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International audience; Iron−sulfur (Fe−S) clusters are prosthetic groups of proteins biosynthesized on scaffold proteins by highly conserved multi-protein machineries. Biosynthesis of Fe−S clusters into the ISCU scaffold protein is initiated by ferrous iron insertion, followed by sulfur acquisition, via a still elusive mechanism. Notably, whether iron initially binds to the ISCU cysteine-rich assembly site or to a cysteine-less auxiliary site via N/O ligands remains unclear. We show here by SEC, circular dichroism (CD), and Mossbauer spectroscopies that iron binds to the assembly site of the monomeric form of prokaryotic and eukaryotic ISCU proteins via either one or two cysteines, referred to the 1-Cys and 2-Cys forms, respectively. The latter predominated at pH 8.0 and correlated with the Fe−S cluster assembly activity, whereas the former increased at a more acidic pH, together with free iron, suggesting that it constitutes an intermediate of the iron insertion process. Iron not binding to the assembly site was non-specifically bound to the aggregated ISCU, ruling out the existence of a structurally defined auxiliary site in ISCU. Characterization of the 2-Cys form by site-directed mutagenesis, CD, NMR, X-ray absorption, Mossbauer, and electron paramagnetic resonance spectroscopies showed that the iron center is coordinated by four strictly conserved amino acids of the assembly site, Cys35, Asp37, Cys61, and His103, in a tetrahedral geometry. The sulfur receptor Cys104 was at a very close distance and apparently bound to the iron center when His103 was missing, which may enable iron-dependent sulfur acquisition. Altogether, these data provide the structural basis to elucidate the Fe−S cluster assembly process and establish that the initiation of Fe−S cluster biosynthesis by insertion of a ferrous iron in the assembly site of ISCU is a conserved mechanism.

Transmembrane receptors are central components of the chemosensory systems by which motile bacteria detect and respond to chemical gradients. An attractant bound to the receptor periplasmic domain generates conformational signals that regulate a histidine kinase interacting with its cytoplasmic domain. Ligand-induced signaling through the periplasmic and transmembrane domains of the receptor involves a piston-like helical displacement, but the nature of this signaling through the >200 A four-helix coiled coil of the cytoplasmic domain had not yet been identified. We performed single-molecule Forster resonance energy transfer measurements on Escherichia coli aspartate receptor homodimers inserted into native phospholipid bilayers enclosed in nanodiscs. The receptors were labeled with fluorophores at diagnostic positions near the middle of the cytoplasmic coiled coil. At these positions, we found that the two N-helices of the homodimer were more distant, that is, less tightly packed and more dynamic than the companion C-helix pair, consistent with previous deductions that the C-helices form a stable scaffold and the N-helices are dynamic. Upon ligand binding, the scaffold pair compacted further, while separation and dynamics of the dynamic pair increased. Thus, ligand binding had asymmetric effects on the two helical pairs, shifting mean distances in opposite directions and increasing the dynamics of one pair. We suggest that this reflects a conformational change in which differential alterations to the packing and dynamics of the two helical pairs are coupled. These coupled changes could represent a previously unappreciated mode of conformational signaling that may well occur in other coiled-coil signaling proteins.

Addition of dioxygen at low temperature to the nonheme ferrous complex, Fe(II)(Me(3)TACN)(OSi(Ph2))(2)O) (1), in 2-MeTHF produces a peroxo-bridged diferric complex, Fe(2)(III)(μ-O(2))(Me(3)TACN)(2)((OSi(Ph2))(2)O)(2) (2), which was characterized by UV-vis, resonance Raman, and variable field Mössbauer spectroscopies. Illumination of a frozen solution of 2 in THF with white light leads to homolytic O–O bond cleavage and generation of a Fe(IV)(O) complex 4 (ν(FeO) = 818 cm(−1); δ = 0.22 mm s(−1), ΔE(Q) = 0.23 mm s(−1)). Variable field Mössbauer spectroscopy measurements show that 4 is a rare example of a high-spin, S = 2 Fe(IV)(O) complex, and the first synthetic example to be generated directly from O(2). Complex 4 is highly reactive, as expected for a high-spin ferryl, and decays rapidly in fluid solution at cryogenic temperatures. This decay process in 2-MeTHF involves C–H cleavage of the solvent. However, the controlled photolysis of 2 in situ with visible light and excess phenol substrate leads to competitive phenol oxidation, via the proposed transient generation of 4 as the active oxidant.

