From cultivation to cancer: formation of N-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 N-nitrosamines (TSNAs), such as N'-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. A key aim of this review is to provide a greater understanding of TSNAs: their precursors, the microbial and chemical mechanisms that contribute to their formation in ST, their mutagenicity leading to cancer due to ST use, and potential means of lowering TSNA levels in tobacco products. TSNAs are not present in harvested tobacco but can form due to nitrosating agents reacting with tobacco alkaloids present in tobacco during certain types of curing. TSNAs can also form during or following ST production when certain microorganisms perform nitrate metabolism, with dissimilatory nitrate reductases converting nitrate to nitrite that is then released into tobacco and reacts chemically with tobacco alkaloids. When ST usage occurs, TSNAs are absorbed and metabolized to reactive compounds that form DNA adducts leading to mutations in critical target genes, including the RAS oncogenes and the p53 tumor suppressor gene. DNA repair mechanisms remove most adducts induced by carcinogens, thus preventing many but not all mutations. Lastly, because TSNAs and other agents cause cancer, previously documented strategies for lowering their levels in ST products are discussed, including using tobacco with lower nornicotine levels, pasteurization and other means of eliminating microorganisms, omitting fermentation and fire-curing, refrigerating ST products, and including nitrite scavenging chemicals as ST ingredients.

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

RirA is a global iron regulator in diverse Alphaproteobacteria 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. The cluster is coordinated by three conserved cysteine residues and an unknown fourth ligand. Considering the lability of one of the irons and the resulting cluster fragility, we hypothesized that the fourth ligand may not be an amino acid residue. To investigate this, we considered that the introduction of an amino acid residue that could coordinate the cluster might stabilize it. A structural model of RirA, based on the Rrf2 family nitrosative stress response regulator NsrR, highlighted residue 8, an Asn in the RirA sequence, as being appropriately positioned to coordinate the cluster. Substitution of Asn8 with Asp, the equivalent, cluster-coordinating residue of NsrR, or with Cys, resulted in proteins that contained a [4Fe-4S] cluster, with N8D RirA exhibiting spectroscopic properties very similar to NsrR. The variant proteins retained the ability to bind RirA-regulated DNA, and could still act as repressors of RirA-regulated genes in vivo. However, they were significantly more stable than wild-type RirA when exposed to O2 and/or low iron. Importantly, they exhibited reduced capacity to respond to cellular iron levels, even abolished in the case of the N8D version, and thus were no longer iron sensing. This work demonstrates the importance of cluster fragility for the iron-sensing function of RirA, and more broadly, how a single residue substitution can alter cluster coordination and functional properties in the Rrf2 superfamily of regulators.

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. We identified a position for installing Bpa in 7D12 that has minimal effect on 7D12-EGFR binding affinity in the absence of light. Upon exposure to 365-nm light, this Bpa-containing 7D12 mutant forms a covalent bond with EGFR in an antigen-specific manner. We then developed a method for site-specific incorporation of pcY and Bpa at two distinct sites in 7D12. Finally, we demonstrated that in the absence of light, this pcY- and Bpa-containing mutant of 7D12 does not bind to EGFR, but irradiation with 365-nm light activates (1) specific binding and (2) covalent bond formation with EGFR.

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 c-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 (Em, +198 mV), making it difficult to rationalize the thiosulfate oxidizing capability. Here, we describe the reaction of Campylobacter jejuni thiosulfate dehydrogenase (TsdA) with sulfite, an analogue of thiosulfate. The reaction leads to stoichiometric conversion of the active site Cys to cysteinyl sulfonate (Cα-CH2-S-SO3-) such that the protein exists in a form closely resembling a proposed intermediate in the pathway for thiosulfate oxidation that carries a cysteinyl thiosulfate (Cα-CH2-S-SSO3-). The active site heme in the stable sulfonated protein displays an Em approximately 200 mV more positive than the Cys-/His-ligated state. This can explain the thiosulfate oxidizing activity of the enzyme and allows us to propose a catalytic mechanism for thiosulfate oxidation. Substrate-driven release of the Cys heme ligand allows that side chain to provide the site of substrate binding and redox transformation; the neighboring heme then simply provides a site for electron relay to an appropriate partner. This chemistry is distinct from that displayed by the Cys-ligated hemes found in gas-sensing hemoproteins and in enzymes such as the cytochromes P450. Thus, a further class of thiolate-ligated hemes is proposed, as exemplified by the TsdA centers that have evolved to catalyze the controlled redox transformations of inorganic oxo anions of sulfur.

