Strategies for O2 activation by copper enzymes were recently expanded to include mononuclear Cu sites, with the discovery of the copper-dependent polysaccharide monooxygenases, also classified as auxiliary-activity enzymes 9-11 (AA9-11). These enzymes are finding considerable use in industrial biofuel production. Crystal structures of polysaccharide monooxygenases have emerged, but experimental studies are yet to determine the solution structure of the Cu site and how this relates to reactivity. From X-ray absorption near edge structure and extended X-ray absorption fine structure spectroscopies, we observed a change from four-coordinate Cu(II) to three-coordinate Cu(I) of the active site in solution, where three protein-derived nitrogen ligands coordinate the Cu in both redox states, and a labile hydroxide ligand is lost upon reduction. The spectroscopic data allowed for density functional theory calculations of an enzyme active site model, where the optimized Cu(I) and (II) structures were consistent with the experimental data. The O2 reactivity of the Cu(I) site was probed by EPR and stopped-flow absorption spectroscopies, and a rapid one-electron reduction of O2 and regeneration of the resting Cu(II) enzyme were observed. This reactivity was evaluated computationally, and by calibration to Cu-superoxide model complexes, formation of an end-on Cu-AA9-superoxide species was found to be thermodynamically favored. We discuss how this thermodynamically difficult one-electron reduction of O2 is enabled by the unique protein structure where two nitrogen ligands from His1 dictate formation of a T-shaped Cu(I) site, which provides an open coordination position for strong O2 binding with very little reorganization energy.

Excited state potential energy surfaces and their interactions in Fe(IV)=O active sites.

The non-heme ferryl active sites are of significant interest for their application in biomedical and green catalysis. These sites have been shown to have an S = 1 or S = 2 ground spin state; the latter is functional in biology. Low-temperature magnetic circular dichroism (LT MCD) spectroscopy probes the nature of the excited states in these species including ligand-field (LF) states that are otherwise difficult to study by other spectroscopies. In particular, the temperature dependences of MCD features enable their unambiguous assignment and thus determination of the low-lying excited states in two prototypical S = 1 and S = 2 NHFe(IV)[double bond, length as m-dash]O complexes. Furthermore, some MCD bands exhibit vibronic structures that allow mapping of excited-state interactions and their effects on the potential energy surfaces (PESs). For the S = 2 species, there is also an unusual spectral feature in both near-infrared absorption and MCD spectra - Fano antiresonance (dip in Abs) and Fano resonance (sharp peak in MCD) that indicates the weak spin-orbit coupling of an S = 1 state with the S = 2 LF state. These experimental data are correlated with quantum-chemical calculations that are further extended to analyze the low-lying electronic states and the evolution of their multiconfigurational characters along the Fe-O PESs. These investigations show that the lowest-energy states develop oxyl Fe(III) character at distances that are relevant to the transition state (TS) for H-atom abstraction and define the frontier molecular orbitals that participate in the reactivity of S = 1 vs. S = 2 non-heme Fe(IV)[double bond, length as m-dash]O active sites. The S = 1 species has only one available channel that requires the C-H bond of a substrate to approach perpendicular to the Fe-oxo bond (the π channel). In contrast, there are three channels (one σ and two π) available for the S = 2 non-heme Fe(IV)[double bond, length as m-dash]O system allowing C-H substrate approach both along and perpendicular to the Fe-oxo bond that have important implications for enzymatic selectivity.

Geometric and electronic structure of the Mn(IV)Fe(III) cofactor in class Ic ribonucleotide reductase: correlation to the class Ia binuclear non-heme iron enzyme.