A five-coordinate, disiloxide-ligated cobalt(II) (

A new nonheme iron(II) complex, FeII (Me3 TACN)((OSiPh2 )2 O) (1), is reported. Reaction of 1 with NO(g) gives a stable mononitrosyl complex Fe(NO)(Me3 TACN)((OSiPh2 )2 O) (2), which was characterized by Mossbauer (δ=0.52 mm s-1 , |ΔEQ |=0.80 mm s-1 ), EPR (S=3/2), resonance Raman (RR) and Fe K-edge X-ray absorption spectroscopies. The data show that 2 is an {FeNO}7 complex with an S=3/2 spin ground state. The RR spectrum (λexc =458 nm) of 2 combined with isotopic labeling (15 N, 18 O) reveals ν(N-O)=1680 cm-1 , which is highly activated, and is a nearly identical match to that seen for the reactive mononitrosyl intermediate in the nonheme iron enzyme FDPnor (ν(NO)=1681 cm-1 ). Complex 2 reacts rapidly with H2 O in THF to produce the N-N coupled product N2 O, providing the first example of a mononuclear nonheme iron complex that is capable of converting NO to N2 O in the absence of an exogenous reductant.

Reaction of the 5-coordinate Fe(II)(N(4)S) complexes, [Fe(II)(iPr(3)TACN)(abt(x))](OTf) (abt = aminobenzenethiolate, X = H, CF(3)) with a one-electron oxidant and an appropriate base leads to net H-atom loss, generating new Fe(III)(iminobenzenethiolate) complexes that were characterized by single-crystal X-ray diffraction (XRD), as well as UV-vis, EPR, and Mössbauer spectroscopies. The spectroscopic data indicate that the iminobenzenethiolate complexes have S = 3/2 ground states. In the absence of a base, oxidation of the Fe(II)(abt) complexes leads to disulfide formation instead of oxidation at the metal center. Bracketing studies with separated proton-coupled electron-transfer (PCET) reagents show that the Fe(II)(aminobenzenethiolate) and Fe(III)(iminobenzenethiolate) forms are readily interconvertible by H(+)/e(−) transfer, and provide a measure of the bond dissociation free energy (BDFE) for the coordinated N–H bond between 64 – 69 kcal mol(−1). This work shows that coordination to the iron center causes a dramatic weakening of the N–H bond, and that Fe- versus S- oxidation in a nonheme iron complex can be controlled by the protonation state of an ancillary amino donor.

A new nonheme iron(II) complex, Fe(II)(Me(3)TACN)((OSi(Ph2))(2)O) (1), is reported. Reaction of 1 with NO((g)) gives a stable mononitrosyl complex Fe(NO)(Me(3)TACN)((OSi(Ph2))(2)O) (2), which was characterized by Mössbauer (δ = 0.52 mm s(−1), |ΔE(Q)| = 0.80 mm s(−1)), EPR (S = 3/2), resonance Raman (RR) and Fe K-edge X-ray absorption spectroscopies. The data show that 2 is an {FeNO}(7) complex with an S = 3/2 spin ground state. The RR spectrum (λ(exc) = 458 nm) of 2 combined with isotopic labeling ((15)N, (18)O) reveals ν(N-O) = 1680 cm(−1), which is highly activated, and is a nearly identical match to that seen for the reactive mononitrosyl intermediate in the nonheme iron enzyme FDPnor (ν(NO) = 1681 cm(−1)). Complex 2 reacts rapidly with H(2)O in THF to produce the N-N coupled product N(2)O, providing the first example of a mononuclear nonheme iron complex that is capable of converting NO to N(2)O in the absence of an exogenous reductant.

