Identification of Genes Involved in Fe–S Cluster Biosynthesis of Nitrogenase in Paenibacillus polymyxa WLY78

NifS and NifU (encoded by nifS and nifU) are generally dedicated to biogenesis of the nitrogenase Fe–S cluster in diazotrophs. However, nifS and nifU are not found in N₂-fixing Paenibacillus strains, and the mechanisms involved in Fe–S cluster biosynthesis of nitrogenase is not clear. Here, we found that the genome of Paenibacillus polymyxa WLY78 contains the complete sufCDSUB operon, a partial sufC2D2B2 operon, a nifS-like gene, two nifU-like genes (nfuA-like and yutI), and two iscS genes. Deletion and complementation studies showed that the sufC, sufD, and sufB genes of the sufCDSUB operon, and nifS-like and yutI genes were involved in the Fe–S cluster biosynthesis of nitrogenase. Heterologous complementation studies demonstrated that the nifS-like gene of P. polymyxa WLY78 is interchangeable with Klebsiella oxytoca nifS, but P. polymyxa WLY78 SufCDB cannot be functionally replaced by K. oxytoca NifU. In addition, K. oxytoca nifU and Escherichia coli nfuA are able to complement the P. polymyxa WLY78 yutI mutant. Our findings thus indicate that the NifS-like and SufCDB proteins are the specific sulfur donor and the molecular scaffold, respectively, for the Fe–S cluster formation of nitrogenase in P. polymyxa WLY78. YutI can be an Fe–S cluster carrier involved in nitrogenase maturation in P. polymyxa WLY78.     

Molecular Basis of Multiple Mitochondrial Dysfunctions Syndrome 2 Caused by CYS59TYR BOLA3 Mutation

Multiple mitochondrial dysfunctions syndrome (MMDS) is a rare neurodegenerative disorder associated with mutations in genes with a vital role in the biogenesis of mitochondrial [4Fe–4S] proteins. Mutations in one of these genes encoding for BOLA3 protein lead to MMDS type 2 (MMDS2). Recently, a novel phenotype for MMDS2 with complete clinical recovery was observed in a patient containing a novel variant (c.176G > A, p.Cys59Tyr) in compound heterozygosity. In this work, we aimed to rationalize this unique phenotype observed in MMDS2. To do so, we first investigated the structural impact of the Cys59Tyr mutation on BOLA3 by NMR, and then we analyzed how the mutation affects both the formation of a hetero-complex between BOLA3 and its protein partner GLRX5 and the iron–sulfur cluster-binding properties of the hetero-complex by various spectroscopic techniques and by experimentally driven molecular docking. We show that (1) the mutation structurally perturbed the iron–sulfur cluster-binding region of BOLA3, but without abolishing [2Fe–2S]²⁺ cluster-binding on the hetero-complex; (2) tyrosine 59 did not replace cysteine 59 as iron–sulfur cluster ligand; and (3) the mutation promoted the formation of an aberrant apo C59Y BOLA3–GLRX5 complex. All these aspects allowed us to rationalize the unique phenotype observed in MMDS2 caused by Cys59Tyr mutation.     

Molecular Details of the Frataxin–Scaffold Interaction during Mitochondrial Fe–S Cluster Assembly

Iron–sulfur clusters are essential to almost every life form and utilized for their unique structural and redox-targeted activities within cells during many cellular pathways. Although there are three different Fe–S cluster assembly pathways in prokaryotes (the NIF, SUF and ISC pathways) and two in eukaryotes (CIA and ISC pathways), the iron–sulfur cluster (ISC) pathway serves as the central mechanism for providing 2Fe–2S clusters, directly and indirectly, throughout the entire cell in eukaryotes. Proteins central to the eukaryotic ISC cluster assembly complex include the cysteine desulfurase, a cysteine desulfurase accessory protein, the acyl carrier protein, the scaffold protein and frataxin (in humans, NFS1, ISD11, ACP, ISCU and FXN, respectively). Recent molecular details of this complex (labeled NIAUF from the first letter from each ISC protein outlined earlier), which exists as a dimeric pentamer, have provided real structural insight into how these partner proteins arrange themselves around the cysteine desulfurase, the core dimer of the (NIAUF)₂ complex. In this review, we focus on both frataxin and the scaffold within the human, fly and yeast model systems to provide a better understanding of the biophysical characteristics of each protein alone and within the FXN/ISCU complex as it exists within the larger NIAUF construct. These details support a complex dynamic interaction between the FXN and ISCU proteins when both are part of the NIAUF complex and this provides additional insight into the coordinated mechanism of Fe–S cluster assembly.     

