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.
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 E p,c values for the reduction of the FeO 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 E p,c values, reflecting the electrophilicity of the FeO unit. In contrast, no correlation with E p,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.
Microtubules (MTs) play a fundamental role in many vital processes such as cell division and neuronal activity. They are key structural and functional elements in axons, supporting neurite ...differentiation and growth, as well as transporting motor proteins along the axons, which use MTs as support tracks. Tau is a stabilizing MT associated protein, whose functions are mainly regulated by phosphorylation. A disruption of the MT network, which might be caused by Tau loss of function, is observed in a group of related diseases called tauopathies, which includes Alzheimer's disease (AD). Tau is found hyperphosphorylated in AD, which might account for its loss of MT stabilizing capacity. Since destabilization of MTs after dissociation of Tau could contribute to toxicity in neurodegenerative diseases, a molecular understanding of this interaction and its regulation is essential.
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 (μ-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 Δ⁹-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.
Picky ferryl: The complex Fe(TpPh² )(BF) (TpPh² =hydrotris(3,5-diphenylpyrazolyl)borate; BF=benzoylformate) reacts with O₂ 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 TpPh² ligand, favoring oblate spheroidal substrates.
Two FeII 2(N-EtHPTB)(μ-O2X)2+ complexes, where N-EtHPTB is the anion of N,N,N′ N′-tetrakis(2-benzimidazolylmethyl)-2-hydroxy-1,3-diaminopropane and O2X is O2PPh2 (1·O2PPh2) or O2AsMe2 (1·O2AsMe2), ...have been synthesized. Their crystal structures both show interiron distances of 3.54 Å that arise from a (μ-alkoxo)diiron(II) core supported by an O2X bridge. These diiron(II) complexes react with O2 at low temperatures in MeCN (−40 °C) and CH2Cl2 (−60 °C) to form long-lived O2 adducts that are best described as (μ-η1:η1-peroxo)diiron(III) species (2·O2X) with νO−O ∼ 850 cm−1. Upon warming to −30 °C, 2·O2PPh2 converts irreversibly to a second (μ-η1:η1-peroxo)diiron(III) intermediate (3·O2PPh2) with νO−O ∼ 900 cm−1, a value which matches that reported for Fe2(N-EtHPTB)(O2)(O2CPh)2+ (3·O2CPh) (Dong et al. J. Am. Chem. Soc. 1993, 115, 1851−1859). Mössbauer spectra of 2·O2PPh2 and 3·O2PPh2 indicate that the iron centers within each species are antiferromagnetically coupled with J ∼ 60 cm−1, while extended X-ray absorption fine structure analysis reveals interiron distances of 3.25 and 3.47 Å for 2·O2PPh2 and 3·O2PPh2, respectively. A similarly short interiron distance (3.27 Å) is found for 2·O2AsMe2. The shorter interiron distance associated with 2·O2PPh2 and 2·O2AsMe2 is proposed to derive from a triply bridged diiron(III) species with alkoxo (from N-EtHPTB), 1,2-peroxo, and 1,3-O2X bridges, while the longer distance associated with 3·O2PPh2 results from the shift of the O2PPh2 bridge to a terminal position on one iron. The differences in νO−O are also consistent with the different interiron distances. It is suggested that the O···O bite distance of the O2X moiety affects the thermal stability of 2·O2X, with the O2X having the largest bite distance (O2AsMe2) favoring the 2·O2X adduct and the O2X having the smallest bite distance (O2CPh) favoring the 3·O2X adduct. Interestingly, neither 3·O2AsMe2 nor the benzoate analog of 2·O2X (2·O2Bz) are observed.
Two methane monooxygenase (MMO) systems have been identified in methanotrophic bacteria, namely, a soluble or cytoplasmic MMO and a membrane-associated or particulate MMO. The active site of the ...well-characterized soluble MMO contains a bis-μ-hydroxo-bridged diiron cluster. X-ray crystallographic studies of the particulate enzyme, pMMO, have identified two copper centers on the α subunit (pmoB) of the αβγ trimer and a site at the interface of the βγ subunits filled by a Zn, apparently from the crystallization buffer. In our hands, pMMO preparations containing 1−2 iron atoms per αβγ show the highest catalytic activity. We have employed Mössbauer spectroscopy to characterize the iron in our preparations. Interestingly, we find in pMMO a component with the same spectral properties as the antiferromagnetically coupled diiron(III) cluster in the soluble enzyme. In whole cells, we find nearly 1 diiron center per αβγ of pMMO; in purified enzyme preparations, only 10% of the sites appear to be occupied. These occupancies correlate well with the measured specific activities of purified pMMO and pMMO in whole cells. We suggest that it is the “Zn site” that accommodates the diiron center in active pMMO.
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.
High versus low: The high-yield generation of a synthetic high-spin oxoiron(IV) complex, FeIV(O)(TMG₃tren)²⁺ (see picture, TMG₃tren = 1,1,1-tris{2-N2-(1,1,3,3-tetramethylguanidino)ethyl}amine), has ...been achieved by using the very bulky tetradentate TMG₃tren 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.
The green complex S = 1 (TPEN)FeO2+ TPEN = N,N,N‘,N‘-tetrakis(2-pyridylmethyl)ethane-1,2-diamine has been obtained by treating the (TPEN)Fe2+ precursor with meta-chloroperoxybenzoic acid (m-CPBA). ...This high-valent complex belongs to the emerging family of synthetic models of FeIVO intermediates invoked during the catalytic cycle of biological systems. This complex exhibits spectroscopic characteristics that are similar to those of other models reported recently with a similar amine/pyridine environment. Thanks to its relative stability, vibrational data in solution have been obtained by Fourier transform infrared. A comparison of the FeO and Fe18O wavenumbers reveals that the Fe−oxo vibration is not a pure one. The ability of the green complex to oxidize small organic molecules has been studied. Mixtures of oxygenated products derived from two- or four-electron oxidations are obtained. The reactivity of this FeO2+ complex is then not straightforward, and different mechanisms may be involved.
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