The activation of dioxygen by Fe(II)(Me(3)TACN)(S(2)SiMe(2)) (1) is reported. Reaction of 1 with O(2) at −135 °C in 2-MeTHF generates a thiolate-ligated (peroxo)diiron complex Fe(III)(2)(O(2))(Me(3)TACN)(2)(S(2)SiMe(2))(2) (2) that was characterized by UV–vis (λ(max) = 300, 390, 530, 723 nm), Mössbauer (δ = 0.53, |ΔE(Q)| = 0.76 mm s(−1)), resonance Raman (RR) (ν(O–O) = 849 cm(−1)), and X-ray absorption (XAS) spectroscopies. Complex 2 is distinct from the outer-sphere oxidation product 1(ox) (UV–vis (λ(max) = 435, 520, 600 nm), Mössbauer (δ = 0.45, ΔE(Q) = 3.6 mm s(−1)), and EPR (S = 5/2, g = [6.38, 5.53, 1.99])), obtained by one-electron oxidation of 1. Cleavage of the peroxo O–O bond can be initiated either photochemically or thermally to produce a new species assigned as an Fe(IV)(O) complex, Fe(IV)(O)(Me(3)TACN)(S(2)SiMe(2)) (3), which was identified by UV–vis (λ(max) = 385, 460, 890 nm), Mössbauer (δ = 0.21, |ΔE(Q)| = 1.57 mm s(−1)), RR (ν(Fe(IV)=O) = 735 cm(−1)), and X-ray absorption spectroscopies, as well as reactivity patterns. Reaction of 3 at low temperature with H atom donors gives a new species, Fe(III)(OH)(Me(3)TACN)(S(2)SiMe(2)) (4). Complex 4 was independently synthesized from 1 by the stoichiometric addition of a one-electron oxidant and a hydroxide source. This work provides a rare example of dioxygen activation at a mononuclear nonheme iron(II) complex that produces both Fe(III)–O–O–Fe(III) and Fe(IV)(O) species in the same reaction with O(2). It also demonstrates the feasibility of forming Fe/O(2) intermediates with strongly donating sulfur ligands while avoiding immediate sulfur oxidation.

The synthesis and characterization of a Co(II) dithiolato complex Co(M(e3)TACN)(S(2)SiMe(2)) (1) is reported. Reaction of 1 with O(2) generates a rare thiolate-ligated cobalt-superoxo species Co(O(2))(M(e3)TACN)(S(2)SiMe(2)) (2) that was characterized spectroscopically and structurally by resonance Raman, EPR, and X-ray absorption spectroscopies as well as density functional theory (DFT). Metal-superoxo species are proposed to S-oxygenate metal-bound thiolate donors in nonheme thiol dioxygenases, but 2 does not lead to S-oxygenation of the intramolecular thiolate donors, and does not react with exogenous sulfur donors. However, complex 2 is capable of oxidizing the O-H bonds of 2,2,6,6-tetramethylpiperidin-1-ol (TEMPOH) derivatives via H-atom abstraction. Complementary proton-coupled electron-transfer (PCET) reactivity is seen for 2 with separated proton/reductant pairs. The reactivity studies indicate that 2 can abstract H-atoms from weak X-H bonds with BDFE ≤ 70 kcal mol-1. DFT calculations predict that the putative Co(OOH) product has an O-H bond dissociation free energy (BDFE) = 67 kcal mol(−1), which matches the observed pattern of reactivity seen for 2. These data provide new information regarding the selectivity of S-oxygenation versus H-atom abstraction in thiolate-ligated nonheme metalloenzymes that react with O(2).

One of the long-standing challenges to performing electrogenerated chemiluminescence (ECL) research is the need for dedicated instrumentation or highly customized cells to achieve reproducibility. This manuscript describes an approach to designing ECL systems through the hyphenation of existing laboratory instruments, which provide innate time correlation of electrochemical and emission data. This design methodology lowers the entry barrier required to obtaining reproducible ECL measurements and provides flexibility in the scope of applications. Uniquely, the simplicity of this system's experimental interface, a spectrochemical quartz cuvette, readily enables collaboration with finite element modeling that simulates ECL occurring in the cuvette-based cell. This combination of empirical and simulation data allowed for the investigation of the intertwined kinetics behind the coreactant ECL mechanism of tris(2,2'-bipyridine)ruthenium(II) (Ru(bpy)32+) and tripropylamine (TPA). The complexity of the system measurable via the hyphenation methodology was further scaled though the addition of tris[2-(4,6-difluorophenyl)pyridinato-C2, N] iridium(III) (Ir(dFppy)3) and the observation of real time multiplexing.

Extended metal atom chain (EMAC) complexes of first-row transition metals with metal-metal bonds have the potential to elicit unique magnetic properties and reactivities. Until now, the library of EMAC complexes with late-first-row transition metals was incomplete because of the omission of a triiron species with Fe-Fe bonding. Herein we report the synthesis and preliminary investigation of the first linear, triiron(II) complex containing close Fe-Fe interactions. The complex is supported by three dianionic 2,6-bis[(trimethylsilyl)amido]pyridine ligands (L), with an overall composition of Fe3L3, and pseudohelical ligand coordination stabilizing the local trigonal-planar geometry at each iron. Fe3L3 was characterized by X-ray diffraction, 1H NMR, cyclic voltammetry, electronic absorption, and Mossbauer spectroscopies. Evans method analysis indicated a large uncompensated spin and an S = 6 ground state, suggesting ferromagnetic coupling in the triiron chain, likely due to direct exchange.