The Arabidopsis Mitochondrial Glutaredoxin GRXS15 Provides [2Fe-2S] Clusters for ISCA-Mediated [4Fe-4S] Cluster Maturation

Iron-sulfur (Fe-S) proteins are crucial for many cellular functions, particularly those involving electron transfer and metabolic reactions. An essential monothiol glutaredoxin GRXS15 plays a key role in the maturation of plant mitochondrial Fe-S proteins. However, its specific molecular function is not clear, and may be different from that of the better characterized yeast and human orthologs, based on known properties. Hence, we report here a detailed characterization of the interactions between Arabidopsis thaliana GRXS15 and ISCA proteins using both in vivo and in vitro approaches. Yeast two-hybrid and bimolecular fluorescence complementation experiments demonstrated that GRXS15 interacts with each of the three plant mitochondrial ISCA1a/1b/2 proteins. UV-visible absorption/CD and resonance Raman spectroscopy demonstrated that coexpression of ISCA1a and ISCA2 resulted in samples with one [2Fe-2S]²⁺ cluster per ISCA1a/2 heterodimer, but cluster reconstitution using as-purified [2Fe-2S]-ISCA1a/2 resulted in a [4Fe-4S]²⁺ cluster-bound ISCA1a/2 heterodimer. Cluster transfer reactions monitored by UV-visible absorption and CD spectroscopy demonstrated that [2Fe-2S]-GRXS15 mediates [2Fe-2S]²⁺ cluster assembly on mitochondrial ferredoxin and [4Fe-4S]²⁺ cluster assembly on the ISCA1a/2 heterodimer in the presence of excess glutathione. This suggests that ISCA1a/2 is an assembler of [4Fe-4S]²⁺ clusters, via two-electron reductive coupling of two [2Fe-2S]²⁺ clusters. Overall, the results provide new insights into the roles of GRXS15 and ISCA1a/2 in effecting [2Fe-2S]²⁺ to [4Fe-4S]²⁺ cluster conversions for the maturation of client [4Fe-4S] cluster-containing proteins in plants.     

Criticality of Surface Characteristics of Intravenous Iron–Carbohydrate Nanoparticle Complexes: Implications for Pharmacokinetics and Pharmacodynamics

Un-complexed polynuclear ferric oxyhydroxide cannot be administered safely or effectively to patients. When polynuclear iron cores are formed with carbohydrates of various structures, stable complexes with surface carbohydrates driven by multiple interacting sites and forces are formed. These complexes deliver iron in a usable form to the body while avoiding the serious adverse effects of un-complexed forms of iron, such as polynuclear ferric oxyhydroxide. The rate and extent of plasma clearance and tissue biodistribution is variable among the commercially available iron–carbohydrate complexes and is driven principally by the surface characteristics of the complexes which dictate macrophage opsonization. The surface chemistry differences between the iron–carbohydrate complexes results in significant differences in in vivo pharmacokinetic and pharmacodynamic profiles as well as adverse event profiles, demonstrating that the entire iron–carbohydrate complex furnishes the pharmacologic action for these complex products. Currently available physicochemical characterization methods have limitations in biorelevant matrices resulting in challenges in defining critical quality attributes for surface characteristics for this class of complex nanomedicines.     

