Search

The trafficking and aggregation of neurodegenerative proteins often involves the interaction between intrinsically disordered domains, stabilized by the inclusion of physiologic metal ions such as copper or zinc. Characterizing the metal ion coordination environment is critical for assessing the stability and organization of these relevant protein-protein interactions but is challenging given the lack of regular molecular order or global structure. The cellular prion protein (PrP C ) binds both monomers and aggregates of Alzheimer's amyloid-beta (Aβ), promoting Aβ internalization and aberrant signaling, respectively. Both proteins bind Cu 2+ with high affinity, opening the potential for copper to form an intermolecular bridge. We describe here a novel approach utilizing multiple EPR experiments to investigate the simultaneous Cu 2+ coordination of PrP C and Aβ in a 1:1:1 mixture. Uniformly 15 N-labeled PrP C is used in conjunction with natural abundance 14 N Aβ, the combination of which leads to distinct energy manifolds for paramagnetic Cu 2+ and resolved by the pulsed EPR experiments ESEEM and HYSCORE. We develop acquisition parameters to simultaneously optimize 14 N (I = 1) and 15 N (I = ½) pulsed EPR signals and we also advance the theory of ESEEM and HYSCORE to quantitatively describe multiple 15 N imidazole coordination. This unique approach provides compelling evidence of a copper-stabilized ternary complex, with equatorial Cu 2+ coordination formed by one histidine imidazole from Aβ and three from PrP. Moreover, the methodologies developed here provide a framework for assessing the copper environment in other interacting neurodegenerative proteins., Competing Interests: Conflict of Interest The authors declare no conflicts of interest., (Copyright © 2025 The Authors. Published by Elsevier Inc. All rights reserved.)

[FeFe]-hydrogenases are enzymes that catalyze the redox interconversion of H + and H 2 using a six-iron active site, known as the H-cluster, which consists of a structurally unique [2Fe] H subcluster linked to a [4Fe-4S] H subcluster. A set of enzymes, HydG, HydE, and HydF, are responsible for the biosynthesis of the [2Fe] H subcluster. Among them, it is well established that HydG cleaves tyrosine into CO and CN - and forms a mononuclear [Fe(II)(Cys)(CO) 2 (CN)] complex. Recent work using EPR spectroscopy and X-ray crystallography show that HydE uses this organometallic Fe complex as its native substrate. The low spin Fe(II) center is reduced into an adenosylated Fe(I) species, which is proposed to form an Fe(I)Fe(I) dimer within HydE. The highly unusual transformation catalyzed by HydE draws interest in both biochemistry and organometallic chemistry. Due to the instability of the substrate, the intermediates, and the proposed product, experimental characterization of the detailed HydE mechanism and its final product is challenging. Herein, the catalytic mechanism of HydE is studied using hybrid quantum mechanics/molecular mechanics (QM/MM) molecular dynamics simulations. A radical relay mechanism was found for the cleavage of the cysteine S-Cβ bond that is energetically favored with respect to a closed-shell mechanism involving unconventional proton transfer. In addition, we propose a pathway for the dimerization of two Fe(I) complexes within the HydE hydrophobic cavity, which is consistent with the recent experimental result that HydF can perform [FeFe]-hydrogenase maturation with a synthetic dimer complex as the substrate. These simulation results take us further down the path to a more complete understanding of these enzymes that synthesize one of Nature's most efficient energy conversion catalysts.

Background: CFTR modulator therapies have positive clinical outcomes, yet chronic inflammation and bacterial infections persist in people with CF (pwCF). How elexacaftor-tezacaftor-ivacaftor (ETI) fails to improve innate immune signaling responsible for bacterial clearance and inflammation resolution remains unknown., Methods: We used an unbiased proteomics approach to measure the effect of ETI on inflammatory proteins. Plasma from 20 pediatric pwCF and 20 non-CF (NCF) was collected during routine examination and 3 months after ETI initiation. Protein screening was performed with an inflammation panel (Target 96, Olink ® ). Bioinformatics analysis was used to determine changes in protein expression., Results: There were significantly fewer pulmonary exacerbations after ETI initiation, along with sustained improvement in lung function and reduced bacterial colonization. Unpaired analysis of CF pre-ETI and NCF resulted in 34 significantly different proteins. Of these, CCL20, MMP-10, EN-RAGE, and AXIN1 had a log 2 fold change of 1.2 or more. There was a modest shift in overall CF protein profiles post-ETI toward the NCF cluster. Unpaired analysis of protein differential expression between NCF and CF post-ETI identified a total of 24 proteins significantly impacted by ETI therapy ( p -value ≤ 0.05), with only CCL20 having a log 2 fold change higher than 1.2. Paired analysis (CF pre- and CF post-ETI) of differential protein expression demonstrated significant expression changes of MMP-10, EN-RAGE, and IL-17A. Pathway analysis identified significantly impacted pathways such as the NF-κB pathway., Conclusion: This study showed that ETI in a pediatric cohort had a modest effect on several inflammatory proteins with potential as biomarkers. Pathways significantly impacted by ETI can be further studied for future therapies to combat persistent inflammation and dysregulated immunity., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2025 Ozuna, Bojja, Partida-Sanchez, Hall-Stoodley, Amer, Britt, Sheikh, Frank, Wang, Kang, Miralda, Durfey and Kopp.)

Dioxygen (O 2 ) is a potent oxidant used by aerobic organisms for energy transduction and critical biosynthetic processes. Numerous metalloenzymes harness O 2 to mediate C-H bond hydroxylation reactions, but most commonly feature iron or copper ions in their active site cofactors. In contrast, many manganese-activated enzymes─such as glutamine synthetase and isocitrate lyase─perform redox neutral chemical transformations and very few are known to activate O 2 or C-H bonds. Here, we report that the dimanganese-metalated form of the cambialistic monooxygenase SfbO (Mn 2 -SfbO) can efficiently mediate enzymatic C-H bond hydroxylation. The activity of the dimanganese form of SfbO toward substrate hydroxylation is comparable to that of its heterobimetallic Mn/Fe form but exhibits distinct kinetic profiles. Kinetic, spectroscopic, and structural studies invoke a mixed-valent dimanganese cofactor (Mn II Mn III ) in O 2 activation and evidence a stoichiometric role for superoxide in maturing an O 2 -inert Mn II 2 cofactor. Computational studies support a hypothesis wherein superoxide addition to the Mn II 2 cofactor installs a critical bridging hydroxide ligand that stabilizes higher-valent manganese oxidation states. These findings establish the viability of proteinaceous dimanganese cofactors in mediating complex, multistep redox transformations.

The low reduction potentials required for the reduction of dinitrogen (N 2 ) render metal-based nitrogen-fixation catalysts vulnerable to irreversible damage by dioxygen (O 2 ) 1-3 . Such O 2 sensitivity represents a major conundrum for the enzyme nitrogenase, as a large fraction of nitrogen-fixing organisms are either obligate aerobes or closely associated with O 2 -respiring organisms to support the high energy demand of catalytic N 2 reduction 4 . To counter O 2 damage to nitrogenase, diazotrophs use O 2 scavengers, exploit compartmentalization or maintain high respiration rates to minimize intracellular O 2 concentrations 4-8 . A last line of damage control is provided by the 'conformational protection' mechanism 9 , in which a [2Fe:2S] ferredoxin-family protein termed FeSII (ref.  10 ) is activated under O 2 stress to form an O 2 -resistant complex with the nitrogenase component proteins 11,12 . Despite previous insights, the molecular basis for the conformational O 2 protection of nitrogenase and the mechanism of FeSII activation are not understood. Here we report the structural characterization of the Azotobacter vinelandii FeSII-nitrogenase complex by cryo-electron microscopy. Our studies reveal a core complex consisting of two molybdenum-iron proteins (MoFePs), two iron proteins (FePs) and one FeSII homodimer, which polymerize into extended filaments. In this three-protein complex, FeSII mediates an extensive network of interactions with MoFeP and FeP to position their iron-sulphur clusters in catalytically inactive but O 2 -protected states. The architecture of the FeSII-nitrogenase complex is confirmed by solution studies, which further indicate that the activation of FeSII involves an oxidation-induced conformational change., Competing Interests: Competing interests: The authors declare no competing interests., (© 2025. The Author(s), under exclusive licence to Springer Nature Limited.)