The Di-Iron Protein YtfE Is a Nitric Oxide-Generating Nitrite Reductase Involved in the Management of Nitrosative Stress.

Previously characterized nitrite reductases fall into three classes: siroheme-containing enzymes (NirBD), cytochrome c hemoproteins (NrfA and NirS), and copper-containing enzymes (NirK). We show here that the di-iron protein YtfE represents a physiologically relevant new class of nitrite reductases. Several functions have been previously proposed for YtfE, including donating iron for the repair of iron-sulfur clusters that have been damaged by nitrosative stress, releasing nitric oxide (NO) from nitrosylated iron, and reducing NO to nitrous oxide (N2O). Here, in vivo reporter assays confirmed that Escherichia coli YtfE increased cytoplasmic NO production from nitrite. Spectroscopic and mass spectrometric investigations revealed that the di-iron site of YtfE exists in a mixture of forms, including nitrosylated and nitrite-bound, when isolated from nitrite-supplemented, but not nitrate-supplemented, cultures. Addition of nitrite to di-ferrous YtfE resulted in nitrosylated YtfE and the release of NO. Kinetics of nitrite reduction were dependent on the nature of the reductant; the lowest Km, measured for the di-ferrous form, was ∼90 µM, well within the intracellular nitrite concentration range. The vicinal di-cysteine motif, located in the N-terminal domain of YtfE, was shown to function in the delivery of electrons to the di-iron center. Notably, YtfE exhibited very low NO reductase activity and was only able to act as an iron donor for reconstitution of apo-ferredoxin under conditions that damaged its di-iron center. Thus, YtfE is a high-affinity, low-capacity nitrite reductase that we propose functions to relieve nitrosative stress by acting in combination with the co-regulated NO-consuming enzymes Hmp and Hcp.

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. Mutations in certain components of the human system lead to disease, and so further studies of this system could be important for developing strategies for medical treatments. We have studied two core components of the Isc biogenesis system: IscS, a cysteine desulfurase; and IscU, a scaffold protein on which clusters are built before subsequent transfer onto recipient apo-proteins. Fe2+-binding, sulfur transfer, and formation of a [2Fe-2S] was followed by a range of techniques, including time-resolved mass spectrometry, and intermediate and product species were unambiguously identified through isotopic substitution experiments using 57Fe and 34S. Under cluster synthesis conditions, sulfur adducts and the [2Fe-2S] cluster product readily accumulated on IscU, but iron adducts (other than the cluster itself) were not observed at physiologically relevant Fe2+ concentrations. Our data indicate that either Fe2+ or sulfur transfer can occur first, but that the transfer of sulfane sulfur (S0) to IscU must occur first if Zn2+ is bound to IscU, suggesting that it is the key step that initiates cluster assembly. Following this, [2Fe-2S] cluster formation is a largely concerted reaction once Fe2+ is introduced.

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, O2, 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. Here, we provide practical guidance for the study of iron-sulfur proteins by native MS, illustrated by examples where it has been used to unambiguously determine the type of cluster coordinated to the protein framework. We also describe the use of time-resolved native MS to follow the kinetics of cluster conversion, allowing the elucidation of the precise series of molecular events for all species involved. Finally, we provide advice on a unique approach to a typical thermodynamic titration, uncovering early, quasi-stable, intermediates in the reaction of a cluster with nitric oxide, resulting in cluster nitrosylation.

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. While mass spectrometry is very widely used in biological research, it is normally employed under denaturing conditions where non-covalently attached cofactors are lost. However, mass spectrometry under conditions where the protein retains its native structure and, thus, cofactors, is now itself a flourishing field, and the application of such 'native' mass spectrometry to study metalloproteins is now relatively widespread. Here we describe recent advances in using native MS to study Fe-S cluster proteins. Through its ability to accurately measure mass changes that reflect chemistry occurring at the cluster, this approach has yielded a remarkable richness of information that is not accessible by other, more traditional techniques.