Electron Paramagnetic Resonance and Magnetic Circular Dichroism Spectra of the Nitrogenase M Cluster Precursor Suggest Sulfur Migration upon Oxidation: A Proposal for Substrate and Inhibitor Binding

The active site of the nitrogen-fixing enzyme Mo-nitrogenase is the M cluster ([MoFe₇S₉C ⋅ R-homocitrate]), also known as the FeMo cofactor or FeMoco. The biosynthesis of this highly complex metallocluster involves a series of proteins. Among them, NifB, a radical-SAM enzyme, is instrumental in the assembly of the L cluster ([Fe₈S₉C]), a precursor and all-iron core of the M cluster. In the absence of sulfite, NifB assembles a precursor form of the L cluster called the L* cluster ([Fe₈S₈C]), which lacks the final ninth sulfur. EPR and MCD spectroscopies are used to probe the electronic structures of the paramagnetic, oxidized forms of both the L and L* clusters, labeled L^Ox and [ L* ] ^Ox . This study shows that both L^Ox and [ L* ] ^Ox have nearly identical EPR and MCD spectra, thus suggesting that the two clusters have identical structures upon oxidation; in other words, a sulfur migrates away from L^Ox following oxidation, thereby rendering the cluster identical to [ L* ] ^Ox . It is proposed that a similar migration could occur to the M cluster upon oxidation, and that this is an instrumental part of both M cluster formation and nitrogenase substrate/inhibitor binding.     

Interferon Beta Activity Is Modulated via Binding of Specific S100 Proteins

Interferon-β (IFN-β) is a pleiotropic cytokine used for therapy of multiple sclerosis, which is also effective in suppression of viral and bacterial infections and cancer. Recently, we reported a highly specific interaction between IFN-β and S100P lowering IFN-β cytotoxicity to cancer cells (Int J Biol Macromol. 2020; 143: 633–639). S100P is a member of large family of multifunctional Ca²⁺-binding proteins with cytokine-like activities. To probe selectivity of IFN-β—S100 interaction with respect to S100 proteins, we used surface plasmon resonance spectroscopy, chemical crosslinking, and crystal violet assay. Among the thirteen S100 proteins studied S100A1, S100A4, and S100A6 proteins exhibit strictly Ca²⁺-dependent binding to IFN-β with equilibrium dissociation constants, K_d, of 0.04–1.5 µM for their Ca²⁺-bound homodimeric forms. Calcium depletion abolishes the S100—IFN-β interactions. Monomerization of S100A1/A4/A6 decreases K_d values down to 0.11–1.0 nM. Interferon-α is unable of binding to the S100 proteins studied. S100A1/A4 proteins inhibit IFN-β-induced suppression of MCF-7 cells viability. The revealed direct influence of specific S100 proteins on IFN-β activity uncovers a novel regulatory role of particular S100 proteins, and opens up novel approaches to enhancement of therapeutic efficacy of IFN-β.     

The Se–S Bond Formation in the Covalent Inhibition Mechanism of SARS-CoV-2 Main Protease by Ebselen-like Inhibitors: A Computational Study

The inhibition mechanism of the main protease (M^pro) of SARS-CoV-2 by ebselen (EBS) and its analog with a hydroxyl group at position 2 of the benzisoselenazol-3(2H)-one ring (EBS-OH) was studied by using a density functional level of theory. Preliminary molecular dynamics simulations on the apo form of M^pro were performed taking into account both the hydrogen donor and acceptor natures of the Nδ and Nε of His41, a member of the catalytic dyad. The potential energy surfaces for the formation of the Se–S covalent bond mediated by EBS and EBS-OH on M^pro are discussed in detail. The EBS-OH shows a distinctive behavior with respect to EBS in the formation of the noncovalent complex. Due to the presence of canonical H-bonds and noncanonical ones involving less electronegative atoms, such as sulfur and selenium, the influence on the energy barriers and reaction energy of the Minnesota hybrid meta-GGA functionals M06, M06-2X and M08HX, and the more recent range-separated hybrid functional wB97X were also considered. The knowledge of the inhibition mechanism of M^pro by the small protease inhibitors EBS or EBS-OH can enlarge the possibilities for designing more potent and selective inhibitor-based drugs to be used in combination with other antiviral therapies.     