The epidermal growth factor receptor (EGFR) is critical to normal cellular signaling pathways. Moreover, it has been implicated in a range of pathologies, including cancer. As a result, it is the primary target of many anticancer drugs. One limitation to the design and development of these drugs has been the lack of molecular-level information about the interactions and conformational dynamics of EGFR. To overcome this limitation, this work reports the construction and characterization of functional, fluorescently labeled, and full-length EGFR in model membrane nanolipoprotein particles (NLPs) for in vitro fluorescence studies. To demonstrate the utility of the system, we investigate ATP-EGFR interactions. We observe that ATP binds at the catalytic site providing a means to measure a range of distances between the catalytic site and the C-terminus via Förster resonance energy transfer (FRET). These ATP-based experiments suggest a range of conformations of the C-terminus that may be a function of the phosphorylation state for EGFR. This work is a proof-of-principle demonstration of single-molecule studies as a noncrystallographic assay for EGFR interactions in real-time and under near-physiological conditions. The diverse nature of EGFR interactions means that new tools at the molecular level have the potential to significantly enhance our understanding of receptor pathology and are of utmost importance for cancer-related drug discovery.

A new tetradentate, monoanionic, mixed N/O donor ligand (BNPA(Ph2)O(−)) with second coordination sphere H-bonding groups has been synthesized for stabilization of terminal Fe(III)(OH)(X) complexes. The complex [Fe(II)(BNPA(Ph2)O)(OTf)] (1) reacts with O(2) to give a mononuclear terminal Fe(III)(OH) complex, [Fe(III)(OH)(BNPA(Ph2)O)(OTf)] (2), both of which were characterized by X-ray diffraction, electrospray ionization mass spectrometry, UV–vis, (1)H and (19)F nuclear magnetic resonance, (57)Fe Mössbauer, and electron paramagnetic resonance spectroscopies. Treatment of 2 with carbon radicals (Ar(3)C·) gives Ar(3)COH and the Fe(II) complex 1, in direct analogy with the elusive radical “rebound” process proposed for nonheme iron enzymes.

Biological systems are subjected to continuous environmental fluctuations, and therefore, flexibility in the structure and function of their protein building blocks is essential for survival. Protein dynamics are often local conformational changes, which allows multiple dynamical processes to occur simultaneously and rapidly in individual proteins. Experiments often average over these dynamics and their multiplicity, preventing identification of the molecular origin and impact on biological function. Green plants survive under high light by quenching excess energy, and Light-Harvesting Complex Stress Related 1 (LHCSR1) is the protein responsible for quenching in moss. Here, we expand an analysis of the correlation function of the fluorescence lifetime by improving the estimation of the lifetime states and by developing a multicomponent model correlation function, and we apply this analysis at the single-molecule level. Through these advances, we resolve previously hidden rapid dynamics, including multiple parallel processes. By applying this technique to LHCSR1, we identify and quantitate parallel dynamics on hundreds of microseconds and tens of milliseconds timescales, likely at two quenching sites within the protein. These sites are individually controlled in response to fluctuations in sunlight, which provides robust regulation of the light-harvesting machinery. Considering our results in combination with previous structural, spectroscopic, and computational data, we propose specific pigments that serve as the quenching sites. These findings, therefore, provide a mechanistic basis for quenching, illustrating the ability of this method to uncover protein function.

The synthesis and characterization of trinuclear GeII and SnII chlorides and a trialuminum complex supported by a trinucleating tris(β-diketiminate) cyclophane ligand (L3–) are reported. The in situ deprotonation of H3L with benzylpotassium and subsequent reaction with GeCl2·dioxane or SnCl2 afforded (GeCl)3L (1) and (SnCl)3L (2) in 42 and 60% yields, respectively. Each GeII and SnII atom is three-coordinate and exhibit pseudotrigonal pyramidal geometry as anticipated for three-coordinate divalent group 14 cations. UV/visible spectra collected on THF solutions of 1 and 2 display a bathochromic shift in the absorption from 1 to 2 (from 361 to 375 nm). Addition of AlMe3 to toluene solutions of H3L resulted in the formation of (AlMe2)2AlMe3HL (3), which possesses two NCCCN chelated AlMe2 moieties. The third β-diketimine arm remains protonated and adopts an atypical trans conformation with an AlMe3 coordinated to the solvent exposed imine N atom.