In Nature, the four-electron reduction of O 2 is catalyzed at preorganized multimetallic active sites. These complex active sites often feature low-coordinate, redox-active metal centers precisely positioned to facilitate rapid O 2 activation processes that obviate the generation of toxic, partially reduced oxygen species. Very few biomimetic constructs simultaneously recapitulate the complexity and reactivity of these biological cofactors. Herein, we report solid-state O 2 activation at a triiron(II) active site templated by phosphinimide ligands. Insight into the structure of the O 2 reduction intermediates was obtained via in crystallo O 2 dosing experiments in conjunction with spectroscopic, structural, magnetic, and computational studies. These data support the in situ formation of an Fe 2 III Fe IV -dioxo intermediate upon exposure to O 2 that participates in oxygen atom and hydrogen atom transfer reactivity with exogenous substrates to furnish a stable Fe II Fe 2 III -oxo species. Combined, these studies provide an extraordinary level of detail into the dynamics of bond-forming and -breaking processes operative at complex multimetallic active sites.

The selective synthesis of biomimetic Fe/Mn complexes able to mimic the geometry and catalytic activity of enzymes possessing this cofactor is still a challenge. Herein, we discuss the stepwise synthesis, characterization, and magnetic properties of a Fe(II)/Mn(II) species and related Fe(II)/Fe(II) complexes.

Visible light-driven pyridoxal radical biocatalysis has emerged as a promising strategy for the stereoselective synthesis of valuable noncanonical amino acids (ncAAs). Previously, the use of well-tailored photoredox catalysts represented the key to enable efficient pyridoxal phosphate (PLP) enzyme-catalyzed radical reactions. Here, we report a PLP-dependent threonine aldolase-catalyzed asymmetric α-C-H alkylation of abundant amino acids using Katritzky pyridinium salts as alkylating agents. The use of engineered threonine aldolases allowed for this redox-neutral radical alkylation to proceed efficiently, giving rise to challenging α-trisubstituted and -tetrasubstituted ncAA products in a protecting-group-free fashion with excellent enantiocontrol. Mechanistically, this enantioselective α-alkylation capitalizes on the unique reactivity of the persistent enzymatic quinonoid intermediate derived from the PLP cofactor and the amino acid substrate to allow for novel radical C-C coupling. Surprisingly, this photobiocatalytic process does not require the use of well-established photoredox catalysts and operates through an unconventional photoinduced radical generation involving a PLP-derived aldimine. The ability to develop photobiocatalytic reactions without relying on classic photocatalysts or photoenzymes opens up new avenues for advancing stereoselective intermolecular radical reactions that are not known in either organic chemistry or enzymology.

Ozone (O 3 ) inhalation triggers asthmatic airway hyperresponsiveness (AHR), but the mechanisms by which this occurs are unknown. Previously, we developed a murine model of dust mite, ragweed, and aspergillus (DRA)-induced allergic lung inflammation followed by O 3 exposure for mechanistic investigation. The present study used single cell RNA-sequencing for unbiased profiling of immune cells within the lungs of mice exposed to DRA, O 3 , or DRA+O 3 , to identify the components of the immune cell niche that contribute to AHR. Alveolar macrophages (AMs) had the greatest number of differentially expressed genes following DRA+O 3 , most of which were unique to the 2-hit exposure. Following DRA+O 3 , AMs activated transcriptional pathways related to cholesterol biosynthesis, degradation of the extracellular matrix, endosomal TLR processing, and various cytokine signals. We also identified AM and monocyte subset populations that were unique to the DRA+O 3 group. These unique AMs activated gene pathways related to inflammation, sphingolipid metabolism, and bronchial constriction. The unique monocyte population had a gene signature that suggested phospholipase activation and increased degradation of the extracellular matrix. Flow cytometry analysis of BAL immune cells showed recruited monocyte-derived AMs after DRA and DRA+O 3 , but not after O 3 exposure alone. O 3 alone increased BAL neutrophils but this response was attenuated in DRA+O 3 mice. DRA-induced changes in the airspace immune cell profile were reflected in elevated BAL cytokine/chemokine levels following DRA+O 3 compared to O 3 alone. The present work highlights the role of monocytes and AMs in the response to O 3 and suggests that the presence of distinct subpopulations following allergic inflammation may contribute to O 3 -induced AHR.

ConspectusNature's prototypical hydrogen-forming catalysts─hydrogenases─have attracted much attention because they catalyze hydrogen evolution at near zero overpotential and ambient conditions. Beyond any possible applications in the energy sphere, the hydrogenases feature complicated active sites, which implies novel biosynthetic pathways. In terms of the variety of cofactors, the [FeFe]-hydrogenase is among the most complex.For more than a decade, we have worked on the biosynthesis of the active site of [FeFe] hydrogenases. This site, the H-cluster, is a six-iron ensemble consisting of a [4Fe-4S] H cluster linked to a [2Fe] H cluster that is coordinated to CO, cyanide, and a unique organic azadithiolate ligand. Many years ago, three enzymes, namely, HydG, HydE, and HydF, were shown to be required for the biosynthesis and the in vitro maturation of [FeFe] hydrogenases. The structures of the maturases were determined crystallographically, but still little progress was made on the biosynthetic pathway. As described in this Account, the elucidation of the biosynthetic pathway began in earnest with the identification of a molecular iron-cysteinate complex produced within HydG.In this Account, we present our most recent progress toward the molecular mechanism of [2Fe] H biosynthesis using a collaborative approach involving cell-free biosynthesis, isotope and element-sensitive spectroscopies, as well as inorganic synthesis of purported biosynthetic intermediates. Our study starts from the radical SAM enzyme HydG that lyses tyrosine into CO and cyanide and forms an Fe(CO) 2 (CN)-containing species. Crystallographic identification of a unique auxiliary 5Fe-4S cluster in HydG leads to a proposed catalytic cycle in which a free cysteine-chelated "dangler" Fe serves as the platform for the stepwise formation of a [4Fe-4S][Fe(CO)(CN)(cysteinate)] intermediate, which releases the [Fe(CO) 2 (CN)(cysteinate)] product, Complex B. Since Complex B is unstable, we applied synthetic organometallic chemistry to make an analogue, syn-B, and showed that it fully replaces HydG in the in vitro maturation of the H-cluster. Syn-B serves as the substrate for the next radical SAM enzyme HydE, where the low-spin Fe(II) center is activated by 5'-dAdo • to form an adenosylated Fe(I) intermediate. We propose that this Fe(I) species strips the carbon backbone and dimerizes in HydE to form a [Fe 2 (SH) 2 (CO) 4 (CN) 2 ] 2- product. This mechanistic scenario is supported by the use of a synthetic version of this dimer complex, syn-dimer, which allows for the formation of active hydrogenase with only the HydF maturase. Further application of this semisynthesis strategy shows that an [Fe 2 (SCH 2 NH 2 ) 2 (CO) 4 (CN) 2 ] 2- complex can activate the apo hydrogenase, marking it as the last biosynthetic intermediate en route to the H-cluster. This combined enzymatic and semisynthetic approach greatly accelerates our understanding of H-cluster biosynthesis. We anticipate additional mechanistic details regarding H-cluster biosynthesis to be gleaned, and this methodology may be further applied in the study of other complex metallocofactors.

