The transport activity of the multidrug ABC transporter BmrA does not require a wide separation of the nucleotide-binding domains.

ATP-binding cassette (ABC) transporters are ubiquitous membrane proteins responsible for the translocation of a wide diversity of substrates across biological membranes. Some of them confer multidrug or antimicrobial resistance to cancer cells and pathogenic microorganisms, respectively. Despite a wealth of structural data gained in the last two decades, the molecular mechanism of these multidrug efflux pumps remains elusive, including the extent of separation between the two nucleotide-binding domains (NBDs) during the transport cycle. Based on recent outward-facing structures of BmrA, a homodimeric multidrug ABC transporter from Bacillus subtilis, we introduced a cysteine mutation near the C-terminal end of the NBDs to analyze the impact of disulfide-bond formation on BmrA function. Interestingly, the presence of the disulfide bond between the NBDs did not prevent the ATPase, nor did it affect the transport of Hoechst 33342 and doxorubicin. Yet, the 7-amino-actinomycin D was less efficiently transported, suggesting that a further opening of the transporter might improve its ability to translocate this larger compound. We solved by cryo-EM the apo structures of the cross-linked mutant and the WT protein. Both structures are highly similar, showing an intermediate opening between their NBDs while their C-terminal extremities remain in close proximity. Distance measurements obtained by electron paramagnetic resonance spectroscopy support the intermediate opening found in these 3D structures. Overall, our data suggest that the NBDs of BmrA function with a tweezers-like mechanism distinct from the related lipid A exporter MsbA.

Structural insights into the semiquinone form of human Cytochrome P450 reductase by DEER distance measurements between a native flavin and a spin labelled non-canonical amino acid.

The flavoprotein Cytochrome P450 reductase (CPR) is the unique electron pathway from NADPH to Cytochrome P450 (CYPs). The conformational dynamics of human CPR in solution, which involves transitions from a "locked/closed" to an "unlocked/open" state, is crucial for electron transfer. To date, however, the factors guiding these changes remain unknown. By Site-Directed Spin Labelling coupled to Electron Paramagnetic Resonance spectroscopy, we have incorporated a non-canonical amino acid onto the flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) domains of soluble human CPR, and labelled it with a specific nitroxide spin probe. Taking advantage of the endogenous FMN cofactor, we successfully measured for the first time, the distance distribution by DEER between the semiquinone state FMNH• and the nitroxide. The DEER data revealed a salt concentration-dependent distance distribution, evidence of an "open" CPR conformation at high salt concentrations exceeding previous reports. We also conducted molecular dynamics simulations which unveiled a diverse ensemble of conformations for the "open" semiquinone state of the CPR at high salt concentration. This study unravels the conformational landscape of the one electron reduced state of CPR, which had never been studied before.

Guidelines for the Simulations of Nitroxide X-Band cw EPR Spectra from Site-Directed Spin Labeling Experiments Using SimLabel.

Site-directed spin labeling (SDSL) combined with continuous wave electron paramagnetic resonance (cw EPR) spectroscopy is a powerful technique to reveal, at the local level, the dynamics of structural transitions in proteins. Here, we consider SDSL-EPR based on the selective grafting of a nitroxide on the protein under study, followed by X-band cw EPR analysis. To extract valuable quantitative information from SDSL-EPR spectra and thus give a reliable interpretation on biological system dynamics, a numerical simulation of the spectra is required. However, regardless of the numerical tool chosen to perform such simulations, the number of parameters is often too high to provide unambiguous results. In this study, we have chosen SimLabel to perform such simulations. SimLabel is a graphical user interface (GUI) of Matlab, using some functions of Easyspin. An exhaustive review of the parameters used in this GUI has enabled to define the adjustable parameters during the simulation fitting and to fix the others prior to the simulation fitting. Among them, some are set once and for all (gy, gz) and others are determined (Az, gx) thanks to a supplementary X-band spectrum recorded on a frozen solution. Finally, we propose guidelines to perform the simulation of X-band cw-EPR spectra of nitroxide labeled proteins at room temperature, with no need of uncommon higher frequency spectrometry and with the minimal number of variable parameters.