Solvent-Induced Formation of Novel Ni(II) Complexes Derived from Bis-Thiosemicarbazone Ligand: An Insight from Experimental and Theoretical Investigations

In this work, we report solvent-induced complexation properties of a new N₂S₂ tetradentate bis-thiosemicarbazone ligand (H₂L^I), prepared by the condensation of 4-phenylthiosemicarbazide with bis-aldehyde, namely 2,2’-(ethane-1,2-diylbis(oxy)dibenzaldehyde, towards nickel(II). Using ethanol as a reaction medium allowed the isolation of a discrete mononuclear homoleptic complex [NiL^I] (1), for which its crystal structure contains three independent molecules, namely 1-I, 1-II, and 1-III, in the asymmetric unit. The doubly deprotonated ligand L^I in the structure of 1 is coordinated in a cis-manner through the azomethine nitrogen atoms and the thiocarbonyl sulfur atoms. The coordination geometry around metal centers in all the three crystallographically independent molecules of 1 is best described as the seesaw structure. Interestingly, using methanol as a reaction medium in the same synthesis allowed for the isolation of a discrete mononuclear homoleptic complex [Ni(L^II)₂] (2), where L^II is a monodeprotonated ligand 2-(2-(2-(2-(dimethoxymethyl)phenoxy)ethoxy)benzylidene)-N-phenylhydrazine-1-carbothioamide (HL^II). The ligand L^II was formed in situ from the reaction of L^I with methanol upon coordination to the metal center under synthetic conditions. In the structure of 2, two ligands L^II are coordinated in a trans-manner through the azomethine nitrogen atom and the thiocarbonyl sulfur atom, also yielding a seesaw coordination geometry around the metal center. The charge and energy decomposition scheme ETS-NOCV allows for the conclusion that both structures are stabilized by a bunch of London dispersion-driven intermolecular interactions, including predominantly N–H∙∙∙S and N–H∙∙∙O hydrogen bonds in 1 and 2, respectively; they are further augmented by less typical C–H∙∙∙X (where X = S, N, O, π), CH∙∙∙HC, π∙∙∙π stacking and the most striking, attractive long-range intermolecular C–H∙∙∙Ni preagostic interactions. The latter are found to be determined by both stabilizing Coulomb forces and an exchange-correlation contribution as revealed by the IQA energy decomposition scheme. Interestingly, the analogous long-range C–H∙∙∙S interactions are characterized by a repulsive Coulomb contribution and the prevailing attractive exchange-correlation constituent. The electron density of the delocalized bonds (EDDB) method shows that the nickel(II) atom shares only ~0.8|e| due to the σ-conjugation with the adjacent in-plane atoms, demonstrating a very weak σ-metalloaromatic character.     

Proteomic Insights into Sulfur Metabolism in the Hydrogen-Producing Hyperthermophilic Archaeon Thermococcus onnurineus NA1

The hyperthermophilic archaeon Thermococcus onnurineus NA1 has been shown to produce H₂ when using CO, formate, or starch as a growth substrate. This strain can also utilize elemental sulfur as a terminal electron acceptor for heterotrophic growth. To gain insight into sulfur metabolism, the proteome of T. onnurineus NA1 cells grown under sulfur culture conditions was quantified and compared with those grown under H₂-evolving substrate culture conditions. Using label-free nano-UPLC-MS^E-based comparative proteomic analysis, approximately 38.4% of the total identified proteome (589 proteins) was found to be significantly up-regulated (≥1.5-fold) under sulfur culture conditions. Many of these proteins were functionally associated with carbon fixation, Fe–S cluster biogenesis, ATP synthesis, sulfur reduction, protein glycosylation, protein translocation, and formate oxidation. Based on the abundances of the identified proteins in this and other genomic studies, the pathways associated with reductive sulfur metabolism, H₂-metabolism, and oxidative stress defense were proposed. The results also revealed markedly lower expression levels of enzymes involved in the sulfur assimilation pathway, as well as cysteine desulfurase, under sulfur culture condition. The present results provide the first global atlas of proteome changes triggered by sulfur, and may facilitate an understanding of how hyperthermophilic archaea adapt to sulfur-rich, extreme environments.     