Dynamic wireframe DNA structures have gained significant attention in recent years, with research aimed toward using these architectures for sensing and encapsulation applications. For these assemblies to reach their full potential, however, knowledge of the rates of strand displacement and hybridization on these constructs is required. Herein, we report the use of single-molecule fluorescence methodologies to observe the reversible switching between double- and single-stranded forms of triangular wireframe DNA nanotubes. Specifically, by using fluorescently labeled DNA strands, we were able to monitor changes in intensity over time as we introduced different sequences. This allowed us to extract detailed kinetic information on the strand displacement and hybridization processes. Due to the polymeric nanotube structure, the ability to individually address each of the three sides, and the inherent polydispersity of our samples as a result of the step polymerization by which they are formed, a library of compounds could be studied independently yet simultaneously. Kinetic models relying on mono-exponential decays, multi-exponential decays, or sigmoidal behavior were adjusted to the different constructs to retrieve erasing and refilling kinetics. Correlations were made between the kinetic behavior observed, the site accessibility, the nanotube length, and the structural robustness of wireframe DNA nanostructures, including fully single-stranded analogs. Overall, our results reveal how the length, morphology, and rigidity of the DNA framework modulate the kinetics of strand displacement and hybridization as well as the overall addressability and structural stability of the structures under study.

A nonheme iron(III) terminal methoxide complex, [Fe(III)(N3PyO(2Ph))(OCH(3))]ClO(4), was synthesized. Reaction of this complex with the triphenylmethyl radical (Ph(3)C•) leads to formation of Ph(3)COCH(3) and the one-electron reduced iron(II) center, as seen by UV-vis, EPR, (1)H NMR, and Mössbauer spectroscopy. These results indicate that homolytic Fe-O bond cleavage occurs together with C-O bond formation, providing a direct observation of the “radical rebound” process proposed for both biological and synthetic nonheme iron centers.

Using a simple metathesis approach, the triiron(II) tribromide complex Fe3Br3L (1) reacts with tetrabutylammonium azide to afford the monoazide dibromide analogue Fe3(Br)2(N3)L (2) in high yield. The inclusion of azide was confirmed by IR spectroscopy with a ν(N3) = 2082 cm(-1) as well as combustion analysis and X-ray crystallography. Heating 2 in the solid state results in the complete loss of the azide vibration in the IR spectra and the isolation of the olive-green mononitride complex Fe3(Br)2(N)L (3). Solution magnetic susceptibility measurements support that the trimetallic core within 2 is oxidized upon generation of 3 (5.07 vs 3.09 μB). Absorption maxima in the UV-visible-near-IR (NIR) spectra of 2 and 3 support the azide-to-nitride conversion, and a broad NIR absorption centered at 1117 nm is similar to that previously reported for the intervalence charge-transfer band for a mixed-valent nitridodiiron cluster. The cyclic voltammograms recorded for 3 are comparable to those of 1 with no reductive waves observed between ∼0 and -2.5 V (vs Fc/Fc(+)), whereas a reversible one-electron redox process is observed for Fe3(NH2)3L (4). These results suggest that intercluster cooperativity is unlikely to predominate the dinitrogen reduction mechanism when 1 is treated with KC8 under N2.

International audience; A series of tri- and dimetallic metal complexes of pyridine dicarboxamide cryptates are reported in which changes to the base and metal source result in diverse structure types. Addition of strong bases, such as KH or KN(SiMe3)2, followed by divalent metal halides allows direct access to trinuclear complexes in which each metal center is coordinated by a dianionic N,N,N-chelate of each arm. These complexes bind a guest K+ cation within the central cavity in a trigonal planar coordination environment. Minor changes to the solvent and equivalents of base used in the syntheses of the triiron(II) and tricobalt(II) complexes affords two trinuclear clusters with atypical O,N,O-coordination by each pyridine dicarboxamide arm; the amide carbonyl O atoms are oriented toward the interior of the cavity to coordinate to each metal center. Finally, varying the base enables the selective synthesis of dinuclear nickel(II) and copper(II) complexes in which one pyridine dicarboxamide arm remains protonated. These amide protons are at one end of a hydrogen bonding network that extends throughout the internal cavity and terminates at a metal bound hydroxide, carbonate, or bicarbonate donor. In the dinickel complex, the bicarbonate cannot be liberated as CO2 either thermally or upon sparging with N2, which differs from previously reported monometallic complexes. The carbonate or bicarbonate ligands likely arise from sequestration of atmospheric CO2 based on the observed reaction of the di(hydroxonickel) analog.