The regulatory protein NsrR, a member of the Rrf2 family transcription repressors, is specifically dedicated to sensing nitric oxide (NO) in variety pathogenic and non-pathogenic bacteria. It has been proposed that NO directly modulates NsrR activity by interacting with predicted [Fe-S] cluster protein, but no experimental evidence published support this hypothesis. Here we report purification from obligate aerobe Streptomyces coelicolor. We demonstrate using UV-visible, near UV CD EPR...

The reactivity of protein bound iron−sulfur clusters with nitric oxide (NO) is well documented, but little known about the actual mechanism cluster nitrosylation. Here, we report studies members Wbl family [4Fe−4S] containing proteins, which play key roles in regulating developmental processes actinomycetes, including Streptomyces and Mycobacteria, have been shown to be NO responsive. coelicolor WhiD Mycobacterium tuberculosis WhiB1 react extremely rapidly a multiphasic reaction involving,...

Mycobacterium tuberculosis is a major pathogen that has the ability to establish, and emerge from, persistent state. Wbl family proteins are associated with developmental processes in actinomycetes, M. seven such proteins. In present study it shown H37Rv whiB1 gene essential. The WhiB1 protein possesses [4Fe-4S]2+ cluster stable air but reacts rapidly eight equivalents of nitric oxide yield two dinuclear dinitrosyl-iron thiol complexes. [4Fe-4S] form did not bind promoter DNA, reduced...

Fumarate and nitrate reduction (FNR) regulatory proteins are O 2 -sensing bacterial transcription factors that control the switch between aerobic anaerobic metabolism. Under conditions [4Fe-4S] 2+ -FNR exists as a DNA-binding homodimer. In response to elevated oxygen levels, cluster undergoes rapid conversion [2Fe-2S] cluster, resulting in dimer-to-monomer transition loss of site-specific DNA binding. this work, resonance Raman UV-visible absorption/CD spectroscopies MS were used...

The facultative anaerobe <i>Escherichia coli</i> adopts different metabolic modes in response to the availability of oxygen. global transcriptional regulator FNR (fumarate-nitrate reduction) monitors oxygen environment. Binding as a homodimer palindromic sequences DNA, carries sensory domain, remote from DNA binding helix-turn-helix motif, which responds sensing mechanism involves transformation [4Fe-4S]²⁺ cluster into [2Fe-2S] form <i>in vitro</i> on reaction with Evidence is...

In Escherichia coli, the switch between aerobic and anaerobic metabolism is controlled primarily by FNR (regulator of fumarate nitrate reduction), protein that regulates transcription >100 genes in response to oxygen. Under oxygen-limiting conditions, binds a [4Fe-4S]2+ cluster, generating transcriptionally active dimeric form. Upon exposure oxygen cluster converts [2Fe-2S]2+ form, leading dissociation into monomers, which are incapable binding DNA with high affinity. The mechanism...

Abstract NsrR from Streptomyces coelicolor ( Sc) regulates the expression of three genes through progressive degradation its [4Fe–4S] cluster on nitric oxide (NO) exposure. We report 1.95 Å resolution crystal structure dimeric holo-ScNsrR and show that is coordinated by invariant Cys residues one monomer and, unexpectedly, Asp8 other. A cavity map suggests NO displaces as a ligand while D8A D8C variants remain sensitive, DNA binding affected. structural comparison with an apo-IscR-DNA...

Abstract 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 photocaged tyrosine (pcY) for p -benzoyl-ʟ-phenylalanine (Bpa) We identified a position installing Bpa 7D12 has minimal effect on 7D12–EGFR binding affinity the absence light....

Fumarate and nitrate reduction regulatory (FNR) proteins are bacterial transcription factors that coordinate the switch between aerobic anaerobic metabolism. In absence of O(2), FNR binds a [4Fe-4S](2+) cluster (ligated by Cys-20, 23, 29, 122) promoting formation transcriptionally active dimer. presence is converted into monomeric, non-DNA-binding form containing [2Fe-2S](2+) cluster. The reaction with O(2) has been shown to proceed via 2-step process, an O(2)-dependent 1-electron oxidation...

WhiD, a member of the WhiB-like (Wbl) family iron−sulfur proteins found exclusively within actinomycetes, is required for late stages sporulation in Streptomyces coelicolor. Like all other Wbl proteins, WhiD has not so far been purified soluble form that contains significant amount cluster, and characterization relied on cluster-reconstituted protein. Thus, major goal research to obtain characterize native protein containing clusters. Here we report analysis S. coelicolor anaerobically from...

