Binding of a single nitric oxide molecule is sufficient to disrupt DNA binding of the nitrosative stress regulator NsrR.

The regulatory protein NsrR, a member of the Rrf2 protein superfamily, plays a major role in the cellular response to nitrosative stress in many benign and pathogenic bacteria. The homodimeric protein binds a [4Fe-4S] cluster in each subunit (termed holo NsrR), and represses transcription of genes primarily involved in NO detoxification. Holo NsrR reacts rapidly with multiple NO molecules per [4Fe-4S] cluster, a complex reaction, with loss of DNA binding and formation of NsrR-bound iron-nitrosyl species.

Synergy of native mass spectrometry and other biophysical techniques in studies of iron‑sulfur cluster proteins and their assembly.

The application of mass spectrometric methodologies has revolutionised biological chemistry, from identification through to structural and conformational studies of proteins and other macromolecules. Native mass spectrometry (MS), in which proteins retain their native structure, is a rapidly growing field. This is particularly the case for studies of metalloproteins, where non-covalently bound cofactors remain bound following ionisation.

Polyurethane infused with heparin capped silver nanoparticles dressing for wound healing application: Synthesis, characterization and antimicrobial studies.

Burn and diabetic wounds present significant challenges due to their complex nature, delayed healing, pain, and high susceptibility to bacterial infections. In this study, we developed and evaluated polyurethane (PU) nanofibers embedded with heparin-functionalized silver nanoparticles (hep-AgNPs) using an electrospinning technique. The choice to functionalize silver nanoparticles with heparin was based on heparin's established role in modulating inflammation and promoting angiogenesis.

CyaY and TusA regulate ISC- and SUF-mediated l-cysteine desulfurase activity.

CyaY, the frataxin homolog of , plays an important role in ISC iron-sulfur cluster assembly through interactions with the cysteine desulfurase IscS, which regulate the supply of sulfur. IscS is not exclusive for ISC Fe-S cluster assembly, as it functions as a hub for the supply of sulfur to a number of other sulfur-requiring pathways, such as for the biosynthesis of Moco and thiolated tRNAs. How the balance of sulfur supply to the various competing pathways is achieved is not fully understood, but a network of protein-protein interactions plays a key role.

Binding of IscU and TusA to different but competing sites of IscS influences the activity of IscS and directs sulfur to the respective biomolecular synthesis pathway.

All sulfur transfer pathways generally have in common an l-cysteine desulfurase as the initial sulfur-mobilizing enzyme, which serves as a sulfur donor for the biosynthesis of numerous sulfur-containing biomolecules in the cell. In , the housekeeping l-cysteine desulfurase IscS functions as a hub for sulfur transfer through interactions with several partner proteins, which bind at different sites on IscS. So far, the interaction sites of IscU, Fdx, CyaY, and IscX involved in iron sulfur (Fe-S) cluster assembly, TusA, required for molybdenum cofactor biosynthesis and mnmsU34 transfer RNA (tRNA) modifications, and ThiI, involved in both the biosynthesis of thiamine and sU8 tRNA modifications, have been mapped.

From cultivation to cancer: formation of -nitrosamines and other carcinogens in smokeless tobacco and their mutagenic implications.

Tobacco use is a major cause of preventable morbidity and mortality globally. Tobacco products, including smokeless tobacco (ST), generally contain tobacco-specific -nitrosamines (TSNAs), such as '-nitrosonornicotine (NNN) and 4-(methylnitrosamino)-1-(3-pyridyl)-butanone (NNK), which are potent carcinogens that cause mutations in critical genes in human DNA. This review covers the series of biochemical and chemical transformations, related to TSNAs, leading from tobacco cultivation to cancer initiation.

Stabilisation of the RirA [4Fe-4S] cluster results in loss of iron-sensing function.

RirA is a global iron regulator in diverse that belongs to the Rrf2 superfamily of transcriptional regulators, which can contain an iron-sulfur (Fe-S) cluster. Under iron-replete conditions, RirA contains a [4Fe-4S] cluster, enabling high-affinity binding to RirA-regulated operator sequences, thereby causing the repression of cellular iron uptake. Under iron deficiency, one of the cluster irons dissociates, generating an unstable [3Fe-4S] form that subsequently degrades to a [2Fe-2S] form and then to apo RirA, resulting in loss of high-affinity DNA-binding.

Liquid-chromatography mass spectrometry describes post-translational modification of Shewanella outer membrane proteins.