The Hunt for the Closed Conformation of the Fruit-Ripening Enzyme 1-Aminocyclopropane-1-carboxylic Oxidase: A Combined Electron Paramagnetic Resonance and Molecular Dynamics Study.

1-Aminocyclopropane-1-carboxylic oxidase (ACCO) is a non-heme iron(II)-containing enzyme involved in the biosynthesis of the phytohormone ethylene, which regulates fruit ripening and flowering in plants. The active conformation of ACCO, and in particular that of the C-terminal part, remains unclear and open and closed conformations have been proposed. In this work, a combined experimental and computational study to understand the conformation and dynamics of the C-terminal part is reported. Site-directed spin-labeling coupled to electron paramagnetic resonance (SDSL-EPR) spectroscopy was used. Mutagenesis experiments were performed to generate active enzymes bearing two paramagnetic labels (nitroxide radicals) anchored on cysteine residues, one in the main core and one in the C-terminal part. Inter-spin distance distributions were measured by pulsed EPR spectroscopy and compared with the results of molecular dynamics simulations. The results reveal the existence of a flexibility of the C-terminal part. This flexibility generates several conformations of the C-terminal part of ACCO that correspond neither to the existing crystal structures nor to the modelled structures. This highly dynamic region of ACCO raises questions on its exact function during enzymatic activity.

Dimerization interface and dynamic properties of yeast IF1 revealed by Site-Directed Spin Labeling EPR spectroscopy.

The mitochondrial ATPase inhibitor, IF1, regulates the activity of the mitochondrial ATP synthase. The oligomeric state of IF1 related to pH is crucial for its inhibitory activity. Although extensive structural studies have been performed to characterize the oligomeric states of bovine IF1, only little is known concerning those of yeast IF1. While bovine IF1 can be found as an inhibitory dimer at low pH and a non-inhibitory tetramer at high pH, a monomer/dimer equilibrium has been described for yeast IF1, high pH values favoring the monomeric state. Combining different strategies involving the grafting of nitroxide spin labels combined with Electron Paramagnetic Resonance (EPR) spectroscopy, the present study brings the first structural characterization, at the residue level, of yeast IF1 in its dimeric form. The results show that the dimerization interface involves the central region of the peptide revealing that the dimer corresponds to a non-inhibitory state. Moreover, we demonstrate that the C-terminal region of the peptide is highly dynamic and that this segment is probably folded back onto the central region. Finally, the pH-dependence of the inter-label distance distribution has been observed indicating a conformational change between two structural states in the dimer.

Characterization of Cu(II)-reconstituted ACC Oxidase using experimental and theoretical approaches.

1-Aminocyclopropane-1-carboxylic acid oxidase (ACCO) is a non heme iron(II) containing enzyme that catalyzes the final step of the ethylene biosynthesis in plants. The iron(II) ion is bound in a facial triad composed of two histidines and one aspartate (H177, D179 and H234). Several active site variants were generated to provide alternate binding motifs and the enzymes were reconstituted with copper(II). Continuous wave (cw) and pulsed Electron Paramagnetic Resonance (EPR) spectroscopies as well as Density Functional Theory (DFT) calculations were performed and models for the copper(II) binding sites were deduced. In all investigated enzymes, the copper ion is equatorially coordinated by the two histidine residues (H177 and H234) and probably two water molecules. The copper-containing enzymes are inactive, even when hydrogen peroxide is used in peroxide shunt approach. EPR experiments and DFT calculations were undertaken to investigate substrate's (ACC) binding on the copper ion and the results were used to rationalize the lack of copper-mediated activity.

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.

Temperature Effect on Ionic Current and ssDNA Transport through Nanopores.