The Fumarate nitrate reduction (FNR) regulator from Escherichia coli controls expression of >300 genes in response to O2 through reaction with its [4Fe-4S] cluster cofactor. FNR is the master switch for transition between anaerobic and aerobic respiration. In physiological concentrations nitric oxide (NO), also regulates genes, including reductase (nar) operon, a major source endogenous cellular NO, hmp, which encodes an NO-detoxifying enzyme. Here we show that reacts rapidly multiphasic...

Significance The transcriptional regulator Fumarate and Nitrate Reduction is the master switch for transition between anaerobic aerobic respiration in many other bacteria. It fulfills this role by controlling expression of &gt;300 genes response to O 2 . senses through a [4Fe-4S] cluster cofactor, which undergoes conversion [2Fe-2S] on reaction with , leading loss DNA binding. By using time-resolved electrospray ionization mass spectrometry, we gained insight into detection intermediates...

Nar1 is an essential eukaryotic protein proposed to function as iron-sulfur (Fe/S) cluster trafficking factor in the cytosolic assembly (CIA) machinery. However, such a role has remained unclear due difficulties purifying adequate amounts of cofactor-bound protein. The [FeFe]-hydrogenase-like two conserved binding sites for [4Fe-4S] clusters, one which predicted be labile site transfer downstream targets. Here, we report new preparation procedure that facilitated studies by UV-Vis, EPR, and...

Mycobacterium tuberculosis causes pulmonary (TB) and claims ~1.8 million human lives per annum. Host nitric oxide (NO) is important in controlling TB infection. M. WhiB1 a NO-responsive Wbl protein (actinobacterial iron-sulfur proteins first identified the 1970s). Until now, structure of has not been available. Here NMR structural model reveals that are four-helix bundles with core three α-helices held together by [4Fe-4S] cluster. The cluster required for formation complex major sigma...

Abstract Members of the Rrf2 superfamily transcription factors are widespread in bacteria but their functions largely unexplored. The few that have been characterized detail sense nitric oxide (NsrR), iron limitation (RirA), cysteine availability (CymR) and sulfur (Fe-S) cluster status cell (IscR). In this study we combined ChIP- dRNA-seq with vitro biochemistry to characterize a putative NsrR homologue Streptomyces venezuelae . ChIP-seq analysis revealed rather than regulating nitrosative...

RirA is a global regulator of iron homeostasis in Rhizobium and related α-proteobacteria. In its [4Fe-4S] cluster-bound form it represses uptake by binding to IRO Box sequences upstream RirA-regulated genes. Under low and/or aerobic conditions, undergoes cluster conversion/degradation apo-RirA, which can no longer bind sequences. Here, we apply time-resolved mass spectrometry electron paramagnetic resonance spectroscopy determine how the senses O2. The data indicate that key iron-sensing...

Previously characterized nitrite reductases fall into three classes: siroheme-containing enzymes (NirBD), cytochrome c hemoproteins (NrfA and NirS), copper-containing (NirK). We show here that the di-iron protein YtfE represents a physiologically relevant new class of reductases. Several functions have been previously proposed for YtfE, including donating iron repair iron–sulfur clusters damaged by nitrosative stress, releasing nitric oxide (NO) from nitrosylated iron, reducing NO to nitrous...

The Escherichia coli FNR protein regulates the transcription of >100 genes in response to environmental O2, thereby coordinating anoxia. Under O2-limiting conditions, binds a [4Fe-4S]2+ cluster through four cysteine residues (Cys20, Cys23, Cys29, Cys122). acquisition converts into transcriptionally active dimeric form. Upon exposure [2Fe-2S]2+ form, generating monomers that no longer bind DNA with high affinity. mechanism conversion reaction and nature released iron sulfur are considerable...

In Escherichia coli, the switch between aerobic and anaerobic metabolism is primarily controlled by fumarate nitrate reduction transcriptional regulator FNR. absence of O2, FNR binds a [4Fe-4S]2+ cluster, generating transcriptionally active dimeric form. Exposure to O2 results in conversion cluster [2Fe-2S]2+ form, leading dissociation protein into inactive monomers. The proceeds two steps. Step 1 involves one-electron oxidation resulting release Fe2+, [3Fe-4S]1+ intermediate, superoxide...