The Ferroxidase Centre of Escherichia coli Bacterioferritin Plays a Key Role in the Reductive Mobilisation of the Mineral Iron Core.

Ferritins are multimeric cage-forming proteins that play a crucial role in cellular iron homeostasis. All H-chain-type ferritins harbour a diiron site, the ferroxidase centre, at the centre of a 4 α-helical bundle, but bacterioferritins are unique in also binding 12 hemes per 24 meric assembly. The ferroxidase centre is known to be required for the rapid oxidation of Fe2+ during deposition of an immobilised ferric mineral core within the protein's hollow interior. In contrast, the heme of bacterioferritin is required for the efficient reduction of the mineral core during iron release, but has little effect on the rate of either oxidation or mineralisation of iron. Thus, the current view is that these two cofactors function in iron uptake and release, respectively, with no functional overlap. However, rapid electron transfer between the heme and ferroxidase centre of bacterioferritin from Escherichia coli was recently demonstrated, suggesting that the two cofactors may be functionally connected. Here we report absorbance and (magnetic) circular dichroism spectroscopies, together with in vitro assays of iron-release kinetics, which demonstrate that the ferroxidase centre plays an important role in the reductive mobilisation of the bacterioferritin mineral core, which is dependent on the heme-ferroxidase centre electron transfer pathway.

Probing the mechanism of the dedicated NO sensor [4Fe-4S] NsrR: the effect of cluster ligand environment.

NsrR from Streptomyces coelicolor is a bacterial nitric oxide (NO) sensor/nitrosative stress regulator as its primary function, and has been shown to have differential response at low, mid, and high levels of NO. These must correspond to discrete structural changes at the protein-bound [4Fe-4S] cluster in response to stepwise nitrosylation of the cluster. We have investigated the effect of the monohapto carboxylate ligand in the site differentiated [4Fe-4S] cluster cofactor of the protein NsrR on modulating its reactivity to NO with a focus on indentifying mechanistic intermediates. We have prepared a synthetic model [4Fe-4S] cluster complex with tripodal ligand and one single site differentiated site occupied by either thiolate or carboxylate ligand. We report here the mechanistic details of sequential steps of nitrosylation as observed by ESI MS and IR spectroscopy. Parallel non-denaturing mass spectrometry analyses were performed using site-differentiated variants of NsrR with the native aspartic acid, cysteine, or alanine in the position of the forth ligand to the cluster. A mono-nitrosylated synthetic [4Fe-4S] cluster was observed for the first time in a biologically-relevant thiolate-based coordination environment. Combined synthetic and protein data give unprecedented clarity in the modulation of nitrosylation of a [4Fe-4S] cluster.

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.

Whole-cell studies of substrate and inhibitor specificity of isoprene monooxygenase and related enzymes.

Co-oxidation of a range of alkenes, dienes, and aromatic compounds by whole cells of the isoprene-degrading bacterium Rhodococcus sp. AD45 expressing isoprene monooxygenase was investigated, revealing a relatively broad substrate specificity for this soluble diiron centre monooxygenase. A range of 1-alkynes (C2 -C8 ) were tested as potential inhibitors. Acetylene, a potent inhibitor of the related enzyme soluble methane monooxygenase, had little inhibitory effect, whereas 1-octyne was a potent inhibitor of isoprene monooxygenase, indicating that 1-octyne could potentially be used as a specific inhibitor to differentiate between isoprene consumption by bona fide isoprene degraders and co-oxidation of isoprene by other oxygenase-containing bacteria, such as methanotrophs, in environmental samples. The isoprene oxidation kinetics of a variety of monooxygenase-expressing bacteria were also investigated, revealing that alkene monooxygenase from Xanthobacter and soluble methane monooxygenases from Methylococcus and Methylocella, but not particulate methane monooxygenases from Methylococcus or Methylomicrobium, could co-oxidise isoprene at appreciable rates. Interestingly the ammonia monooxygenase from the nitrifier Nitrosomonas europaea could also co-oxidise isoprene at relatively high rates, suggesting that co-oxidation of isoprene by additional groups of bacteria, under the right conditions, might occur in the environment.