Mechanisms of iron- and O2-sensing by the [4Fe-4S] cluster of the global iron regulator RirA.

RirA is a global regulator of iron homeostasis in Rhizobium 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. Here, we apply time-resolved mass spectrometry and electron paramagnetic resonance spectroscopy to determine how the RirA cluster senses iron and O2. The data indicate that the key iron-sensing step is the O2-independent, reversible dissociation of Fe2+ from [4Fe-4S]2+ to form [3Fe-4S]0. The dissociation constant for this process was determined as Kd = ~3 µM, which is consistent with the sensing of 'free' iron in the cytoplasm. O2-sensing occurs through enhanced cluster degradation under aerobic conditions, via O2-mediated oxidation of the [3Fe-4S]0 intermediate to form [3Fe-4S]1+. This work provides a detailed mechanistic/functional view of an iron-responsive regulator.

Mass Spectrometric Identification of [4Fe-4S](NO)x 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. Although information about the products of cluster nitrosylation is now emerging, detection and identification of intermediates remains a major challenge, due to their transient nature and the difficulty in distinguishing spectroscopically similar iron-NO species. Here we report studies of the NO-sensing Fe-S cluster regulators NsrR and WhiD using non-denaturing mass spectrometry, in which non-covalent interactions between the protein and Fe/S/NO species are preserved. The data provide remarkable insight into the nitrosylation reactions, permitting identification, for the first time, of protein-bound mono-, di- and tetranitrosyl [4Fe-4S] cluster complexes ([4Fe-4S](NO), [4Fe-4S])(NO)2 and [4Fe-4S](NO)4 ) as intermediates along pathways to formation of product Roussin's red ester (RRE) and Roussin's black salt (RBS)-like species. The data allow the nitrosylation mechanisms of NsrR and WhiD to be elucidated and clearly distinguished.

Generation of 34S-substituted protein-bound [4Fe-4S] clusters using 34S-L-cysteine.

The ability to specifically label the sulphide ions of protein-bound iron-sulphur (FeS) clusters with 34S 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 O-acetyl-l-serine sulfhydrylase CysK to generate 34S-substituted l-cysteine and subsequently use it as a substrate for the l-cysteine desulfurase NifS to gradually supply 34S2- for in vitro FeS cluster assembly in an otherwise standard cluster reconstitution protocol.

Mass spectrometric detection of iron nitrosyls, sulfide oxidation and mycothiolation during nitrosylation of the NO sensor [4Fe-4S] NsrR.

The bacterial nitric oxide (NO)-sensing transcriptional regulator NsrR binds a [4Fe-4S] cluster that enables DNA-binding and thus repression of the cell's NO stress response. Upon exposure to NO, the cluster undergoes a complex nitrosylation reaction resulting in a mixture of iron-nitrosyl species, which spectroscopic studies have indicated are similar to well characterized low molecular weight dinitrosyl iron complex (DNIC), Roussin's Red Ester (RRE) and Roussin's Black Salt (RBS). Here we report mass spectrometric studies that enable the unambiguous identification of NsrR-bound RRE-type species, including a persulfide bound form that results from the oxidation of cluster sulfide. In the presence of the low molecular weight thiols glutathione and mycothiol, glutathionylated and mycothiolated forms of NsrR were readily formed.

Redox-Sensing Iron-Sulfur Cluster Regulators.

SIGNIFICANCE: Iron-sulfur cluster proteins carry out multiple functions, including as regulators of gene transcription/translation in response to environmental stimuli. In all known cases, the cluster acts as the sensory module, where the inherent reactivity/fragility of iron-sulfur clusters with small/redox-active molecules is exploited to effect conformational changes that modulate binding to DNA regulatory sequences. This promotes an often substantial reprogramming of the cellular proteome that enables the organism or cell to adapt to, or counteract, its changing circumstances. Recent Advances: Significant progress has been made recently in the structural and mechanistic characterization of iron-sulfur cluster regulators and, in particular, the O2 and NO sensor FNR, the NO sensor NsrR, and WhiB-like proteins of Actinobacteria. These are the main focus of this review. CRITICAL ISSUES: Striking examples of how the local environment controls the cluster sensitivity and reactivity are now emerging, but the basis for this is not yet fully understood for any regulatory family. FUTURE DIRECTIONS: Characterization of iron-sulfur cluster regulators has long been hampered by a lack of high-resolution structural data. Although this still presents a major future challenge, recent advances now provide a firm foundation for detailed understanding of how a signal is transduced to effect gene regulation. This requires the identification of often unstable intermediate species, which are difficult to detect and may be hard to distinguish using traditional techniques. Novel approaches will be required to solve these problems.