We expand upon the synthetic utility of anionic rhenium complex Na[(BDI)ReCp] ( 1 , BDI = N,N' -bis(2,6-diisopropylphenyl)-3,5-dimethyl-β-diketiminate) to generate several rhenium-phosphorus complexes. Complex 1 reacts in a metathetical manner with chlorophosphines Ph 2 PCl, Me NHP-Cl, and OHP-Cl to generate XL-type phosphido complexes 2 , 3 , and 4 , respectively ( Me NHP-Cl = 2-chloro-1,3-dimethyl-1,3,2-diazaphospholidine; OHP-Cl = 2-chloro-1,3,2-dioxaphospholane). Crystallographic and computational investigations of phosphido triad 2 , 3 , and 4 reveal that increasing the electronegativity of the phosphorus substituent (C < N < O) results in a shortening and strengthening of the rhenium-phosphorus bond. Complex 1 reacts with iminophosphane Mes*NPCl (Mes* = 2,4,6-tri tert -butylphenyl) to generate linear iminophosphanyl complex 5 . In the presence of a suitable halide abstraction reagent, 1 reacts with the dichlorophosphine i Pr 2 NPCl 2 to afford cationic phosphinidene complex 6 + . Complex 6 + may be reduced by one electron to form 6 • , a rare example of a stable, paramagnetic phosphinidene complex. Spectroscopic and structural investigations, as well as computational analyses, are employed to elucidate the influence of the phosphorus substituent on the nature of the rhenium-phosphorus bond in 2 through 6 . Furthermore, we examine several common analogies employed to understand metal phosphido, phosphinidene, and iminophosphanyl complexes.

Electronic (e-) cigarette formulations containing nicotine salts from a range of organic acid conjugates and pH values have dominated the commercial market. The acids in the nicotine salt formulations may alter the redox environment in e-cigarettes, impacting free radical formation in e-cigarette aerosol. Here, the generation of aerosol mass and free radicals from a fourth-generation e-cigarette device was evaluated at 2 wt % nicotine salts (pH 7, 30:70 mixture propylene glycol to vegetable glycerin) across eight organic acids used in e-liquids: benzoic acid (BA), salicylic acid (SLA), lactic acid (LA), levulinic acid (LVA), succinic acid (SA), malic acid (MA), tartaric acid (TA), and citric acid (CA). Furthermore, 2 wt % BA nicotine salts were studied at the following nicotine to acid ratios: 1:2 (pH 4), 1:1 (pH 7), and 2:1 (pH 8), in comparison with freebase nicotine (pH 10). Radical yields were quantified by spin-trapping and electron paramagnetic resonance (EPR) spectroscopy. The EPR spectra of free radicals in the nicotine salt aerosol matched those generated from the Fenton reaction, which are primarily hydroxyl (OH) radicals and other reactive oxygen species (ROS). Although the aerosol mass formation was not significantly different for most of the tested nicotine salts and acid concentrations, notable ROS yields were observed only from BA, CA, and TA under the study conditions. The e-liquids with SLA, LA, LVA, SA, and MA produced less ROS than the 2 wt % freebase nicotine e-liquid, suggesting that organic acids may play dual roles in the production and scavenging of ROS. For BA nicotine salts, it was found that the ROS yield increased with a higher acid concentration (or a lower nicotine to acid ratio). The observation that BA nicotine salts produce the highest ROS yield in aerosol generated from a fourth-generation vape device, which increases with acid concentration, has important implications for ROS-mediated health outcomes that may be relevant to consumers, manufacturers, and regulatory agencies.

Electrocatalytic water oxidation is a key transformation in many strategies designed to harness solar energy and store it as chemical fuels. Understanding the mechanism(s) of the best electrocatalysts for water oxidation has been a fundamental chemical challenge for decades. Here, we quantitate evolved dioxygen isotopologue composition via gas-phase EPR spectroscopy to elucidate the mechanisms of water oxidation on metal oxide electrocatalysts with high precision. Isotope fractionation is paired with computational and kinetic modeling, showing that this technique is sensitive enough to differentiate O-O bond-forming steps. Strong agreement between experiment and theory indicates that for the nickel-iron layered double hydroxide─one of the best earth-abundant electrocatalysts to be studied─water oxidation proceeds via a dioxo coupling mechanism to form a side-bound peroxide rather than a hydroxide attack to form an end-bound peroxide.

The paper aims to elucidate the final stages in the biosynthesis of the [2Fe] H active site of the [FeFe]-hydrogenases. The recently hypothesized intermediate [Fe 2 (SCH 2 NH 2 ) 2 (CN) 2 (CO) 4 ] 2- ([1] 2- ) was prepared by a multistep route from [Fe 2 (S 2 )(CN)(CO) 5 ] - . The following synthetic intermediates were characterized in order: [Fe 2 (SCH 2 NHFmoc) 2 (CNBEt 3 )(CO) 5 ] - , [Fe 2 (SCH 2 NHFmoc) 2 (CN)-(CO) 5 ] - , and [Fe 2 (SCH 2 NHFmoc) 2 (CN) 2 (CO) 4 ] 2- , where Fmoc is fluorenylmethoxycarbonyl). Derivatives of these anions include [K(18-crown-6)] + , PPh 4 + and PPN + salts as well as the 13 CD 2 -isotopologues. These Fe 2 species exist as a mixture of two isomers attributed to diequatorial (ee) and axial-equatorial (ae) stereochemistry at sulfur. In vitro experiments demonstrate that [1] 2- maturates HydA1 in the presence of HydF and a cocktail of reagents. HydA1 can also be maturated using a highly simplified cocktail, omitting HydF and other proteins. This result is consistent with HydA1 participating in the maturation process and refines the roles of HydF., (© 2024 The Authors. Angewandte Chemie International Edition published by Wiley-VCH GmbH.)

Airway smooth muscle cell (ASM) is renowned for its involvement in airway hyperresponsiveness through impaired ASM relaxation and bronchoconstriction in asthma, which poses a significant challenge in the field. Recent studies have explored different targets in ASM to alleviate airway hyperresponsiveness, however, a sizeable portion of patients with asthma still experience poor control. In our study, we explored protein phosphatase 2 A (PP2A) in ASM as it has been reported to regulate cellular contractility by controlling intracellular calcium ([Ca 2+ ] i ), ion channels, and respective regulatory proteins. We obtained human ASM cells and lung tissues from healthy and patients with asthma and evaluated PP2A expression using RNA-Seq data, immunofluorescence, and immunoblotting. We further investigated the functional importance of PP2A by determining its role in bronchoconstriction using mouse bronchus and human ASM cell [Ca 2+ ] i regulation. We found robust expression of PP2A isoforms in human ASM cells with PP2Aα being highly expressed. Interestingly, PP2Aα was significantly downregulated in asthmatic tissue and human ASM cells exposed to proinflammatory cytokines. Functionally, FTY720 (PP2A agonist) inhibited acetylcholine- or methacholine-induced bronchial contraction in mouse bronchus and further potentiated isoproterenol-induced bronchial relaxation. Mechanistically, FTY720 inhibited histamine-evoked [Ca 2+ ] i response and myosin light chain (MLC) phosphorylation in the presence of interleukin-13 (IL-13) in human ASM cells. To conclude, we for the first time established PP2A signaling in ASM, which can be further explored to develop novel therapeutics to alleviate airway hyperresponsiveness in asthma. NEW & NOTEWORTHY This novel study deciphered the expression and function of protein phosphatase 2Aα (PP2Aα) in airway smooth muscle (ASM) during asthma and/or inflammation. We showed robust expression of PP2Aα in human ASM while its downregulation in asthmatic ASM. Similarly, we demonstrated reduced PP2Aα expression in ASM exposed to proinflammatory cytokines. PP2Aα activation inhibited bronchoconstriction of isolated mouse bronchi. In addition, we unveiled that PP2Aα activation inhibits the intracellular calcium release and myosin light chain phosphorylation in human ASM.