The class Ic ribonucleotide reductase (RNR) from Chlamydia trachomatis (Ct) utilizes a Mn/Fe heterobinuclear cofactor, rather than the Fe/Fe cofactor found in the ß (R2) subunit of the class Ia enzymes, to react with O2. This reaction produces a stable Mn(IV)Fe(III) cofactor that initiates a radical, which transfers to the adjacent α (R1) subunit and reacts with the substrate. We have studied the Mn(IV)Fe(III) cofactor using nuclear resonance vibrational spectroscopy (NRVS) and absorption (Abs)/circular dichroism (CD)/magnetic CD (MCD)/variable temperature, variable field (VTVH) MCD spectroscopies to obtain detailed insight into its geometric/electronic structure and to correlate structure with reactivity; NRVS focuses on the Fe(III), whereas MCD reflects the spin-allowed transitions mostly on the Mn(IV). We have evaluated 18 systematically varied structures. Comparison of the simulated NRVS spectra to the experimental data shows that the cofactor has one carboxylate bridge, with Mn(IV) at the site proximal to Phe127. Abs/CD/MCD/VTVH MCD data exhibit 12 transitions that are assigned as d-d and oxo and OH(-) to metal charge-transfer (CT) transitions. Assignments are based on MCD/Abs intensity ratios, transition energies, polarizations, and derivative-shaped pseudo-A term CT transitions. Correlating these results with TD-DFT calculations defines the Mn(IV)Fe(III) cofactor as having a µ-oxo, µ-hydroxo core and a terminal hydroxo ligand on the Mn(IV). From DFT calculations, the Mn(IV) at site 1 is necessary to tune the redox potential to a value similar to that of the tyrosine radical in class Ia RNR, and the OH(-) terminal ligand on this Mn(IV) provides a high proton affinity that could gate radical translocation to the α (R1) subunit.

Geometric and electronic structure contributions to function in non-heme iron enzymes.

Mononuclear non-heme Fe (NHFe) enzymes play key roles in DNA repair, the biosynthesis of antibiotics, the response to hypoxia, cancer therapy, and many other biological processes. These enzymes catalyze a diverse range of oxidation reactions, including hydroxylation, halogenation, ring closure, desaturation, and electrophilic aromatic substitution (EAS). Most of these enzymes use an Fe(II) site to activate dioxygen, but traditional spectroscopic methods have not allowed researchers to insightfully probe these ferrous active sites. We have developed a methodology that provides detailed geometric and electronic structure insights into these NHFe(II) active sites. Using these data, we have defined a general mechanistic strategy that many of these enzymes use: they control O2 activation (and limit autoxidation and self-hydroxylation) by allowing Fe(II) coordination unsaturation only in the presence of cosubstrates. Depending on the type of enzyme, O2 activation either involves a 2e(-) reduced Fe(III)-OOH intermediate or a 4e(-) reduced Fe(IV)═O intermediate. Nuclear resonance vibrational spectroscopy (NRVS) has provided the geometric structure of these intermediates, and magnetic circular dichroism (MCD) has defined the frontier molecular orbitals (FMOs), the electronic structure that controls reactivity. This Account emphasizes that experimental spectroscopy is critical in evaluating the results of electronic structure calculations. Therefore these data are a key mechanistic bridge between structure and reactivity. For the Fe(III)-OOH intermediates, the anticancer drug activated bleomycin (BLM) acts as the non-heme Fe analog of compound 0 in heme (e.g., P450) chemistry. However BLM shows different reactivity: the low-spin (LS) Fe(III)-OOH can directly abstract a H atom from DNA. The LS and high-spin (HS) Fe(III)-OOHs have fundamentally different transition states. The LS transition state goes through a hydroxyl radical, but the HS transition state is activated for EAS without O-O cleavage. This activation is important in one class of NHFe enzymes that utilizes a HS Fe(III)-OOH intermediate in dioxygenation. For Fe(IV)═O intermediates, the LS form has a π-type FMO activated for attack perpendicular to the Fe-O bond. However, the HS form (present in the NHFe enzymes) has a π FMO activated perpendicular to the Fe-O bond and a σ FMO positioned along the Fe-O bond. For the NHFe enzymes, the presence of π and σ FMOs enables enzymatic control in determining the type of reactivity: EAS or H-atom extraction for one substrate with different enzymes and halogenation or hydroxylation for one enzyme with different substrates.

Elucidation of the Fe(IV)=O intermediate in the catalytic cycle of the halogenase SyrB2.