Redox-sensing iron-sulfur cluster regulators.

SIGNIFICANCE: Iron-sulfur cluster proteins carry out a wide range of functions, including as regulators of gene transcription/translation in response to environmental stimuli. In all known cases, the cluster acts as the sensory module, where the inherent reactivity/fragility of iron-sulfur clusters towards small/redox active molecules is exploited to effect conformational changes that modulate binding to DNA regulatory sequences. This promotes an often substantial re-programming of the cellular proteome that enables the organism or cell to adapt to, or counteract, its changing circumstances. Recent Advances. Significant progress has been made recently in the structural and mechanistic characterization of iron-sulfur cluster regulators and, in particular, the O2 and NO sensor FNR, the NO sensor NsrR, and WhiB-like proteins of Actinobacteria. These are the main focus of this review. CRITICAL ISSUES: Striking examples of how the local environment controls the cluster sensitivity and reactivity are now emerging, but the basis for this is not yet fully understood for any regulatory family. FUTURE DIRECTIONS: Characterization of iron-sulfur cluster regulators has long been hampered by a lack of high resolution structural data. Though this still presents a major future challenge, recent advances now provide a firm foundation for detailed understanding of how a signal is transduced to effect gene regulation. This requires the identification of often unstable intermediate species, which are difficult to detect and may be hard to distinguish using traditional techniques. Novel approaches will be required to solve these problems.

Cmr is a redox-responsive regulator of DosR that contributes to M. tuberculosis virulence.

Mycobacterium tuberculosis (MTb) is the causative agent of pulmonary tuberculosis (TB). MTb colonizes the human lung, often entering a non-replicating state before progressing to life-threatening active infections. Transcriptional reprogramming is essential for TB pathogenesis. In vitro, Cmr (a member of the CRP/FNR super-family of transcription regulators) bound at a single DNA site to act as a dual regulator of cmr transcription and an activator of the divergent rv1676 gene. Transcriptional profiling and DNA-binding assays suggested that Cmr directly represses dosR expression. The DosR regulon is thought to be involved in establishing latent tuberculosis infections in response to hypoxia and nitric oxide. Accordingly, DNA-binding by Cmr was severely impaired by nitrosation. A cmr mutant was better able to survive a nitrosative stress challenge but was attenuated in a mouse aerosol infection model. The complemented mutant exhibited a ∼2-fold increase in cmr expression, which led to increased sensitivity to nitrosative stress. This, and the inability to restore wild-type behaviour in the infection model, suggests that precise regulation of the cmr locus, which is associated with Region of Difference 150 in hypervirulent Beijing strains of Mtb, is important for TB pathogenesis.

Crystal structures of the NO sensor NsrR reveal how its iron-sulfur cluster modulates DNA binding.

NsrR from Streptomyces coelicolor (Sc) regulates the expression of three genes through the progressive degradation of its [4Fe-4S] cluster on nitric oxide (NO) exposure. We report the 1.95 Å resolution crystal structure of dimeric holo-ScNsrR and show that the cluster is coordinated by the three invariant Cys residues from one monomer and, unexpectedly, Asp8 from the other. A cavity map suggests that NO displaces Asp8 as a cluster ligand and, while D8A and D8C variants remain NO sensitive, DNA binding is affected. A structural comparison of holo-ScNsrR with an apo-IscR-DNA complex shows that the [4Fe-4S] cluster stabilizes a turn between ScNsrR Cys93 and Cys99 properly oriented to interact with the DNA backbone. In addition, an apo ScNsrR structure suggests that Asn97 from this turn, along with Arg12, which forms a salt-bridge with Asp8, are instrumental in modulating the position of the DNA recognition helix region relative to its major groove.