Pyrrolysine, the 22nd amino acid encoded by the natural genetic code, is essential for methanogenic archaea to catabolize methylamines into methane. The structure of pyrrolysine consists of a methylated pyrroline carboxylate that is linked to the ε-amino group of the l-lysine via an amide bond. The biosynthesis of pyrrolysine requires three enzymes: PylB, PylC, and PylD. PylB is a radical S -adenosyl-l-methionine (SAM) enzyme and catalyzes the first biosynthetic step, the isomerization of l-lysine into methylornithine. PylC catalyzes an ATP-dependent ligation of methylornithine and a second l-lysine to form l-lysine-N ε -methylornithine. The last biosynthetic step is catalyzed by PylD via oxidation of the PylC product to form pyrrolysine. While enzymatic reactions of PylC and PylD have been well characterized by X-ray crystallography and in vitro studies, mechanistic understanding of PylB is still relatively limited. Here, we report the first in vitro activity of PylB to form methylornithine via the isomerization of l-lysine. We also identify a lysyl C4 radical intermediate that is trapped, with its electronic structure and geometric structure well characterized by EPR and ENDOR spectroscopy. In addition, we demonstrate that SAM functions as a catalytic cofactor in PylB catalysis rather than canonically as a cosubstrate. This work provides detailed mechanistic evidence for elucidating the carbon backbone rearrangement reaction catalyzed by PylB during the biosynthesis of pyrrolysine.

Dinuclear monooxygenases mediate challenging C-H bond oxidation reactions throughout nature. Many of these enzymes are presumed to exclusively utilize diiron cofactors. Herein we report the bioinformatic discovery of an orphan dinuclear monooxygenase that preferentially utilizes a heterobimetallic manganese-iron (Mn/Fe) cofactor to mediate an O 2 -dependent C-H bond hydroxylation reaction. Unlike the structurally similar Mn/Fe-dependent monooxygenase AibH2, the diiron form of this enzyme (SfbO) exhibits a nascent enzymatic activity. This behavior raises the possibility that many other dinuclear monooxygenases may be endowed with the capacity to harness cofactors with a variable metal content.

Introduction: Premature infants have an increased risk of respiratory morbidity, including the development of recurrent wheezing. We sought to determine perinatal factors in late preterm infants associated with an increased risk of recurrent wheezing in the first 3 years of life., Methods: A retrospective chart review of infants born between 32 and 36 weeks gestational age at a tertiary hospital from 2013 to 2016 was performed. Infants with any co-morbid medical conditions were excluded. Recurrent wheezing was identified by two or more visit diagnoses for reactive airway disease, wheezing-associated respiratory infection, wheezing, or asthma during the first 3 years of life. Those with recurrent wheezing were compared to matched preterm infants who did not develop wheezing., Results: Three hundred and fourteen late preterm infants were included in this study; 210 infants developed recurrent wheezing while 104 did not. Gender, sex, and race were comparable between both groups. Development of wheezing was associated with positive family history of asthma (p = .014), receiving antibiotics during the neonatal period (p < .001), requiring continuous positive airway pressure for <24 h (p = .019), and receiving supplemental oxygen during the newborn period (p = .023)., Conclusion: This study retrospectively identified risk factors associated with development of wheezing in late preterm infants. Prospective studies are needed to determine whether these factors will predict recurrent wheeze in this patient population., (© 2023 The Authors. Pediatric Pulmonology published by Wiley Periodicals LLC.)

In nature, nonheme iron enzymes use dioxygen to generate high-spin iron(IV)=O species for a variety of oxygenation reactions. Although synthetic chemists have long sought to mimic this reactivity, the enzyme-like activation of O 2 to form high-spin iron(IV) = O species remains an unrealized goal. Here, we report a metal-organic framework featuring iron(II) sites with a local structure similar to that in α-ketoglutarate-dependent dioxygenases. The framework reacts with O 2 at low temperatures to form high-spin iron(IV) = O species that are characterized using in situ diffuse reflectance infrared Fourier transform, in situ and variable-field Mössbauer, Fe Kβ x-ray emission, and nuclear resonance vibrational spectroscopies. In the presence of O 2 , the framework is competent for catalytic oxygenation of cyclohexane and the stoichiometric conversion of ethane to ethanol.

[FeFe]-hydrogenases employ a catalytic H-cluster, consisting of a [4Fe-4S] H cluster linked to a [2Fe] H subcluster with CO, CN - ligands, and an azadithiolate bridge, which mediates the rapid redox interconversion of H + and H 2 . In the biosynthesis of this H-cluster active site, the radical S -adenosyl-l-methionine (radical SAM, RS) enzyme HydG plays the crucial role of generating an organometallic [Fe(II)(CN)(CO) 2 (cysteinate)] - product that is en route to forming the H-cluster. Here, we report direct observation of this diamagnetic organometallic Fe(II) complex through Mössbauer spectroscopy, revealing an isomer shift of δ = 0.10 mm s -1 and quadrupole splitting of Δ E Q = 0.66 mm s -1 . These Mössbauer values are a change from the starting values of δ = 1.15 mm s -1 and Δ E Q = 3.23 mm s -1 for the ferrous "dangler" Fe in HydG. These values of the observed product complex B are in good agreement with Mössbauer parameters for the low-spin Fe 2+ ions in synthetic analogues, such as 57 Fe Syn-B, which we report here. These results highlight the essential role that HydG plays in converting a resting-state high-spin Fe(II) to a low-spin organometallic Fe(II) product that can be transferred to the downstream maturase enzymes.

In this letter, a 263 GHz traveling wave tube for electron paramagnetic resonance spectroscopy is designed, fabricated and tested. A periodic permanent magnet focused pencil beam electron optical system is adopted. A folded waveguide slow-wave structure with modified serpentine bends is optimized to provide high-power wideband performance and stable operation. An experiment has been performed to verify the analysis results and confirm the amplifier stability. The device provides a maximum 11.9 W saturation output power and 25.5 dB saturation gain. Although the available solid-state signal source is unable to drive the amplifier to saturation beyond 260 - 264 GHz, 10 W output power over 5.6 GHz bandwidth has been measured.

[FeFe] hydrogenases contain a 6-Fe cofactor that serves as the active site for efficient redox interconversion between H 2 and protons. The biosynthesis of the so-called H-cluster involves unusual enzymatic reactions that synthesize organometallic Fe complexes containing azadithiolate, CO, and CN - ligands. We have previously demonstrated that specific synthetic [Fe(CO) x (CN) y ] complexes can be used to functionally replace proposed Fe intermediates in the maturation reaction. Here, we report the results from performing such cluster semisynthesis in the context of a recent fully defined cluster maturation procedure, which eliminates unknown components previously employed from Escherichia coli cell lysate and demonstrate this provides a concise route to H-cluster synthesis. We show that formaldehyde can be used as a simple reagent as the carbon source of the bridging adt ligand of H-cluster in lieu of serine/serine hydroxymethyltransferase. In addition to the actual H-cluster, we observe the formation of several H-cluster-like species, the identities of which are probed by cryogenic photolysis combined with EPR/ENDOR spectroscopy.

Copper complexes are widely used in the synthesis of fine chemicals and materials to catalyze couplings of heteroatom nucleophiles with aryl halides. We show that cross-couplings catalyzed by some of the most active catalysts occur by a mechanism not previously considered. Copper(II) [Cu(II)] complexes of oxalamide ligands catalyze Ullmann coupling to form the C-O bond in aryl ethers by concerted oxidative addition of an aryl halide to Cu(II) to form a high-valent species that is stabilized by radical character on the oxalamide ligand. This mechanism diverges from those involving Cu(I) and Cu(III) intermediates that have been posited for other Ullmann-type couplings. The stability of the Cu(II) state leads to high turnover numbers, >1000 for the coupling of phenoxide with aryl chloride electrophiles, as well as an ability to run the reactions in air.