We have investigated the role of electrostatic interactions in the transport of nucleic acids and ions through nanopores. The passage of DNA through nanopores has so far been conjectured to involve a free-energy barrier for entry, followed by a downhill translocation where the driving voltage accelerates the polymer. We have tested the validity of this conjecture by using two toxins, α-hemolysin and aerolysin, which differ in their shape, size, and charge. The characteristic timescales in each toxin as a function of temperature show that the entry barrier is ∼15 kBT and the translocation barrier is ∼35 kBT, although the electrical force in the latter step is much stronger. Resolution of this fact, using a theoretical model, reveals that the attraction between DNA and the charges inside the barrel of the pore is the most dominant factor in determining the translocation speed and not merely the driving electrochemical potential gradient.

Diversification of EPR signatures in Site Directed Spin Labeling using a ß-phosphorylated nitroxide.

Site Directed Spin Labeling (SDSL) combined with EPR spectroscopy is a very powerful approach to investigate structural transitions in proteins in particular flexible or even disordered ones. Conventional spin labels are based on nitroxide derivatives leading to classical 3-line spectra whose spectral shapes are indicative of the environment of the labels and thus constitute good reporters of structural modifications. However, the similarity of these spectral shapes precludes probing two regions of a protein or two partner proteins simultaneously. To overcome the limitation due to the weak diversity of nitroxide label EPR spectral shapes, we designed a new spin label based on a ß-phosphorylated nitroxide giving 6-line spectra. This paper describes the synthesis of this new spin label, its grafting at four different positions of a model disordered protein able to undergo an induced α-helical folding and its characterization by EPR spectroscopy. For comparative purposes, a classical nitroxide has been grafted at the same positions of the model protein. The ability of the new label to report on structural transitions was evaluated by analyzing the spectral shape modifications induced either by the presence of a secondary structure stabilizer (trifluoroethanol) or by the presence of a partner protein. Taken together the results demonstrate that the new phosphorylated label gives a very distinguishable signature which is able to report from subtle to larger structural transitions, as efficiently as the classical spin label. As a complementary approach, molecular dynamics (MD) calculations were performed to gain further insights into the binding process between the labeled NTAIL and PXD. MD calculations revealed that the new label does not disturb the interaction between the two partner proteins and reinforced the conclusion on its ability to probe different local environments in a protein. Taken together this study represents an important step forward in the extension of the panoply of SDSL-EPR approaches.

Structural Flexibility of Tau in Its Interaction with Microtubules as Viewed by Site-Directed Spin Labeling EPR Spectroscopy.

Tau is a microtubule-associated protein that belongs to the Intrinsically Disordered Proteins (IDPs) family. IDPs or Intrinsically Disordered Regions (IDRs) play key roles in protein interaction networks and their dysfunctions are often related to severe diseases. Defined by their lack of stable secondary and tertiary structures in physiological conditions while being functional, these proteins use their inherent structural flexibility to adapt to and interact with various binding partners. Knowledges on the structural dynamics of IDPs and their different conformers are crucial to finely decipher fundamental biological processes controlled by mechanisms such as conformational adaptations or switches, induced fit, or conformational selection events. Different mechanisms of binding have been proposed: among them, the so-called folding-upon-binding in which the IDP adopts a certain conformation upon interacting with a partner protein, or the formation of a "fuzzy" complex in which the IDP partly keeps its dynamical character at the surface of its partner. The dynamical nature and physicochemical properties of unbound as well as bound IDPs make this class of proteins particularly difficult to characterize by classical bio-structural techniques and require specific approaches for the fine description of their inherent dynamics.Among other techniques, Site-Directed Spin Labeling combined with Electron Paramagnetic Resonance (SDSL-EPR) spectroscopy has gained much interest in this last decade for the study of IDPs. SDSL-EPR consists in grafting a paramagnetic label (mainly a nitroxide radical) at selected site(s) of the macromolecule under interest followed by its observation using and/or combining different EPR strategies. These nitroxide spin labels detected by continuous wave (cw) EPR spectroscopy are used as perfect reporters or "spy spins" of their local environment, being able to reveal structural transitions, folding/unfolding events, etc. Another approach is based on the measurement of inter-label distance distributions in the 1.5-8.0 nm range using pulsed dipolar EPR experiments, such as Double Electron-Electron Resonance (DEER) spectroscopy. The technique is then particularly well suited to study the behavior of Tau in its interaction with its physiological partner: microtubules (MTs). In this chapter we provide a detailed experimental protocol for the labeling of Tau protein and its EPR study while interacting with preformed (Paclitaxel-stabilized) MTs, or using Tau as MT inducer. We show how the choice of nitroxide label can be crucial to obtain functional information on Tau/tubulin complexes.