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.

Protein encapsulation within the internal cavity of a bacterioferritin.

The thermal and chemical stability of 24mer ferritins has led to attempts to exploit their naturally occurring nanoscale (8 nm) internal cavities for biotechnological applications. An area of increasing interest is the encapsulation of molecules either for medical or biocatalysis applications. Encapsulation requires ferritin dissociation, typically induced using high temperature or acidic conditions (pH ≥ 2), which generally precludes the inclusion of fragile cargo such as proteins or peptide fragments. Here we demonstrate that minimizing salt concentration combined with adjusting the pH to ≤8.5 (i.e. low proton/metal ion concentration) reversibly shifts the naturally occurring equilibrium between dimeric and 24meric assemblies of Escherichia coli bacterioferritin (Bfr) in favour of the disassembled form. Interconversion between the different oligomeric forms of Bfr is sufficiently slow under these conditions to allow the use of size exclusion chromatography to obtain wild type protein in the purely dimeric and 24meric forms. This control over association state was exploited to bind heme at natural sites that are not accessible in the assembled protein. The potential for biotechnological applications was demonstrated by the encapsulation of a small, acidic [3Fe-4S] cluster-containing ferredoxin within the Bfr internal cavity. The capture of ∼4-6 negatively charged ferredoxin molecules per cage indicates that charge complementarity with the inner protein surface is not an essential determinant of successful encapsulation.

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. Important structural differences reside in the variation of the DNA minor and major groove widths. In addition, different DNA curvatures and different interactions with the protein sensors are observed. We also report studies of NsrR binding to four hmpA1 variants, which indicate that flexibility in the central region is not a key binding determinant. Our study explores the promotor binding specificities of three closely related transcriptional regulators.

Insights into methionine S-methylation in diverse organisms.

Dimethylsulfoniopropionate (DMSP) is an important marine anti-stress compound, with key roles in global nutrient cycling, chemotaxis and, potentially, climate regulation. Recently, diverse marine Actinobacteria, α- and γ-proteobacteria were shown to initiate DMSP synthesis via the methionine (Met) S-methyltransferase enzyme (MmtN), generating S-methyl-Met (SMM). Here we characterize a roseobacterial MmtN, providing structural and mechanistic insights into this DMSP synthesis enzyme. We propose that MmtN uses the proximity and desolvation mechanism for Met S-methylation with two adjacent MmtN monomers comprising the Met binding site. We also identify diverse functional MmtN enzymes in potentially symbiotic archaeal Candidatus Woesearchaeota and Candidate Phyla Radiation (CPR) bacteria, and the animalcule Adineta steineri, not anticipated to produce SMM and/or DMSP. These diverse MmtN enzymes, alongside the larger plant MMT enzyme with an N-terminus homologous to MmtN, likely utilize the same proximity and desolvation mechanism. This study provides important insights into the catalytic mechanism of SMM and/or DMSP production, and proposes roles for these compounds in secondary metabolite production, and SMM cycling in diverse organisms and environments.

Second Coordination Sphere Effects on the Mechanistic Pathways for Dioxygen Activation by a Ferritin: Involvement of a Tyr Radical and the Identification of a Cation Binding Site.

Ferritins are ubiquitous diiron enzymes involved in iron(II) detoxification and oxidative stress responses and can act as metabolic iron stores. The overall reaction mechanisms of ferritin enzymes are still unclear, particularly concerning the role of the conserved, near catalytic center Tyr residue. Thus, we carried out a computational study of a ferritin using a large cluster model of well over 300 atoms including its first- and second-coordination sphere. The calculations reveal important insight into the structure and reactivity of ferritins. Specifically, the active site Tyr residue delivers a proton and electron in the catalytic cycle prior to iron(II) oxidation. In addition, the calculations highlight a likely cation binding site at Asp65 , which through long-range electrostatic interactions, influences the electronic configuration and charge distributions of the metal center. The results are consistent with experimental observations but reveal novel detail of early mechanistic steps that lead to an unusual mixed-valent iron(III)-iron(II) center.