The oxidation of thianthrene and 10-phenylphenothiazine into cation radicals has been examined using redox-active Lewis acids. The reaction of titanium(IV) tetrachloride with thianthrene in toluene produces a solution with an EPR spectrum indicative of oxidation of thianthrene to a cation radical, but the molecular compound ( 1 ) (μ-thianthrene)Ti 2 (μ-Cl 2 )Cl 6 crystallized exclusively. Red crystalline ( 2 ) (μ-thianthrene)Ti 2 (μ-Br 2 )Br 6 formed similarly from titanium(IV) tetrabromide. In contrast, the reaction of antimony(V) pentachloride with thianthrene in toluene yielded crystalline ( 3 ) (thianthrene · + ) 2 (Sb 2 (μ-Cl) 2 Cl 6 2- ) · (SbCl 3 ), while the same reaction in acetonitrile produced crystals of ( 4 ) (thianthrene · + )(SbCl 6 - ). The two paramagnetic salts differ in that in ( 3 ), the folded (thianthrene · + ) cation radicals self-associate, whereas in ( 4 ), the (thianthrene · + ) cation radicals are isolated from one another and are planar. The reaction of 10-phenylphenothiazine with titanium(IV) tetrachloride in toluene solution resulted in the formation of crystalline paramagnetic ( 5 ) (10-phenylphenothiazine · + )(Ti(μ-Cl) 3 Cl 6 - ) and the reaction of 10-phenylphenothiazine with tin(IV) tetrachloride produced paramagnetic ( 6 ) (10-phenylphenothiazine · + )(SnCl 5 - ).

Peptide hormones are essential signaling molecules with therapeutic importance. Identifying regulatory factors that drive their activity gives important insight into their mode of action and clinical development. In this work, we demonstrate the combined impact of Cu(II) and the serum protein albumin on the activity of C-peptide, a 31-mer peptide derived from the same prohormone as insulin. C-peptide exhibits beneficial effects, particularly in diabetic patients, but its clinical use has been hampered by a lack of mechanistic understanding. We show that Cu(II) mediates the formation of ternary complexes between albumin and C-peptide and that the resulting species depend on the order of addition. These ternary complexes notably alter peptide activity, showing differences from the peptide or Cu(II)/peptide complexes alone in redox protection as well as in cellular internalization of the peptide. In standard clinical immunoassays for measuring C-peptide levels, the complexes inflate the quantitation of the peptide, suggesting that such adducts may affect biomarker quantitation. Altogether, our work points to the potential relevance of Cu(II)-linked C-peptide/albumin complexes in the peptide's mechanism of action and application as a biomarker.

The aerobic oxidation of carbon-hydrogen (C-H) bonds in biology is currently known to be accomplished by a limited set of cofactors that typically include heme, nonheme iron, and copper. While manganese cofactors perform difficult oxidation reactions, including water oxidation within Photosystem II, they are generally not known to be used for C-H bond activation, and those that do catalyze this important reaction display limited intrinsic reactivity. Here we report that the 2-aminoisobutyric acid hydroxylase from Rhodococcus wratislaviensis , AibH1H2, requires manganese to functionalize a strong, aliphatic C-H bond (BDE = 100 kcal/mol). Structural and spectroscopic studies of this enzyme reveal a redox-active, heterobimetallic manganese-iron active site at the locus of O 2 activation and substrate coordination. This result expands the known reactivity of biological manganese-iron cofactors, which was previously restricted to single-electron transfer or stoichiometric protein oxidation. Furthermore, the AibH1H2 cofactor is supported by a protein fold distinct from typical bimetallic oxygenases, and bioinformatic analyses identify related proteins abundant in microorganisms. This suggests that many uncharacterized monooxygenases may similarly require manganese to perform oxidative biochemical tasks.

In multicopper oxidases (MCOs), the type 1 (T1) Cu accepts electrons from the substrate and transfers these to the trinuclear Cu cluster (TNC) where O 2 is reduced to H 2 O. The T1 potential in MCOs varies from 340 to 780 mV, a range not explained by the existing literature. This study focused on the ∼350 mV difference in potential of the T1 center in Fet3p and Trametes versicolor laccase (TvL) that have the same 2His1Cys ligand set. A range of spectroscopies performed on the oxidized and reduced T1 sites in these MCOs shows that they have equivalent geometric and electronic structures. However, the two His ligands of the T1 Cu in Fet3p are H-bonded to carboxylate residues, while in TvL they are H-bonded to noncharged groups. Electron spin echo envelope modulation spectroscopy shows that there are significant differences in the second-sphere H-bonding interactions in the two T1 centers. Redox titrations on type 2-depleted derivatives of Fet3p and its D409A and E185A variants reveal that the two carboxylates (D409 and E185) lower the T1 potential by 110 and 255-285 mV, respectively. Density functional theory calculations uncouple the effects of the charge of the carboxylates and their difference in H-bonding interactions with the His ligands on the T1 potential, indicating 90-150 mV for anionic charge and ∼100 mV for a strong H-bond. Finally, this study provides an explanation for the generally low potentials of metallooxidases relative to the wide range of potentials of the organic oxidases in terms of different oxidized states of their TNCs involved in catalytic turnover.

Asthma is a heterogenous chronic inflammatory lung disease with endotypes that manifest different immune system profiles, severity, and responses to current therapies. Regardless of endotype, asthma features increased immune cell infiltration, inflammatory cytokine release, and airway remodeling. Lung macrophages are also heterogenous in that there are separate subsets and, depending on the environment, different effector functions. Lung macrophages are important in recruitment of immune cells such as eosinophils, neutrophils, and monocytes that enhance allergic inflammation and initiate T helper cell responses. Persistent lung remodeling including mucus hypersecretion, increased airway smooth muscle mass, and airway fibrosis contributes to progressive lung function decline that is insensitive to current asthma treatments. Macrophages secrete inflammatory mediators that induce airway inflammation and remodeling. Additionally, lung macrophages are instrumental in protecting against pathogens and play a critical role in resolution of inflammation and return to homeostasis. This review summarizes current literature detailing the roles and existing knowledge gaps for macrophages as key inflammatory orchestrators in asthma pathogenesis. We also raise the idea that modulating inflammatory responses in lung macrophages is important for alleviating asthma.

The residue D1-D170 bridges Mn4 with the Ca ion in the O 2 -evolving Mn 4 CaO 5 cluster of Photosystem II. Recently, the D1-D170E mutation was shown to substantially alter the S n+1 -minus-S n FTIR difference spectra [Debus RJ (2021) Biochemistry 60:3841-3855]. The mutation was proposed to alter the equilibrium between different Jahn-Teller conformers of the S 1 state such that (i) a different S 1 state conformer is stabilized in D1-D170E than in wild-type and (ii) the S 1 to S 2 transition in D1-D170E produces a high-spin form of the S 2 state rather than the low-spin form that is produced in wild-type. In this study, we employed EPR spectroscopy to test if a high-spin form of the S 2 state is formed preferentially in D1-D170E PSII. Our data show that illumination of dark-adapted D1-D170E PSII core complexes does indeed produce a high-spin form of the S 2 state rather than the low-spin multiline form that is produced in wild-type. This observation provides further experimental support for a change in the equilibrium between S state conformers in both the S 1 and S 2 states in a site-directed mutant that retains substantial O 2 evolving activity., (© 2023. The Author(s), under exclusive licence to Springer Nature B.V.)