Electrogenic bacteria deliver excess respiratory electrons to externally located metal oxide particles and electrodes. The biochemical basis for this process is arguably best understood for species of Shewanella where the integral membrane complex termed MtrCAB is key to electron transfer across the bacterial outer membranes. A crystal structure was recently resolved for MtrCAB from S.

Site-specific encoding of photoactivity and photoreactivity into antibody fragments.

Design of biomolecules that perform two or more distinct functions in response to light remains challenging. Here, we have introduced concurrent photoactivity and photoreactivity into an epidermal growth factor receptor (EGFR)-targeting antibody fragment, 7D12. This was achieved by site-specific incorporation of photocaged tyrosine (pcY) for photoactivity and p-benzoyl-ʟ-phenylalanine (Bpa) for photoreactivity into 7D12.

Native mass spectrometric studies of IscSU reveal a concerted, sulfur-initiated mechanism of iron-sulfur cluster assembly.

Iron-sulfur (Fe-S) clusters are cofactors essential for life. Though the proteins that function in the assembly of Fe-S clusters are well known, details of the molecular mechanism are less well established. The Isc (iron-sulfur cluster) biogenesis apparatus is widespread in bacteria and is the closest homologue to the human system.

Reaction of Thiosulfate Dehydrogenase with a Substrate Mimic Induces Dissociation of the Cysteine Heme Ligand Giving Insights into the Mechanism of Oxidative Catalysis.

Thiosulfate dehydrogenases are bacterial cytochromes that contribute to the oxidation of inorganic sulfur. The active sites of these enzymes contain low-spin -type heme with Cys/His axial ligation. However, the reduction potentials of these hemes are several hundred mV more negative than that of the thiosulfate/tetrathionate couple (, +198 mV), making it difficult to rationalize the thiosulfate oxidizing capability.

Structural determinants of DNA recognition by the NO sensor NsrR and related Rrf2-type [FeS]-transcription factors.

Several transcription factors of the Rrf2 family use an iron-sulfur cluster to regulate DNA binding through effectors such as nitric oxide (NO), cellular redox status and iron levels. [4Fe-4S]-NsrR from Streptomyces coelicolor (ScNsrR) modulates expression of three different genes via reaction and complex formation with variable amounts of NO, which results in detoxification of this gas. Here, we report the crystal structure of ScNsrR complexed with an hmpA1 gene operator fragment and compare it with those previously reported for [2Fe-2S]-RsrR/rsrR and apo-IscR/hyA complexes.

Mechanistic insights into the key marine dimethylsulfoniopropionate synthesis enzyme DsyB/DSYB.

Marine algae and bacteria produce approximately eight billion tonnes of the organosulfur molecule dimethylsulfoniopropionate (DMSP) in Earth's surface oceans annually. DMSP is an antistress compound and, once released into the environment, a major nutrient, signaling molecule, and source of climate-active gases. The methionine transamination pathway for DMSP synthesis is used by most known DMSP-producing algae and bacteria.

Native Mass Spectrometry of Iron-Sulfur Proteins.

Iron-sulfur clusters constitute a large and widely distributed group of protein cofactors that play key roles in a wide range of metabolic processes. The inherent reactivity of iron-sulfur clusters toward small molecules, for example, O, NO, or free Fe, makes them ideal for sensing changes in the cellular environment. Nondenaturing, or native, MS is unique in its ability to preserve the noncovalent interactions of many (if not all) species, including stable intermediates, while providing accurate mass measurements in both thermodynamic and kinetic experimental regimes.

Sensing mechanisms of iron-sulfur cluster regulatory proteins elucidated using native mass spectrometry.

The ability to sense and respond to various key environmental cues is important for the survival and adaptability of many bacteria, including pathogens. The particular sensitivity of iron-sulfur (Fe-S) clusters is exploited in nature, such that multiple sensor-regulator proteins, which coordinate the detection of analytes with a (in many cases) global transcriptional response, are Fe-S cluster proteins. The fragility and sensitivity of these Fe-S clusters make studying such proteins difficult, and gaining insight of what they sense, and how they sense it and transduce the signal to affect transcription, is a major challenge.

Interaction of the Wbl protein WhiD with the principal sigma factor σ depends on the WhiD [4Fe-4S] cluster.

The bacterial protein WhiD belongs to the Wbl family of iron-sulfur [Fe-S] proteins present only in the actinomycetes. In , it is required for the late stages of sporulation, but precisely how it functions is unknown. Here, we report results from and experiments with WhiD from (WhiD), which differs from WhiD (WhiD) only at the C terminus.