Cytochrome c prompts the recruitment of its nuclear partners SET/TAF-Iß and NPM1 into biomolecular condensates.

Compartmentalization of proteins by liquid-liquid phase separation (LLPS) is used by cells to control biochemical reactions spatially and temporally. Among them, the recruitment of proteins to DNA foci and nucleolar trafficking occur by biomolecular condensation. Within this frame, the oncoprotein SET/TAF-Iß plays a key role in both chromatin remodeling and DNA damage response, as does nucleophosmin (NPM1) which indeed participates in nucleolar ribosome synthesis. Whereas phase separation by NPM1 is widely characterized, little is known about that undergone by SET/TAF-Iß. Here, we show that SET/TAF-Iß experiences phase separation together with respiratory cytochrome c (Cc), which translocates to the nucleus upon DNA damage. Here we report the molecular mechanisms governing Cc-induced phase separation of SET/TAF-Iß and NPM1, where two lysine-rich clusters of Cc are essential to recognize molecular surfaces on both partners in a specific and coordinated manner. Cc thus emerges as a small, globular protein with sequence-encoded heterotypic phase-separation properties.

Enlarging the panoply of site-directed spin labeling electron paramagnetic resonance (SDSL-EPR): sensitive and selective spin-labeling of tyrosine using an isoindoline-based nitroxide.

Site-directed spin labeling (SDSL) combined with electron paramagnetic resonance (EPR) spectroscopy has emerged as a powerful approach to study structure and dynamics in proteins. One limitation of this approach is the fact that classical spin labels are functionalized to be grafted on natural or site-directed mutagenesis generated cysteine residues. Despite the widespread success of cysteine-based modification strategies, the technique becomes unsuitable when cysteine residues play a functional or structural role in the protein under study. To overcome this limitation, we propose an isoindoline-based nitroxide to selectively target tyrosine residues using a Mannich type reaction, the feasibility of which has been demonstrated in a previous study. This nitroxide has been synthesized and successfully grafted successively on p-cresol, a small tetrapeptide and a model protein: a small chloroplastic protein CP12 having functional cysteines and a single tyrosine. Studying the association of the labeled CP12 with its partner protein, we showed that the isoindoline-based nitroxide is a good reporter to reveal changes in its local environment contrary to the previous study where the label was poorly sensitive to probe structural changes. The successful targeting of tyrosine residues with the isoindoline-based nitroxide thus offers a highly promising approach, complementary to the classical cysteine-SDSL one, which significantly enlarges the field of applications of the technique for probing protein dynamics.

Thermal unfolding of proteins probed at the single molecule level using nanopores.

The nanopore technique has great potential to discriminate conformations of proteins. It is a very interesting system to mimic and understand the process of translocation of biomacromolecules through a cellular membrane. In particular, the unfolding and folding of proteins before and after going through the nanopore are not well understood. We study the thermal unfolding of a protein, probed by two protein nanopores: aerolysin and α-hemolysin. At room temperature, the native folded protein does not enter into the pore. When we increase the temperature from 25 to 50 °C, the molecules unfold and the event frequency of current blockade increases. A similar sigmoid function fits the normalized event frequency evolution for both nanopores, thus the unfolding curve does not depend on the structure and the net charge of the nanopore. We performed also a circular dichroism bulk experiment. We obtain the same melting temperature (around 45 °C) using the bulk and single molecule techniques.