Electrochemical conversion of CO 2 requires selective catalysts and high solubility of CO 2 in the electrolyte to reduce the energy requirement and increase the current efficiency. In this study, the CO 2 reduction reaction (CO 2 RR) over Ag electrodes in acetonitrile-based electrolytes containing 0.1 M [EMIM][2-CNpyr] (1-ethyl-3-methylimidazolium 2-cyanopyrolide), a reactive ionic liquid (IL), is shown to selectively (>94%) convert CO 2 to CO with a stable current density (6 mA·cm -2 ) for at least 12 h. The linear sweep voltammetry experiments show the onset potential of CO 2 reduction in acetonitrile shifts positively by 240 mV when [EMIM][2-CNpyr] is added. This is attributed to the pre-activation of CO 2 through the carboxylate formation via the carbene intermediate of the [EMIM] + cation and the carbamate formation via binding to the nucleophilic [2-CNpyr] - anion. The analysis of the electrode-electrolyte interface by surface-enhanced Raman spectroscopy (SERS) confirms the catalytic role of the functionalized IL where the accumulation of the IL-CO 2 adduct between -1.7 and -2.3 V vs Ag/Ag + and the simultaneous CO formation are captured. This study reveals the electrode surface species and the role of the functionalized ions in lowering the energy requirement of CO 2 RR for the design of multifunctional electrolytes for the integrated capture and conversion., Competing Interests: The authors declare no competing financial interest., (© 2023 The Authors. Published by American Chemical Society.)

The recent discovery of metal-metal bonding and valence delocalization in the dilanthanide complexes (Cp iPr5 ) 2 Ln 2 I 3 (Cp iPr5 = pentaisopropylcyclopentadienyl; Ln = Y, Gd, Tb, Dy) opened up the prospect of harnessing the 4f n 5d z 2 1 electron configurations of non-traditional divalent lanthanide ions to access molecules with novel bonding motifs and magnetism. Here, we report the trinuclear mixed-valence clusters (Cp iPr5 ) 3 Ln 3 H 3 I 2 ( 1-Ln , Ln = Y, Gd), which were synthesized via potassium graphite reduction of the trivalent clusters (Cp iPr5 ) 3 Ln 3 H 3 I 3 . Structural, computational, and spectroscopic analyses support valence delocalization in 1-Ln resulting from a three-center, one-electron σ bond formed from the 4d z 2 and 5d z 2 orbitals on Y and Gd, respectively. Dc magnetic susceptibility data obtained for 1-Gd reveal that valence delocalization engenders strong parallel alignment of the σ-bonding electron and the 4f electrons of each gadolinium center to afford a high-spin ground state of S = 11. Notably, this represents the first clear instance of metal-metal bonding in a molecular trilanthanide complex, and the large spin-spin exchange constant of J = 168(1) cm -1 determined for 1-Gd is only the second largest coupling constant characterized to date for a molecular lanthanide compound.

Asthma in elderly populations is an increasing health problem that is accompanied by diminished lung function and frequent exacerbations. As potent anti-inflammatory drugs, corticosteroids are commonly used to reduce lung inflammation, improve lung function, and manage disease symptoms in asthma. Although effective for most individuals, older patients are more insensitive to corticosteroids, making it difficult to manage asthma in this population. With the number of individuals older than 65 continuing to increase, it is important to understand the distinct mechanisms that promote corticosteroid insensitivity in the aging lung. In this review, we discuss corticosteroid insensitivity in asthma with an emphasis on mechanisms that contribute to persistent inflammation and diminished lung function in older individuals.

Fungi and laccase mediator systems (LMSs) have a proven track record of oxidizing recalcitrant organic compounds. There has been considerable interest in applying LMSs to the treatment of perfluoroalkyl acids (PFAAs), a class of ubiquitous and persistent environmental contaminants. Some laboratory experiments have indicated modest losses of PFAAs over extended periods, but there have been no clear demonstrations of a transformation mechanism or the kinetics that would be needed for remediation applications. We set out to determine if this was a question of identifying and optimizing a rate-limiting step but discovered that observed losses of PFAAs were experimental artifacts. While unable to replicate the oxidation of PFAAs, we show that interactions of the PFAA compounds with laccase and laccase mediator mixtures could cause an artifact that mimics transformation (≲60%) of PFAAs. Furthermore, we employed a surrogate compound, carbamazepine (CBZ), and electron paramagnetic resonance spectroscopy to probe the formation of the radical species that had been proposed to be responsible for contaminant oxidation. We confirmed that under conditions where sufficient radical concentrations were produced to oxidize CBZ, no PFAA removal took place., Competing Interests: The authors declare no competing financial interest., (© 2023 The Authors. Published by American Chemical Society.)

Manganese cofactors activate strong chemical bonds in many essential enzymes. Yet very few manganese-dependent enzymes are known to functionalize ubiquitous carbon-hydrogen (C-H) bonds, and those that catalyze this important reaction display limited intrinsic reactivity. Herein, we report that the 2-aminoisobutyric acid hydroxylase from Rhodococcus wratislaviensis requires manganese to functionalize a C-H bond possessing a bond dissociation enthalpy (BDE) exceeding 100 kcal/mol. Structural and spectroscopic studies of this enzyme reveal a redox-active, heterobimetallic manganese-iron active site that utilizes a manganese ion at the locus for O 2 activation and substrate coordination. Accordingly, this enzyme represents the first documented Mn-dependent monooxygenase in biology. Related proteins are widespread in microorganisms suggesting that many uncharacterized monooxygenases may utilize manganese-containing cofactors to accomplish diverse biological tasks.

Background: Cystic fibrosis (CF) is a multisystem disease with progressive deterioration. Recently, CF transmembrane conductance regulator (CFTR) modulator therapies were introduced that repair underlying protein defects. Objective of this study was to determine the impact of elexacaftor-tezacaftor-ivacaftor (ETI) on clinical parameters and inflammatory responses in people with CF (pwCF)., Methods: Lung function (FEV 1 ), body mass index (BMI) and microbiologic data were collected at initiation and 3-month intervals for 1 year. Blood was analyzed at baseline and 6 months for cytokines and immune cell populations via flow cytometry and compared to non-CF controls., Results: Sample size was 48 pwCF, 28 (58.3%) males with a mean age of 28.8 ± 10.7 years. Significant increases in %predicted FEV 1 and BMI were observed through 6 months of ETI therapy with no change thereafter. Changes in FEV 1 and BMI at 3 months were significantly correlated (r = 57.2, p < 0.01). There were significant reductions in Pseudomonas and Staphylococcus positivity (percent of total samples) in pwCF through 12 months of ETI treatment. Healthy controls (n = 20) had significantly lower levels of circulating neutrophils, interleukin (IL)-6, IL-8, and IL-17A and higher levels of IL-13 compared to pwCF at baseline (n = 48). After 6 months of ETI, pwCF had significant decreases in IL-8, IL-6, and IL-17A levels and normalization of peripheral blood immune cell composition., Conclusions: In pwCF, ETI significantly improved clinical outcomes, reduced systemic pro-inflammatory cytokines, and restored circulating immune cell composition after 6 months of therapy., (© 2022 The Authors. Pediatric Pulmonology published by Wiley Periodicals LLC.)

The synthesis of bimetallic molecular silicide complexes is reported, based on the use of multiple Si-H bond activations in SiH 4 at the metal centers of 14-electron LCo I fragments (L = Tp″, HB(3,5-diisopropylpyrazolyl) 3 - ; [BP 2 t Bu Pz], PhB(CH 2 P t Bu 2 ) 2 (pyrazolyl)). Upon exposure of (Tp″Co) 2 (μ-N 2 ) ( 1 ) to SiH 4 , a mixture of (Tp″Co) 2 (μ-H) ( 2 ) and (Tp″Co) 2 (μ-H) 2 ( 3 ) was formed and no evidence for Si-H oxidative addition products was observed. In contrast, [BP 2 t Bu Pz]-supported Co complexes led to Si-H oxidative additions with the generation of silylene and silicide complexes as products. Notably, the reaction of ([BP 2 t Bu Pz]Co) 2 (μ-N 2 ) ( 5 ) with SiH 4 gave the dicobalt silicide complex [BP 2 t Bu Pz](H) 2 Co═Si═Co(H) 2 [BP 2 t Bu Pz] ( 8 ) in high yield, representing the first direct route to a symmetrical bimetallic silicide. The effect of the [BP 2 t Bu Pz] ligand on Co-Si bonding in 7 and 8 was explored by analysis of solid-state molecular structures and density functional theory (DFT) investigations. Upon exposure to CO or DMAP (DMAP = 4-dimethylaminopyridine), 8 converted to the corresponding [BP 2 t Bu Pz]Co(L) x adducts (L = CO, x = 2; L = DMAP, x = 1) with concomitant loss of SiH 4 , despite the lack of significant Si-H interactions in the starting complex. On heating to 60 °C, 8 underwent reaction with MeCl to produce small quantities of Me x SiH 4- x ( x = 1-3), demonstrating functionalization of the μ-silicon atom in a molecular silicide to form organosilanes.