Mononuclear non-haem iron (NHFe) enzymes catalyse a broad range of oxidative reactions, including halogenation, hydroxylation, ring closure, desaturation and aromatic ring cleavage reactions. They are involved in a number of biological processes, including phenylalanine metabolism, the production of neurotransmitters, the hypoxic response and the biosynthesis of secondary metabolites. The reactive intermediate in the catalytic cycles of these enzymes is a high-spin S = 2 Fe(IV)=O species, which has been trapped for a number of NHFe enzymes, including the halogenase SyrB2 (syringomycin biosynthesis enzyme 2). Computational studies aimed at understanding the reactivity of this Fe(IV)=O intermediate are limited in applicability owing to the paucity of experimental knowledge about its geometric and electronic structure. Synchrotron-based nuclear resonance vibrational spectroscopy (NRVS) is a sensitive and effective method that defines the dependence of the vibrational modes involving Fe on the nature of the Fe(IV)=O active site. Here we present NRVS structural characterization of the reactive Fe(IV)=O intermediate of a NHFe enzyme, namely the halogenase SyrB2 from the bacterium Pseudomonas syringae pv. syringae. This intermediate reacts via an initial hydrogen-atom abstraction step, performing subsequent halogenation of the native substrate or hydroxylation of non-native substrates. A correlation of the experimental NRVS data to electronic structure calculations indicates that the substrate directs the orientation of the Fe(IV)=O intermediate, presenting specific frontier molecular orbitals that can activate either selective halogenation or hydroxylation.

Nuclear resonance vibrational spectroscopic and computational study of high-valent diiron complexes relevant to enzyme intermediates.

High-valent intermediates of binuclear nonheme iron enzymes are structurally unknown despite their importance for understanding enzyme reactivity. Nuclear resonance vibrational spectroscopy combined with density functional theory calculations has been applied to structurally well-characterized high-valent mono- and di-oxo bridged binuclear Fe model complexes. Low-frequency vibrational modes of these high-valent diiron complexes involving Fe motion have been observed and assigned. These are independent of Fe oxidation state and show a strong dependence on spin state. It is important to note that they are sensitive to the nature of the Fe2 core bridges and provide the basis for interpreting parallel nuclear resonance vibrational spectroscopy data on the high-valent oxo intermediates in the binuclear nonheme iron enzymes.

π-Frontier molecular orbitals in S = 2 ferryl species and elucidation of their contributions to reactivity.

S = 2 Fe(IV) ═ O species are key intermediates in the catalysis of most nonheme iron enzymes. This article presents detailed spectroscopic and high-level computational studies on a structurally-defined S = 2 Fe(IV) ═ O species that define its frontier molecular orbitals, which allow its high reactivity. Importantly, there are both π- and σ-channels for reaction, and both are highly reactive because they develop dominant oxyl character at the transition state. These π- and σ-channels have different orientation dependences defining how the same substrate can undergo different reactions (H-atom abstraction vs. electrophilic aromatic attack) with Fe(IV) ═ O sites in different enzymes, and how different substrates can undergo different reactions (hydroxylation vs. halogenation) with an Fe(IV) ═ O species in the same enzyme.

Definition of the intermediates and mechanism of the anticancer drug bleomycin using nuclear resonance vibrational spectroscopy and related methods.

Bleomycin (BLM) is a glycopeptide anticancer drug capable of effecting single- and double-strand DNA cleavage. The last detectable intermediate prior to DNA cleavage is a low spin Fe(III) peroxy level species, termed activated bleomycin (ABLM). DNA strand scission is initiated through the abstraction of the C-4' hydrogen atom of the deoxyribose sugar unit. Nuclear resonance vibrational spectroscopy (NRVS) aided by extended X-ray absorption fine structure spectroscopy and density functional theory (DFT) calculations are applied to define the natures of Fe(III)BLM and ABLM as (BLM)Fe(III)─OH and (BLM)Fe(III)(η(1)─OOH) species, respectively. The NRVS spectra of Fe(III)BLM and ABLM are strikingly different because in ABLM the δFe─O─O bending mode mixes with, and energetically splits, the doubly degenerate, intense O─Fe─N(ax) transaxial bends. DFT calculations of the reaction of ABLM with DNA, based on the species defined by the NRVS data, show that the direct H-atom abstraction by ABLM is thermodynamically favored over other proposed reaction pathways.

Peroxo and oxo intermediates in mononuclear nonheme iron enzymes and related active sites.

Fe(III)OOH and Fe(IV)O intermediates have now been documented in a number of nonheme iron active sites. In this Current Opinion we use spectroscopy combined with electronic structure calculations to define the frontier molecular orbitals (FMOs) of these species and their contributions to reactivity. For the low-spin Fe(III)OOH species in activated bleomycin we show that the reactivity of this nonheme iron intermediate is very different from that of the analogous Compound 0 of cytochrome P450. For Fe(IV)O S=1 model species we experimentally define the electronic structure and its contribution to reactivity, and computationally evaluate how this would change for the Fe(IV)O S=2 intermediates found in nonheme iron enzymes.