Human deoxyhypusine hydroxylase, an enzyme involved in regulating cell growth, activates O2 with a nonheme diiron center.

Deoxyhypusine hydroxylase is the key enzyme in the biosynthesis of hypusine containing eukaryotic translation initiation factor 5A (eIF5A), which plays an essential role in the regulation of cell proliferation. Recombinant human deoxyhypusine hydroxylase (hDOHH) has been reported to have oxygen- and iron-dependent activity, an estimated iron/holoprotein stoichiometry of 2, and a visible band at 630 nm responsible for the blue color of the as-isolated protein. EPR, Mössbauer, and XAS spectroscopic results presented herein provide direct spectroscopic evidence that hDOHH has an antiferromagnetically coupled diiron center with histidines and carboxylates as likely ligands, as suggested by mutagenesis experiments. Resonance Raman experiments show that its blue chromophore arises from a (mu-1,2-peroxo)diiron(III) center that forms in the reaction of the reduced enzyme with O2, so the peroxo form of hDOHH is unusually stable. Nevertheless we demonstrate that it can carry out the hydroxylation of the deoxyhypusine residue present in the elF5A substrate. Despite a lack of sequence similarity, hDOHH has a nonheme diiron active site that resembles both in structure and function those found in methane and toluene monooxygenases, bacterial and mammalian ribonucleotide reductases, and stearoyl acyl carrier protein Delta9-desaturase from plants, suggesting that the oxygen-activating diiron motif is a solution arrived at by convergent evolution. Notably, hDOHH is the only example thus far of a human hydroxylase with such a diiron active site.

PP2A is activated by cytochrome c upon formation of a diffuse encounter complex with SET/TAF-Iß.

Intrinsic protein flexibility is of overwhelming relevance for intermolecular recognition and adaptability of highly dynamic ensemble of complexes, and the phenomenon is essential for the understanding of numerous biological processes. These conformational ensembles-encounter complexes-lack a unique organization, which prevents the determination of well-defined high resolution structures. This is the case for complexes involving the oncoprotein SET/template-activating factor-Iß (SET/TAF-Iß), a histone chaperone whose functions and interactions are significantly affected by its intrinsic structural plasticity. Besides its role in chromatin remodeling, SET/TAF-Iß is an inhibitor of protein phosphatase 2A (PP2A), which is a key phosphatase counteracting transcription and signaling events controlling the activity of DNA damage response (DDR) mediators. During DDR, SET/TAF-Iß is sequestered by cytochrome c (Cc) upon migration of the hemeprotein from mitochondria to the cell nucleus. Here, we report that the nuclear SET/TAF-Iß:Cc polyconformational ensemble is able to activate PP2A. In particular, the N-end folded, globular region of SET/TAF-Iß (a.k.a. SET/TAF-Iß ΔC)-which exhibits an unexpected, intrinsically highly dynamic behavior-is sufficient to be recognized by Cc in a diffuse encounter manner. Cc-mediated blocking of PP2A inhibition is deciphered using an integrated structural and computational approach, combining small-angle X-ray scattering, electron paramagnetic resonance, nuclear magnetic resonance, calorimetry and molecular dynamics simulations.

Dividing to unveil protein microheterogeneities: traveling wave ion mobility study.