Heme-copper oxidases (HCOs) utilize tyrosine (Tyr) to donate one of the four electrons required for the reduction of O 2 to water in biological respiration, while tryptophan (Trp) is speculated to fulfill the same role in cyt bd oxidases. We previously engineered myoglobin into a biosynthetic model of HCOs and demonstrated the critical role that Tyr serves in the oxygen reduction reaction (ORR). To address the roles of Tyr and Trp in these oxidases, we herein report the preparation of the same biosynthetic model with the Tyr replaced by Trp and further demonstrate that Trp can also promote the ORR, albeit with lower activity. An X-ray crystal structure of the Trp variant shows a hydrogen-bonding network involving two water molecules that are organized by Trp, similar to that in the Tyr variant, which is absent in the crystal structure with the native Phe residue. Additional electron paramagnetic resonance measurements are consistent with the formation of a Trp radical species upon reacting with H 2 O 2 . We attribute the lower activity of the Trp variant to Trp's higher reduction potential relative to Tyr. Together, these findings demonstrate, for the first time, that Trp can indeed promote the ORR and provides a structural basis for the observation of varying activities. The results support a redox role for the conserved Trp in bd oxidase while suggesting that HCOs use Tyr instead of Trp to achieve higher reactivity.

The field of sterol and oxysterol biology in lung disease has recently gained attention, revealing a unique need for sterol uptake and metabolism in the lung. The presence of cholesterol transport, biosynthesis, and sterol/oxysterol-mediated signaling in immune cells suggests a role in immune regulation. In support of this idea, statin drugs that inhibit the cholesterol biosynthesis rate-limiting step enzyme, hydroxymethyl glutaryl coenzyme A reductase, show immunomodulatory activity in several models of inflammation. Studies in human asthma reveal contradicting results, whereas promising retrospective studies suggest benefits of statins in severe asthma. Here, we provide a timely review by discussing the role of sterols in immune responses in asthma, analytical tools to evaluate the role of sterols in disease, and potential mechanistic pathways and targets relevant to asthma. Our review reveals the importance of sterols in immune processes and highlights the need for further research to solve critical gaps in the field., Competing Interests: Disclosure of potential conflict of interest: M. Guerau-de-Arellano is an inventor of a PRMT5 inhibitor patent. The rest of the authors declare that they have no relevant conflicts of interest.

A small but growing number of molecular compounds have been isolated featuring divalent lanthanides with 4f n 5d z 2 1 electron configurations. While the majority of these possess trigonal coordination geometries, we previously reported the first examples of linear divalent metallocenes Ln(Cp iPr5 ) 2 (Ln = Tb, Dy; Cp iPr5 = pentaisopropylcyclopentadienyl). Here, we report the synthesis and characterization of the remainder of the Ln(Cp iPr5 ) 2 ( 1-Ln ) series (including Y and excluding Pm). The compounds can be synthesized through salt metathesis of LnI 3 and NaCp iPr5 followed by potassium graphite reduction for Ln = Y, La, Ce, Pr, Nd, Gd, Ho, and Er, by in situ reduction during salt metathesis of LnI 3 and NaCp iPr5 for Ln = Tm and Lu, or through salt metathesis from LnI 2 and NaCp iPr5 for Ln = Sm, Eu, and Yb. Single crystal X-ray diffraction analyses of 1-Ln confirm a linear coordination geometry with pseudo- D 5 d symmetry for the entire series. Structural and ultraviolet-visible spectroscopy data support a 4f n +1 electron configuration for Ln 2+ = Sm, Eu, Tm, and Yb and a 4f n 5d z 2 1 configuration for the other lanthanides ([Kr]4d z 2 1 for Y 2+ ). Characterization of 1-Ln (Ln = Y, La) using electron paramagnetic resonance spectroscopy reveals significant s-d orbital mixing in the highest occupied molecular orbital and hyperfine coupling constants that are the largest reported to date for divalent compounds of yttrium and lanthanum. Evaluation of the room temperature magnetic susceptibilities of 1-Ln and comparison with values previously reported for trigonal Ln 2+ compounds suggests that the more pronounced 6s-5d mixing may be associated with weaker 4f-5d spin coupling.

While sterols regulate immune processes key to the pathogenesis of asthma, inhibition of sterols with statin drugs has shown conflicting results in human asthma. Here, a novel understanding of the impact of sterols on type 17 immune responses and asthma lead us to hypothesize that sterols and statins may be relevant to severe asthma endotypes with neutrophil infiltration., Competing Interests: Declaration of interests The authors have no interests to declare., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

S/N crosstalk species derived from the interconnected reactivity of H 2 S and NO facilitate the transport of reactive sulfur and nitrogen species in signaling, transport, and regulatory processes. We report here that simple Fe 2+ ions, such as those that are bioavailable in the labile iron pool (LIP), react with thionitrite (SNO - ) and perthionitrite (SSNO - ) to yield the dinitrosyl iron complex [Fe(NO) 2 (S 5 )] - . In the reaction of FeCl 2 with SNO - we were able to isolate the unstable intermediate hydrosulfido mononitrosyl iron complex [FeCl 2 (NO)(SH)] - , which was characterized by X-ray crystallography. We also show that [Fe(NO) 2 (S 5 )] - is a simple synthon for nitrosylated Fe-S clusters via its reduction with PPh 3 to yield Roussin's Red Salt ([Fe 2 S 2 (NO) 4 ] 2- ), which highlights the role of S/N crosstalk species in the assembly of fundamental Fe-S motifs., (© 2022 Wiley-VCH GmbH.)

Background: Corticosteroids remain a key therapy for treating children with asthma. Patients with severe asthma are insensitive, resistant, or refractory to corticosteroids and have poorly controlled symptoms that involve airway inflammation, airflow obstruction, and frequent exacerbations. While the pathways that mediate corticosteroid insensitivity in asthma remain poorly defined, recent studies suggest that enhanced Th1 pathways, mediated by TNFα and IFNγ, may play a role. We previously reported that the combined effects of TNFα and IFNγ promote corticosteroid insensitivity in developing human airway smooth muscle (ASM)., Methods: To further understand the effects of TNFα and IFNγ on corticosteroid sensitivity in the context of neonatal and pediatric asthma, we performed RNA sequencing (RNA-seq) on human pediatric ASM treated with fluticasone propionate (FP), TNFα, and/or IFNγ., Results: We found that TNFα had a greater effect on gene expression (~ 1000 differentially expressed genes) than IFNγ (~ 500 differentially expressed genes). Pathway and transcription factor analyses revealed enrichment of several pro-inflammatory responses and signaling pathways. Interestingly, treatment with TNFα and IFNγ augmented gene expression with more than 4000 differentially expressed genes. Effects of TNFα and IFNγ enhanced several pro-inflammatory genes and pathways related to ASM and its contributions to asthma pathogenesis, which persisted in the presence of corticosteroids. Co-expression analysis revealed several gene networks related to TNFα- and IFNγ-mediated signaling, pro-inflammatory mediator production, and smooth muscle contractility. Many of the co-expression network hubs were associated with genes that are insensitive to corticosteroids., Conclusions: Together, these novel studies show the combined effects of TNFα and IFNγ on pediatric ASM and implicate Th1-associated cytokines in promoting ASM inflammation and hypercontractility in severe asthma., (© 2022. The Author(s).)

