En la novela Sanguínea (2020), Gabriela Ponce logra la “escritura con el cuerpo”. Lo hace mediante la admisión de fluidos, sobre todo de la sangre menstrual como forma de afinar el sentido y lo ...escrito. Lo sexual y erótico también juegan un papel importante para encontrar un lenguaje particular que crea una gramática del cuerpo. La maternidad es otro de los elementos que permiten pensar la corporeidad como forma de lenguaje y al mismo tiempo como configuración del espacio. El cuerpo es un espacio en sí mismo en esta novela donde la narración parte de un flujo de conciencia de la protagonista.
The quinol oxidation site QD in E. coli respiratory nitrate reductase A (EcNarGHI) reacts with the three isoprenoid quinones naturally synthesized by the bacterium, i.e. ubiquinones (UQ), ...menaquinones (MK) and demethylmenaquinones (DMK). The binding mode of the demethylmenasemiquinone (DMSK) intermediate to the EcNarGHI QD quinol oxidation site is analyzed in detail using 1,2H hyperfine (hf) spectroscopy in combination with H2O/D2O exchange experiments and DFT modeling, and compared to the menasemiquinone one bound to the QD site (MSKD) previously studied by us. DMSKD and MSKD are shown to bind in a similar and strongly asymmetric manner through a short (~1.7 Å) H-bond. The origin of the specific hf pattern resolved on the DMSKD field-swept EPR spectrum is unambiguously ascribed to slightly inequivalent contributions from two β-methylene protons of the isoprenoid side chain. DFT calculations show that their large isotropic hf coupling constants (Aiso ~12 and 15 MHz) are consistent with both (i) a specific highly asymmetric binding mode of DMSKD and (ii) a near in-plane orientation of its isoprenyl chain at Cβ relative to the aromatic ring, which differs by ~90° to that predicted for free or NarGHI-bound MSK. Our results provide new insights into how the conformation and the redox properties of different natural quinones are selectively fine-tuned by the protein environment at a single Q site. Such a fine-tuning most likely contributes to render NarGHI as an efficient and flexible respiratory enzyme to be used upon rapid variations of the Q-pool content.
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•The binding mode of naphthoquinones to NarGHI is resolved by EPR spectroscopy and DFT.•Demethylmenasemiquinone binding to NarGHI is highly asymmetric.•Side chain conformations of NarGHI-bound mena- and demethylmenaquinones differ by 90°.•Protein environment fine tunes the side chain conformation of the bound semiquinones.
Nitrene transfer reactions have emerged as one of the most powerful and versatile ways to insert an amine function to various kinds of hydrocarbon substrates. However, the mechanisms of nitrene ...generation have not been studied in depth albeit their formation is taken for granted in most cases without definitive evidence of their occurrence. In the present work, we compare the generation of tosylimido iron species and NTs transfer from Fe
and Fe
precursors where the metal is embedded in a tetracarbene macrocycle. Catalytic nitrene transfer to reference substrates (thioanisole, styrene, ethylbenzene and cyclohexane) revealed that the same active species was at play, irrespective of the ferrous versus ferric nature of the precursor. Through combination of spectroscopic (UV-visible, Mössbauer), ESI-MS and DFT studies, an Fe
tosylimido species was identified as the catalytically active species and was characterized spectroscopically and computationally. Whereas its formation from the Fe
precursor was expected by a two-electron oxidative addition, its formation from an Fe
precursor was unprecedented. Thanks to a combination of spectroscopic (UV-visible, EPR, Hyscore and Mössbauer), ESI-MS and DFT studies, we found that, when starting from the Fe
precursor, an Fe
tosyliodinane adduct was formed and decomposed into an Fe
tosylimido species which generated the catalytically active Fe
tosylimide through a comproportionation process with the Fe
precursor.