Overexpression of a protein in a foreign host is often the only route toward an exhaustive characterization, especially when purification from the natural source(s) is hardly achievable. The key issue in these studies relies on quality control of the purified recombinant protein to precisely determining its identity as well as any undesirable microheterogeneities. While standard proteomics approaches preclude unbiased search for modifications, the optional technique of top-down tandem mass spectrometry (MSMS) requires the use of highly accurate and highly resolved experiments to reveal subtle sequence modifications. In the present study, the top-down MSMS approach combined with traveling wave ion mobility (TWIM) separation was evaluated for its ability to achieve high sequence coverage and to reveal subtle microheterogeneities that were hitherto only accessible with Fourier-transform ion cyclotron resonance-MS instruments. The power of this approach is herein illustrated in an in-depth analysis of both the wild type and K496C variant of the recombinant X domain (XD; aa's 459-507) of the measles virus phosphoprotein expressed in Escherichia coli . Using top-down MSMS combined with TWIM, we show that XD samples occasionally exhibit a microheterogeneity that could not be anticipated from the nucleotide sequence of the encoding constructs and that likely reflects a genetic drift, neutral or not, occurring during expression. In addition, a 1-oxyl-2,2,5,5-tetramethyl-δ3-pyrroline-3-methyl methanethiosulfonate nitroxide probe that was grafted onto the K496C XD variant was shown to undergo oxidation and/or protonation in the electrospray ionization source, leading to artifactual mass increases.

Mössbauer and DFT study of the ferromagnetically coupled diiron(IV) precursor to a complex with an Fe(IV)(2)O(2) diamond core.

Recently, we reported the reaction of the (mu-oxo)diiron(III) complex 1 ([Fe(III)(2)(mu-O)(mu-O(2)H(3))(L)(2)](3+), L = tris(3,5-dimethyl-4-methoxypyridyl-2-methyl)amine) with 1 equiv of H(2)O(2) to yield a diiron(IV) intermediate, 2 (Xue, G.; Fiedler, A. T.; Martinho, M.; Munck, E.; Que, L., Jr. Proc. Natl. Acad. Sci. U.S.A. 2008, 105, 20615-20). Upon treatment with HClO(4), complex 2 converted to a species with an Fe(IV)(2)(mu-O)(2) diamond core that serves as the only synthetic model to date for the diiron(IV) core proposed for intermediate Q of soluble methane monooxygenase. Here we report detailed Mossbauer and density functional theory (DFT) studies of 2. The Mossbauer studies reveal that 2 has distinct Fe(IV) sites, a and b. Studies in applied magnetic fields show that the spins of sites a and b (S(a) = S(b) = 1) are ferromagnetically coupled to yield a ground multiplet with S = 2. Analysis of the applied field spectra of the exchange-coupled system yields for site b a set of parameters that matches those obtained for the mononuclear [LFe(IV)(O)(NCMe)](2+) complex, showing that site b (labeled Fe(O)) has a terminal oxo group. Using the zero-field splitting parameters of [LFe(IV)(O)(NCMe)](2+) for our analysis of 2, we obtained parameters for site a that closely resemble those reported for the nonoxo Fe(IV) complex [(beta-BPMCN)Fe(IV)(OH)(OO(t)Bu)](2+), suggesting that a (labeled Fe(OH)) coordinates a hydroxo group. A DFT optimization performed on 2 yielded an Fe-Fe distance of 3.39 A and an Fe-(mu-O)-Fe angle of 131 degrees , in good agreement with the results of our previous EXAFS study. The DFT calculations reproduce the Mossbauer parameters (A-tensors, electric field gradient, and isomer shift) of 2 quite well, including the observation that the largest components of the electric field gradients of Fe(O) and Fe(OH) are perpendicular. The ferromagnetic behavior of 2 seems puzzling given that the Fe-(mu-O)-Fe angle is large but can be explained by noting that the orbital structures of Fe(O) and Fe(OH) are such that the unpaired electrons at the two sites delocalize into orthogonal orbitals at the bridging oxygen, rationalizing the ferromagnetic behavior of 2. Thus, inequivalent coordinations at Fe(O) and Fe(OH) define magnetic orbitals favorable for ferromagnetic ineractions.

Characterization of two distinct adducts in the reaction of a nonheme diiron(II) complex with O2.