Phosphorothioate modifications have widespread use in the field of nucleic acids. As substitution of sulfur for oxygen can alter metal coordination preferences, the phosphorothioate metal-rescue experiment is a powerful method for identifying metal coordination sites that influence specific properties in a large RNAs. The A9/G10.1 metal binding site of the hammerhead ribozyme (HHRz) has previously been shown to be functionally important through phosphorothioate rescue experiments. While an A9-S Rp substitution is inhibitory in Mg 2+ , thiophilic Cd 2+ rescues HHRz activity. Mn 2+ is also often used in phosphorothioate metal-rescue studies but does not support activity for the A9-S Rp HHRz. Here, we use EPR, electron spin-echo envelope modulation (ESEEM), and X-ray absorption spectroscopic methods to directly probe the structural consequences of Mn 2+ and Cd 2+ coordination to R p and S p phosphorothioate modifications at the A9/G10.1 site in the truncated hammerhead ribozyme (tHHRz). The results demonstrate that while Cd 2+ does indeed bind to S in the thio-substituted ligand, Mn 2+ coordinates to the non‑sulfur oxo group of this phosphorothioate, regardless of isomer. Computational models demonstrate the energetic preference of MnO over MnS coordination in metal-dimethylthiophosphate models. In the case of the tHHRz, the resulting Mn 2+ coordination preference of oxygen in either R p or S p A9 phosphorothioates differentially tunes catalytic activity, with MnO coordination in the A9-S Rp phosphorothioate enzyme being inhibitory., (Copyright © 2022 Elsevier Inc. All rights reserved.)

Type 2-high severe asthma is described as a distinct endotype with Th2 inflammation, high eosinophil lung infiltration, impaired lung function, and reduced corticosteroid sensitivity. While the inflammatory milieu is similar to mild asthma, patients with type 2-high severe asthma likely have underlying mechanisms that sustain asthma pathophysiology despite corticosteroid treatments. Acute and chronic allergen models induce robust type 2 inflammatory responses, however differences in corticosteroid sensitivity remains poorly understood. In the present study, we sensitized and challenged mice with ovalbumin (OVA; acute model) or mixed allergens (MA; chronic model). Corticosteroid sensitivity was assessed by administering vehicle, 1, or 3 mg/kg fluticasone propionate (FP) and examining key asthmatic features such as airway inflammation, remodeling, hyperresponsiveness, and antioxidant capacity. Both acute and chronic allergen exposure exhibited enhanced AHR, immune cell infiltration, airway inflammation, and remodeling, but corticosteroids were unable to fully alleviate inflammation, AHR, and airway smooth muscle mass in MA-challenged mice. While there were no differences in antioxidant capacity, persistent IL-4+ Th2 cell population suggests the MA model induces type 2 inflammation that is insensitive to corticosteroids. Our data indicate that chronic allergen exposure is associated with more persistent type 2 immune responses and corticosteroid insensitivity. Understanding differences between acute and chronic allergen models could unlock underlying mechanisms related to type 2-high severe asthma., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Lewis, Ford, Khan, Walum and Britt.)

Severe asthma is characterized by steroid insensitivity and poor symptom control and is responsible for most asthma-related hospital costs. Therapeutic options remain limited, in part due to limited understanding of mechanisms driving severe asthma. Increased arginine methylation, catalyzed by protein arginine methyltransferases (PRMTs), is increased in human asthmatic lungs. In this study, we show that PRMT5 drives allergic airway inflammation in a mouse model reproducing multiple aspects of human severe asthma. We find that PRMT5 is required in CD4 + T cells for chronic steroid-insensitive severe lung inflammation, with selective T cell deletion of PRMT5 robustly suppressing eosinophilic and neutrophilic lung inflammation, pathology, airway remodeling, and hyperresponsiveness. Mechanistically, we observed high pulmonary sterol metabolic activity, retinoic acid-related orphan receptor γt (RORγt), and Th17 responses, with PRMT5-dependent increases in RORγt's agonist desmosterol. Our work demonstrates that T cell PRMT5 drives severe allergic lung inflammation and has potential implications for the pathogenesis and therapeutic targeting of severe asthma., (Copyright © 2022 by The American Association of Immunologists, Inc.)

Radical S -adenosylmethionine (radical SAM or rSAM) enzymes use their S -adenosylmethionine cofactor bound to a unique Fe of a [4Fe-4S] cluster to generate the "hot" 5'-deoxyadenosyl radical, which drives highly selective radical reactions via specific interactions with a given rSAM enzyme's substrate. This Perspective focuses on the two rSAM enzymes involved in the biosynthesis of the organometallic H-cluster of [FeFe] hydrogenases. We present here a detailed sequential model initiated by HydG, which lyses a tyrosine substrate via a 5'-deoxyadenosyl H atom abstraction from those amino acid's amino group, initially producing dehydroglycine and an oxidobenzyl radical. In this model, two successive radical cascade reactions lead ultimately to the formation of HydG's product, a mononuclear Fe organometallic complex: [Fe(II)(CN)(CO) 2 (cysteinate)] - , with the iron originating from a unique "dangler" Fe coordinated by a cysteine ligand providing a sulfur bridge to another [4Fe-4S] auxiliary cluster in the enzyme. In turn, in this model, [Fe(II)(CN)(CO) 2 (cysteinate)] - is the substrate for HydE, the second rSAM enzyme in the biosynthetic pathway, which activates this mononuclear organometallic unit for dimerization, forming a [Fe 2 S 2 (CO) 4 (CN) 2 ] precursor to the [2Fe] H component of the H-cluster, requiring only the completion of the bridging azadithiolate (SCH 2 NHCH 2 S) ligand. This model is built upon a foundation of data that incorporates cell-free synthesis, isotope sensitive spectroscopies, and the selective use of synthetic complexes substituting for intermediates in the enzymatic "assembly line". We discuss controversies pertaining to this model and some remaining open issues to be addressed by future work., Competing Interests: The authors declare no competing financial interest., (© 2021 The Authors. Published by American Chemical Society.)

The biosynthesis of the active site of the [FeFe]-hydrogenases (HydA1), the H-cluster, is of interest because these enzymes are highly efficient catalysts for the oxidation and production of H 2 . The biosynthesis of the [2Fe] H subcluster of the H-cluster proceeds from simple precursors, which are processed by three maturases: HydG, HydE, and HydF. Previous studies established that HydG produces an Fe(CO) 2 (CN) adduct of cysteine, which is the substrate for HydE. In this work, we show that by using the synthetic cluster [Fe 2 (μ-SH) 2 (CN) 2 (CO) 4 ] 2- active HydA1 can be biosynthesized without maturases HydG and HydE.

The radical S -adenosyl-l-methionine (SAM) enzyme HydG cleaves tyrosine to generate CO and CN - ligands of the [FeFe] hydrogenase H-cluster, accompanied by the formation of a 4-oxidobenzyl radical (4-OB • ), which is the precursor to the HydG p -cresol byproduct. Native HydG only generates a small amount of 4-OB • , limiting detailed electron paramagnetic resonance (EPR) spectral characterization beyond our initial EPR lineshape study employing various tyrosine isotopologues. Here, we show that the concentration of trapped 4-OB • is significantly increased in reactions using HydG variants, in which the "dangler Fe" to which CO and CN - bind is missing or substituted by a redox-inert Zn 2+ ion. This allows for the detailed characterization of 4-OB • using high-field EPR and electron nuclear double resonance spectroscopy to extract its g -values and 1 H/ 13 C hyperfine couplings. These results are compared to density functional theory-predicted values of several 4-OB • models with different sizes and protonation states, with a best fit to the deprotonated radical anion configuration of 4-OB • . Overall, our results depict a clearer electronic structure of the transient 4-OB • radical and provide new insights into the radical SAM chemistry of HydG.