Nitrene transfer reactions have emerged as one of the most powerful and versatile ways to insert an amine function to various kinds of hydrocarbon substrates. However, the mechanisms of nitrene ...generation have not been studied in depth albeit their formation is taken for granted in most cases without definitive evidence of their occurrence. In the present work, we compare the generation of tosylimido iron species and NTs transfer from FeII and FeIII precursors where the metal is embedded in a tetracarbene macrocycle. Catalytic nitrene transfer to reference substrates (thioanisole, styrene, ethylbenzene and cyclohexane) revealed that the same active species was at play, irrespective of the ferrous versus ferric nature of the precursor. Through combination of spectroscopic (UV‐visible, Mössbauer), ESI‐MS and DFT studies, an FeIV tosylimido species was identified as the catalytically active species and was characterized spectroscopically and computationally. Whereas its formation from the FeII precursor was expected by a two‐electron oxidative addition, its formation from an FeIII precursor was unprecedented. Thanks to a combination of spectroscopic (UV‐visible, EPR, Hyscore and Mössbauer), ESI‐MS and DFT studies, we found that, when starting from the FeIII precursor, an FeIII tosyliodinane adduct was formed and decomposed into an FeV tosylimido species which generated the catalytically active FeIV tosylimide through a comproportionation process with the FeIII precursor.
An FeIV imide of a tetracarbene macrocycle active in nitrene transfer reactions can be generated from phenyltosyliodinane (PhI=NTs) either from the FeII precursor by oxidative addition (right) or from the FeIII precursor by a comproportionation reaction (left) (AN=acetonitrile, bold horizontal bars=macrocycle).
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.
The Mo/W-bisPGD enzyme superfamily comprises a vast number of mononuclear molybdenum and tungsten enzymes that catalyze a great diversity of vital reactions in prokaryotes. In the past decades, much ...attention has been devoted to the immediate surroundings of the metal atom highlighting the importance of the inner coordination sphere but has failed to identify molecular determinants of the reactivity. Here, we report the mechanistic importance of a set of conserved residues that line the substrate entry tunnel in Escherichia coli nitrate reductase A (Nar), a paradigmatic enzyme of the Mo/W-bisPGD superfamily. Using mutagenesis, enzyme kinetics, electron paramagnetic resonance spectroscopy, and molecular dynamics, we unveil the pivotal role of Glu-581 motion and a number of polar residues in its close proximity in substrate affinity and proton transfer to the Mo active site. Motion of the side chain of Glu-581 exhibiting a strong acid–base cooperativity with Asp-801 and surrounded by several polar interactions controls the hydration inside the protein core, proton transfer, and substrate selectivity toward the active site. Overall, we identify an additional determinant that fine-tunes the reactivity and selectivity in Nar and propose that a gating mechanism is at play in several other members of the superfamily.
Respiratory nitrate reductases (Nars), members of the prokaryotic Mo/W-bis Pyranopterin Guanosine dinucleotide (Mo/W-bisPGD) enzyme superfamily, are key players in nitrate respiration, a major ...bioenergetic pathway widely used by microorganisms to cope with the absence of dioxygen. The two-electron reduction of nitrate to nitrite takes place at their active site, where the molybdenum ion cycles between Mo(VI) and Mo(IV) states via a Mo(V) intermediate. The active site shows two distinct pH-dependent Mo(V) electron paramagnetic resonance (EPR) signals whose structure and catalytic relevance have long been debated. In this study, we use EPR and HYSCORE techniques to probe their nuclear environment in Escherichia coli Nar (EcNar). By using samples prepared at different pH and through different enrichment strategies in
Mo and
N nuclei, we demonstrate that each of the two Mo(V) species is coupled to a single nitrogen nucleus with similar quadrupole characteristics. Structure-based density functional theory calculations allow us to propose a molecular model of the low-pH Mo(V) species consistent with EPR spectroscopic data. Our results show that the metal ion is coordinated by a monodentate aspartate ligand and permit the assignment of the coupled nitrogen nuclei to the Nδ of Asn52, a residue located ∼3.9 Å to the Mo atom in the crystal structures. This is confirmed by measurements on selectively
N-Asn labeled EcNar. Further, we propose a Mo-O(H)···HN structure to account for the transfer of spin density onto the interacting nitrogen nucleus deduced from HYSCORE analysis. This work provides a foundation for monitoring the structure of the molybdenum active site in the presence of various substrates or inhibitors in Nars and other molybdenum enzymes.
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