Two [Fe(II)(2)(N-EtHPTB)(mu-O(2)X)](2+) complexes, where N-EtHPTB is the anion of N,N,N'N'-tetrakis(2-benzimidazolylmethyl)-2-hydroxy-1,3-diaminopropane and O(2)X is O(2)PPh(2) (1 x O(2)PPh(2)) or O(2)AsMe(2) (1 x O(2)AsMe(2)), have been synthesized. Their crystal structures both show interiron distances of 3.54 A that arise from a (mu-alkoxo)diiron(II) core supported by an O(2)X bridge. These diiron(II) complexes react with O(2) at low temperatures in MeCN (-40 degrees C) and CH(2)Cl(2) (-60 degrees C) to form long-lived O(2) adducts that are best described as (mu-eta(1):eta(1)-peroxo)diiron(III) species (2 x O(2)X) with nu(O-O) approximately 850 cm(-1). Upon warming to -30 degrees C, 2 x O(2)PPh(2) converts irreversibly to a second (mu-eta(1):eta(1)-peroxo)diiron(III) intermediate (3 x O(2)PPh(2)) with nu(O-O) approximately 900 cm(-1), a value which matches that reported for [Fe(2)(N-EtHPTB)(O(2))(O(2)CPh)](2+) (3 x O(2)CPh) (Dong et al. J. Am. Chem. Soc. 1993, 115, 1851-1859). Mossbauer spectra of 2 x O(2)PPh(2) and 3 x O(2)PPh(2) indicate that the iron centers within each species are antiferromagnetically coupled with J approximately 60 cm(-1), while extended X-ray absorption fine structure analysis reveals interiron distances of 3.25 and 3.47 A for 2 x O(2)PPh(2) and 3 x O(2)PPh(2), respectively. A similarly short interiron distance (3.27 A) is found for 2 x O(2)AsMe(2). The shorter interiron distance associated with 2 x O(2)PPh(2) and 2 x O(2)AsMe(2) is proposed to derive from a triply bridged diiron(III) species with alkoxo (from N-EtHPTB), 1,2-peroxo, and 1,3-O(2)X bridges, while the longer distance associated with 3 x O(2)PPh(2) results from the shift of the O(2)PPh(2) bridge to a terminal position on one iron. The differences in nu(O-O) are also consistent with the different interiron distances. It is suggested that the O...O bite distance of the O(2)X moiety affects the thermal stability of 2 x O(2)X, with the O(2)X having the largest bite distance (O(2)AsMe(2)) favoring the 2 x O(2)X adduct and the O(2)X having the smallest bite distance (O(2)CPh) favoring the 3 x O(2)X adduct. Interestingly, neither 3 x O(2)AsMe(2) nor the benzoate analog of 2 x O(2)X (2 x O(2)Bz) are observed.

Shape-selective interception by hydrocarbons of the O2-derived oxidant of a biomimetic nonheme iron complex.

Picky ferryl: The complex [Fe(Tp(Ph(2)))(BF)] (Tp(Ph(2)) = hydrotris(3,5-diphenylpyrazolyl)borate; BF = benzoylformate) reacts with O(2) to generate an oxidant (see picture; O red, pink; Fe yellow; N blue; C gray; H white) that oxidizes added hydrocarbons shape-selectively. Discrimination derives from a cleft formed by two phenyl groups of the Tp(Ph(2)) ligand, favoring oblate spheroidal substrates.

A synthetic high-spin oxoiron(IV) complex: generation, spectroscopic characterization, and reactivity.

High versus low: The high-yield generation of a synthetic high-spin oxoiron(IV) complex, [Fe(IV)(O)(TMG(3)tren)](2+) (see picture, TMG(3)tren = 1,1,1-tris{2-[N2-(1,1,3,3-tetramethylguanidino)]ethyl}amine), has been achieved by using the very bulky tetradentate TMG(3)tren ligand, in order to both sterically protect the oxoiron(IV) moiety and enforce a trigonal bipyramidal geometry at the iron center, for which an S = 2 ground state is favored.