NBP35 interacts with DRE2 in the maturation of cytosolic iron-sulphur proteins in Arabidopsis thaliana.

Proteins of the cytosolic pathway for iron-sulphur (FeS) cluster assembly are conserved, except that plants lack a gene for CFD1 (Cytosolic FeS cluster Deficient 1). This poses the question of how NBP35 (Nucleotide-Binding Protein 35 kDa), the heteromeric partner of CFD1 in metazoa, functions on its own in plants. Firstly, we created viable mutant alleles of NBP35 in Arabidopsis to overcome embryo lethality of previously reported knockout mutations. RNAi knockdown lines with less than 30% NBP35 protein surprisingly showed no developmental or biochemical differences to wild-type. Substitution of Cys14 to Ala, which destabilized the N-terminal Fe4 S4 cluster in vitro, caused mild growth defects and a significant decrease in the activity of cytosolic FeS enzymes such as aconitase and aldehyde oxidases. The DNA glycosylase ROS1 was only partially decreased in activity and xanthine dehydrogenase not at all. Plants with strongly depleted NBP35 protein in combination with Cys14 to Ala substitution had distorted leaf development and decreased FeS enzyme activities. To find protein interaction partners of NBP35, a yeast-two-hybrid screen was carried out that identified NBP35 and DRE2 (Derepressed for Ribosomal protein S14 Expression). NBP35 is known to form a dimer, and DRE2 acts upstream in the cytosolic FeS protein assembly pathway. The NBP35-DRE2 interaction was not disrupted by Cys14 to Ala substitution. Our results show that NBP35 has a function in the maturation of FeS proteins that is conserved in plants, and is closely allied to the function of DRE2.

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. This competition could suggest a link between the two proteins with a functional significance. Using a hybrid approach based on nuclear magnetic resonance, small angle scattering and biochemical methods, we show here that IscX is a modulator of the inhibitory properties of CyaY: by competing for the same site on IscS, the presence of IscX rescues the rates of enzymatic cluster formation which are inhibited by CyaY. The effect is stronger at low iron concentrations, whereas it becomes negligible at high iron concentrations. These results strongly suggest the mechanism of the dual regulation of iron sulfur cluster assembly under the control of iron as the effector.

Sensing iron availability via 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. Under low iron conditions, [4Fe-4S] RirA undergoes a cluster conversion reaction resulting in a [2Fe-2S] form, which exhibits much lower affinity for DNA. Under prolonged low iron conditions, the [2Fe-2S] cluster degrades to apo-RirA, which does not bind DNA and can no longer function as a repressor of the cell's iron-uptake machinery. [4Fe-4S] RirA was also found to be sensitive to O2, suggesting that both iron and O2 are important signals for iron metabolism. Consistent with this, in vivo data showed that expression of RirA-regulated genes is also affected by O2. These data lead us to propose a novel regulatory model for iron homeostasis, in which RirA senses iron via the incorporation of a fragile iron-sulfur cluster that is sensitive to iron and O2 concentrations.

Nitrosylation of Nitric-Oxide-Sensing Regulatory Proteins Containing [4Fe-4S] Clusters Gives Rise to Multiple Iron-Nitrosyl Complexes.

The reaction of protein-bound iron-sulfur (Fe-S) clusters with nitric oxide (NO) plays key roles in NO-mediated toxicity and signaling. Elucidation of the mechanism of the reaction of NO with DNA regulatory proteins that contain Fe-S clusters has been hampered by a lack of information about the nature of the iron-nitrosyl products formed. Herein, we report nuclear resonance vibrational spectroscopy (NRVS) and density functional theory (DFT) calculations that identify NO reaction products in WhiD and NsrR, regulatory proteins that use a [4Fe-4S] cluster to sense NO. This work reveals that nitrosylation yields multiple products structurally related to Roussin's Red Ester (RRE, [Fe2 (NO)4 (Cys)2 ]) and Roussin's Black Salt (RBS, [Fe4 (NO)7 S3 ]. In the latter case, the absence of 32 S/34 S shifts in the Fe-S region of the NRVS spectra suggest that a new species, Roussin's Black Ester (RBE), may be formed, in which one or more of the sulfide ligands is replaced by Cys thiolates.