Electron and Proton Transfers Modulate DNA Binding by the Transcription Regulator RsrR.

The [FeS]-RsrR gene transcription regulator senses the redox status in bacteria by modulating DNA binding, while its cluster cycles between +1 and +2 states-only the latter binds DNA. We have previously shown that RsrR can undergo remarkable conformational changes involving a 100° rotation of tryptophan 9 between exposed () and buried () states. Here, we have used the chemical modification of Trp9, site-directed mutagenesis, and crystallographic and computational chemical studies to show that (i) the and states correspond to oxidized and reduced RsrR, respectively, (ii) His33 is protonated in the state due to a change in its p caused by cluster reduction, and (iii) Trp9 rotation is conditioned by the response of its dipole moment to environmental electrostatic changes.

Mechanisms of iron- and O-sensing by the [4Fe-4S] cluster of the global iron regulator RirA.

RirA is a global regulator of iron homeostasis in and related α-proteobacteria. In its [4Fe-4S] cluster-bound form it represses iron uptake by binding to IRO Box sequences upstream of RirA-regulated genes. Under low iron and/or aerobic conditions, [4Fe-4S] RirA undergoes cluster conversion/degradation to apo-RirA, which can no longer bind IRO Box sequences.

Generation of S-substituted protein-bound [4Fe-4S] clusters using S-L-cysteine.

The ability to specifically label the sulphide ions of protein-bound iron-sulphur (FeS) clusters with S isotope greatly facilitates structure-function studies. In particular, it provides insight when using either spectroscopic techniques that probe cluster-associated vibrations, or non-denaturing mass spectrometry, where the ∼+2 Da average increase per sulphide enables unambiguous assignment of the FeS cluster and, where relevant, its conversion/degradation products. Here, we employ a thermostable homologue of the -acetyl-l-serine sulfhydrylase CysK to generate S-substituted l-cysteine and subsequently use it as a substrate for the l-cysteine desulfurase NifS to gradually supply S for FeS cluster assembly in an otherwise standard cluster reconstitution protocol.

Crystal Structure of the Transcription Regulator RsrR Reveals a [2Fe-2S] Cluster Coordinated by Cys, Glu, and His Residues.

The recently discovered Rrf2 family transcriptional regulator RsrR coordinates a [2Fe-2S] cluster. Remarkably, binding of the protein to RsrR-regulated promoter DNA sequences is switched on and off through the facile cycling of the [2Fe-2S] cluster between +2 and +1 states. Here, we report high resolution crystal structures of the RsrR dimer, revealing that the [2Fe-2S] cluster is asymmetrically coordinated across the RsrR monomer-monomer interface by two Cys residues from one subunit and His and Glu residues from the other.

Mass Spectrometric Identification of [4Fe-4S](NO) Intermediates of Nitric Oxide Sensing by Regulatory Iron-Sulfur Cluster Proteins.

Nitric oxide (NO) can function as both a cytotoxin and a signalling molecule. In both cases, reaction with iron-sulfur (Fe-S) cluster proteins plays an important role because Fe-S clusters are reactive towards NO and so are a primary site of general NO-induced damage (toxicity). This sensitivity to nitrosylation is harnessed in the growing group of regulatory proteins that function in sensing of NO via an Fe-S cluster.

Sensing iron availability the fragile [4Fe-4S] cluster of the bacterial transcriptional repressor RirA.

Rhizobial iron regulator A (RirA) is a global regulator of iron homeostasis in many nitrogen-fixing Rhizobia and related species of α-proteobacteria. It belongs to the widespread Rrf2 super-family of transcriptional regulators and features three conserved Cys residues that characterise the binding of an iron-sulfur cluster in other Rrf2 family regulators. Here we report biophysical studies demonstrating that RirA contains a [4Fe-4S] cluster, and that this form of the protein binds RirA-regulated DNA, consistent with its function as a repressor of expression of many genes involved in iron uptake.

The Molecular Bases of the Dual Regulation of Bacterial Iron Sulfur Cluster Biogenesis by CyaY and IscX.

IscX (or YfhJ) is a protein of unknown function which takes part in the iron-sulfur cluster assembly machinery, a highly specialized and essential metabolic pathway. IscX binds to iron with low affinity and interacts with IscS, the desulfurase central to cluster assembly. Previous studies have suggested a competition between IscX and CyaY, the bacterial ortholog of frataxin, for the same binding surface of IscS.