Switching Ion Binding Selectivity of Thiacalix[4]arene Monocrowns at Liquid–Liquid and 2D-Confined Interfaces

Understanding the interaction of ions with organic receptors in confined space is of fundamental importance and could advance nanoelectronics and sensor design. In this work, metal ion complexation of conformationally varied thiacalix[4]monocrowns bearing lower-rim hydroxy (type I), dodecyloxy (type II), or methoxy (type III) fragments was evaluated. At the liquid–liquid interface, alkylated thiacalixcrowns-5(6) selectively extract alkali metal ions according to the induced-fit concept, whereas crown-4 receptors were ineffective due to distortion of the crown-ether cavity, as predicted by quantum-chemical calculations. In type-I ligands, alkali-metal ion extraction by the solvent-accessible crown-ether cavity was prevented, which resulted in competitive Ag⁺ extraction by sulfide bridges. Surprisingly, amphiphilic type-I/II conjugates moderately extracted other metal ions, which was attributed to calixarene aggregation in salt aqueous phase and supported by dynamic light scattering measurements. Cation–monolayer interactions at the air–water interface were monitored by surface pressure/potential measurements and UV/visible reflection–absorption spectroscopy. Topology-varied selectivity was evidenced, towards Sr²⁺ (crown-4), K⁺ (crown-5), and Ag⁺ (crown-6) in type-I receptors and Na⁺ (crown-4), Ca²⁺ (crown-5), and Cs⁺ (crown-6) in type-II receptors. Nuclear magnetic resonance and electronic absorption spectroscopy revealed exocyclic coordination in type-I ligands and cation–π interactions in type-II ligands.     

Interferon-β Activity Is Affected by S100B Protein

Interferon-β (IFN-β) is a pleiotropic cytokine secreted in response to various pathological conditions and is clinically used for therapy of multiple sclerosis. Its application for treatment of cancer, infections and pulmonary diseases is limited by incomplete understanding of regulatory mechanisms of its functioning. Recently, we reported that IFN-β activity is affected by interactions with S100A1, S100A4, S100A6, and S100P proteins, which are members of the S100 protein family of multifunctional Ca²⁺-binding proteins possessing cytokine-like activities (Int J Mol Sci. 2020;21(24):9473). Here we show that IFN-β interacts with one more representative of the S100 protein family, the S100B protein, involved in numerous oncological and neurological diseases. The use of chemical crosslinking, intrinsic fluorescence, and surface plasmon resonance spectroscopy revealed IFN-β binding to Ca²⁺-loaded dimeric and monomeric forms of the S100B protein. Calcium depletion blocks the S100B–IFN-β interaction. S100B monomerization increases its affinity to IFN-β by 2.7 orders of magnitude (equilibrium dissociation constant of the complex reaches 47 pM). Crystal violet assay demonstrated that combined application of IFN-β and S100B (5–25 nM) eliminates their inhibitory effects on MCF-7 cell viability. Bioinformatics analysis showed that the direct modulation of IFN-β activity by the S100B protein described here could be relevant to progression of multiple oncological and neurological diseases.     

Identification and Determination of 1,3-Thiazinane-4-carboxylic Acid in Human Urine—Chromatographic Studies

It is well established that homocysteine (Hcy) and its thiolactone (HTL) are reactive towards aldehydes in an aqueous environment, forming substituted thiazinane carboxylic acids. This report provides evidence that Hcy/HTL and formaldehyde (FA) adduct, namely 1,3-thiazinane-4-carboxylic acid (TCA) is formed in vivo in humans. In order to provide definitive proof, a gas chromatography–mass spectrometry (GC–MS) based method was elaborated to identify and quantify TCA in human urine. The GC–MS assay involves chemical derivatization with isobutyl chloroformate (IBCF) in the presence of pyridine as a catalyst, followed by an ethyl acetate extraction of the obtained isobutyl derivative of TCA (TCA-IBCF). The validity of the method has been demonstrated based upon United States Food and Drug Administration recommendations. The assay linearity was observed within a 1–50 µmol L⁻¹ range for TCA in urine, while the lowest concentration on the calibration curve was recognized as the limit of quantification (LOQ). Importantly, the method was successfully applied to urine samples delivered by apparently healthy volunteers (n = 15). The GC–MS assay may provide a new analytical tool for routine clinical analysis of the role of TCA in living systems in the near future.     