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.

Purification and Characterization of the Isoprene Monooxygenase from Rhodococcus sp. Strain AD45.

Isoprene (2-methyl-1,3-butadiene) is a climate-active gas released to the atmosphere in large quantities, comparable to methane in magnitude. Several bacteria have been isolated which can grow on isoprene as a sole carbon and energy source, but very little information is available about the degradation of isoprene by these bacteria at the biochemical level. Isoprene utilization is dependent on a multistep pathway, with the first step being the oxidation of isoprene to epoxy-isoprene. This is catalyzed by a four-component soluble diiron monooxygenase, isoprene monooxygenase (IsoMO). IsoMO is a six-protein complex comprising an oxygenase (IsoABE), containing the di-iron active site, a Rieske-type ferredoxin (IsoC), a NADH reductase (IsoF), and a coupling/effector protein (IsoD), homologous to the soluble methane monooxygenase and alkene/aromatic monooxygenases. Here, we describe the purification of the IsoMO components from Rhodococcus sp. AD45 and reconstitution of isoprene-oxidation activity in vitro. Some IsoMO components were expressed and purified from the homologous host Rhodococcus sp. AD45-ID, a Rhodococcus sp. AD45 strain lacking the megaplasmid which contains the isoprene metabolic gene cluster. Others were expressed in Escherichia coli and purified as fusion proteins. We describe the characterization of these purified components and demonstrate their activity when combined with Rhodococcus sp. AD45 cell lysate. Demonstration of IsoMO activity in vitro provides a platform for further biochemical and biophysical characterization of this novel soluble diiron center monooxygenase, facilitating new insights into the enzymatic basis for the bacterial degradation of isoprene. IMPORTANCE Isoprene is a highly abundant climate-active gas and a carbon source for some bacteria. Analyses of the genes encoding isoprene monooxygenase (IsoMO) indicate this enzyme is a soluble diiron center monooxygenase in the same family of oxygenases as soluble methane monooxygenase, alkene monooxygenase, and toluene monooxygenase. We report the initial biochemical characterization of IsoMO from Rhodococcus, the first from any bacterium, describing the challenging purification and reconstitution of in vitro activity of its four components. This study lays the foundation for future detailed mechanistic studies of IsoMO, a key enzyme in the global isoprene cycle.

Iron-sulfur clusters as inhibitors and catalysts of viral replication.

A virus hijacks host cellular machineries and metabolites in order to reproduce. In response, the innate immune system activates different processes to fight back. Although many aspects of these processes have been well investigated, the key roles played by iron-sulfur [FeS] clusters, which are among the oldest classes of bio-inorganic cofactors, have barely been considered. Here we discuss how several [FeS] cluster-containing proteins activate, support and modulate the innate immune response to restrict viral infections, and how some of these proteins simultaneously support the replication of viruses. We also propose models of function of some proteins in the innate immune response and argue that [FeS] clusters in many of these proteins act as biological 'fuses' to control the response. We hope this overview helps to inspire future research in the emerging field of bio-inorganic virology/immunology and that such studies may reveal new molecular insight into the links between viral infections and diseases like cancer and neurodegeneration.

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.

Key carboxylate residues for iron transit through the prokaryotic ferritin SynFtn.