Golden and Silver–Golden Chitosan Hydrogels and Fabrics Modified with Golden Chitosan Hydrogels

Golden and silver–golden chitosan hydrogels and hydrogel-modified textiles of potential biomedical applications are investigated in this work. The hydrogels are formed by reactions of chitosan with HAuCl₄·xH₂O. For above the critical concentration of chitosan (c*), chitosan–Au hydrogels were prepared. For chitosan concentrations lower than c*, chitosan–Au nano- and microgels were formed. To characterise chitosan–Au structures, sol–gel analysis, UV–Vis spectrophotometry and dynamic light scattering were performed. Au concentration in the hydrogels was determined by the flame atomic absorption spectrophotometry. Colloidal chitosan–Au solutions were used for the modification of fabrics. The Au content in the modified fabrics was quantified by inductively coupled plasma mass spectrometry technique. Scanning electron microscopy with energy dispersion X-ray spectrometer was used to analyse the samples. Reflectance spectrophotometry was applied to examine the colour of the fabrics. The formation of chitosan–Au–Ag hydrogels by the competitive reaction of Au and Ag ions with the chitosan macromolecules is reported.     

Nano–Liposomes Double Loaded with Curcumin and Tetrandrine: Preparation,Characterization, Hepatotoxicity and Anti–Tumor Effects

(1) Background: Curcumin (CUR) and tetrandrine (TET) are natural compounds with various bioactivities, but have problems with low solubility, stability, and absorption rate, resulting in low bioavailability, and limited applications in food, medicine, and other fields. It is very important to improve the solubility while maintaining the high activity of drugs. Liposomes are micro–vesicles synthesized from cholesterol and lecithin. With high biocompatibility and biodegradability, liposomes can significantly improve drug solubility, efficacy, and bioavailability. (2) Methods: In this work, CUR and TET were encapsulated with nano–liposomes and g DSPE–MPEG 2000 (DP)was added as a stabilizer to achieve better physicochemical properties, biosafety, and anti–tumor effects. (3) Results: The nano–liposome (CT–DP–Lip) showed stable particle size (under 100 nm) under different conditions, high solubility, drug encapsulation efficiency (EE), loading capacity (LC), release rate in vitro, and stability. In addition, in vivo studies demonstrated CT–DP–Lip had no significant toxicity on zebrafish. Tumor cytotoxicity test showed that CT–DP–Lip had a strong inhibitory effect on a variety of cancer cells. (4) Conclusions: This work showed that nano–liposomes can significantly improve the physical and chemical properties of CUR and TET and make them safer and more efficient.     

Thiosulfate-Cyanide Sulfurtransferase a Mitochondrial Essential Enzyme: From Cell Metabolism to the Biotechnological Applications

Thiosulfate: cyanide sulfurtransferase (TST), also named rhodanese, is an enzyme widely distributed in both prokaryotes and eukaryotes, where it plays a relevant role in mitochondrial function. TST enzyme is involved in several biochemical processes such as: cyanide detoxification, the transport of sulfur and selenium in biologically available forms, the restoration of iron–sulfur clusters, redox system maintenance and the mitochondrial import of 5S rRNA. Recently, the relevance of TST in metabolic diseases, such as diabetes, has been highlighted, opening the way for research on important aspects of sulfur metabolism in diabetes. This review underlines the structural and functional characteristics of TST, describing the physiological role and biomedical and biotechnological applications of this essential enzyme.