Ferritins are proteins forming 24meric rhombic dodecahedral cages that play a key role in iron storage and detoxification in all cell types. Their function requires the transport of Fe2+ from the exterior of the protein to buried di-iron catalytic sites, known as ferroxidase centres, where Fe2+ is oxidized to form Fe3+-oxo precursors of the ferritin mineral core. The route of iron transit through animal ferritins is well understood: the Fe2+ substrate enters the protein via channels at the threefold axes and conserved carboxylates on the inner surface of the protein cage have been shown to contribute to transient binding sites that guide Fe2+ to the ferroxidase centres. The routes of iron transit through prokaryotic ferritins are less well studied but for some, at least, there is evidence that channels at the twofold axes are the major route for Fe2+ uptake. SynFtn, isolated from the cyanobacterium Synechococcus CC9311, is an atypical prokaryotic ferritin that was recently shown to take up Fe2+ via its threefold channels. However, the transfer site carboxylate residues conserved in animal ferritins are absent, meaning that the route taken from the site of iron entry into SynFtn to the catalytic centre is yet to be defined. Here, we report the use of a combination of site-directed mutagenesis, absorbance-monitored activity assays and protein crystallography to probe the effect of substitution of two residues potentially involved in this pathway. Both Glu141 and Asp65 play a role in guiding the Fe2+ substrate to the ferroxidase centre. In the absence of Asp65, routes for Fe2+ to, and Fe3+ exit from, the ferroxidase centre are affected resulting in inefficient formation of the mineral core. These observations further define the iron transit route in what may be the first characterized example of a new class of ferritins peculiar to cyanobacteria.

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.

Iron Oxidation in Escherichia coli Bacterioferritin Ferroxidase Centre, a Site Designed to React Rapidly with H2 O2 but Slowly with O2.

Both O2 and H2 O2 can oxidize iron at the ferroxidase center (FC) of Escherichia coli bacterioferritin (EcBfr) but mechanistic details of the two reactions need clarification. UV/Vis, EPR, and Mössbauer spectroscopies have been used to follow the reactions when apo-EcBfr, pre-loaded anaerobically with Fe2+ , was exposed to O2 or H2 O2 . We show that O2 binds di-Fe2+ FC reversibly, two Fe2+ ions are oxidized in concert and a H2 O2 molecule is formed and released to the solution. This peroxide molecule further oxidizes another di-Fe2+ FC, at a rate circa 1000 faster than O2 , ensuring an overall 1:4 stoichiometry of iron oxidation by O2 . Initially formed Fe3+ can further react with H2 O2 (producing protein bound radicals) but relaxes within seconds to an H2 O2 -unreactive di-Fe3+ form. The data obtained suggest that the primary role of EcBfr in vivo may be to detoxify H2 O2 rather than sequester iron.

Electron Transfer from Haem to the Di-Iron Ferroxidase Centre in Bacterioferritin.

The iron redox cycle in ferritins is not completely understood. Bacterioferritins are distinct from other ferritins in that they contain haem groups. It is acknowledged that the two iron motifs in bacterioferritins, the di-nuclear ferroxidase centre and the haem B group, play key roles in two opposing processes, iron sequestration and iron mobilisation, respectively, and the two redox processes are independent. Herein, we show that in Escherichia coli bacterioferritin, there is an electron transfer pathway from the haem to the ferroxidase centre suggesting a new role(s) haem might play in bacterioferritins.

Iron Oxidation in Escherichia coli Bacterioferritin Ferroxidase Centre, a Site Designed to React Rapidly with H2O2 but Slowly with O2.

Both O2 and H2O2 can oxidize iron at the ferroxidase center (FC) of Escherichia coli bacterioferritin (EcBfr) but mechanistic details of the two reactions need clarification. UV/Vis, EPR, and Mössbauer spectroscopies have been used to follow the reactions when apo-EcBfr, pre-loaded anaerobically with Fe2+, was exposed to O2 or H2O2. We show that O2 binds di-Fe2+ FC reversibly, two Fe2+ ions are oxidized in concert and a H2O2 molecule is formed and released to the solution. This peroxide molecule further oxidizes another di-Fe2+ FC, at a rate circa 1000 faster than O2, ensuring an overall 1:4 stoichiometry of iron oxidation by O2. Initially formed Fe3+ can further react with H2O2 (producing protein bound radicals) but relaxes within seconds to an H2O2-unreactive di-Fe3+ form. The data obtained suggest that the primary role of EcBfr in vivo may be to detoxify H2O2 rather than sequester iron.