Nitrosylation of Nitric-Oxide-Sensing Regulatory Proteins Containing [4Fe-4S] Clusters Gives Rise to Multiple Iron-Nitrosyl Complexes.
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ABSTRACT:
SUBMITTER: Serrano PN
PROVIDER: S-EPMC5204455 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:In Bacillus subtilis, NsrR is required for the upregulation of ResDE-dependent genes in the presence of nitric oxide (NO). NsrR was shown to bind to the promoters of these genes and inhibit their transcription in vitro. NO relieves this inhibition by an unknown mechanism. Here, we use spectroscopic techniques (UV-vis, resonance Raman, and EPR) to show that anaerobically isolated NsrR from B. subtilis contains a [4Fe-4S](2+) cluster, which reacts with NO to form dinitrosyl iron complexes. This method of NO sensing is analogous to that of the FNR protein of Escherichia coli. The Fe-S cluster of NsrR is also reactive toward other exogenous ligands such as cyanide, dithiothreitol, and O(2). These results, together with the fact that there are only three cysteine residues in NsrR, suggest that the 4Fe-4S cluster contains a noncysteinyl labile ligand to one of the iron atoms, leading to high reactivity. Size exclusion chromatography and cross-linking experiments show that NsrR adopts a dimeric structure in its [4Fe-4S](2+) holo form as well as in the apo form. These findings provide a first stepping stone to investigate the mechanism of NO sensing in NsrR.
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Project description:Although the NO (nitric oxide)-mediated modification of iron-sulfur proteins has been well-documented in bacteria and mammalian cells, specific reactivity of NO with iron-sulfur proteins still remains elusive. In the present study, we report the first kinetic characterization of the reaction between NO and iron-sulfur clusters in protein using the Escherichia coli IlvD (dihydroxyacid dehydratase) [4Fe-4S] cluster as an example. Combining a sensitive NO electrode with EPR (electron paramagnetic resonance) spectroscopy and an enzyme activity assay, we demonstrate that NO is rapidly consumed by the IlvD [4Fe-4S] cluster with the concomitant formation of the IlvD-bound DNIC (dinitrosyl-iron complex) and inactivation of the enzyme activity under anaerobic conditions. The rate constant for the initial reaction between NO and the IlvD [4Fe-4S] cluster is estimated to be (7.0+/-2.0)x10(6) M(-2) x s(-1) at 25 degrees C, which is approx. 2-3 times faster than that of the NO autoxidation by O2 in aqueous solution. Addition of GSH failed to prevent the NO-mediated modification of the IlvD [4Fe-4S] cluster regardless of the presence of O2 in the medium, further suggesting that NO is more reactive with the IlvD [4Fe-4S] cluster than with GSH or O2. Purified aconitase B [4Fe-4S] cluster from E. coli has an almost identical NO reactivity as the IlvD [4Fe-4S] cluster. However, the reaction between NO and the endonuclease III [4Fe-4S] cluster is relatively slow, apparently because the [4Fe-4S] cluster in endonuclease III is less accessible to solvent than those in IlvD and aconitase B. When E. coli cells containing recombinant IlvD, aconitase B or endonuclease III are exposed to NO using the Silastic tubing NO delivery system under aerobic and anaerobic conditions, the [4Fe-4S] clusters in IlvD and aconitase B, but not in endonuclease III, are efficiently modified forming the protein-bound DNICs, confirming that NO has a higher reactivity with the [4Fe-4S] clusters in IlvD and aconitase B than with O2 or GSH. The results suggest that the iron-sulfur clusters in proteins such as IlvD and aconitase B may constitute the primary targets of the NO cytotoxicity under both aerobic and anaerobic conditions.
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Project description:Patients with a congenital bicuspid aortic valve (BAV), a valve with two instead of three aortic leaflets, have an increased risk of developing thoracic aneurysms and aortic dissection. The mechanisms underlying BAV-associated aortopathy are poorly understood. This study examined BAV-associated aortopathy in Nos3-/- mice, a model with congenital BAV formation. A combination of histological examination and in vivo ultrasound imaging was used to investigate aortic dilation and dissections in Nos3-/- mice. Moreover, cell lineage analysis and single-cell RNA sequencing were used to observe the molecular anomalies within vascular smooth muscle cells (VSMCs) of Nos3-/- mice. Spontaneous aortic dissections were found in ascending aortas located at the sinotubular junction in ∼13% of Nos3-/- mice. Moreover, Nos3-/- mice were prone to developing aortic dilations in the proximal and distal ascending aorta during early adulthood. Lower volumes of elastic fibres were found within vessel walls of the ascending aortas of Nos3-/- mice, as well as incomplete coverage of the aortic inner media by neural crest cell (NCC)-derived VSMCs. VSMCs of Nos3-/- mice showed downregulation of 15 genes, of which seven were associated with aortic aneurysms and dissections in the human population. Elastin mRNA was most markedly downregulated, followed by fibulin-5 expression, both primary components of elastic fibres. This study demonstrates that, in addition to congenital BAV formation, disrupted endothelial-mediated nitric oxide (NO) signalling in Nos3-/- mice also causes aortic dilation and dissection, as a consequence of inhibited elastic fibre formation in VSMCs within the ascending aorta.
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Project description:The cleavage of [4Fe-4S]-type clusters is thought to be important in proteins such as Fe-S scaffold proteins and nitrogenase. However, most [4Fe-4S](2+) clusters in proteins have two antiferromagnetically coupled high-spin layers in which a minority spin is delocalized in each layer, thus forming a symmetric Fe(2.5+)-Fe(2.5+) pair, and how cleavage occurs between the irons is puzzling because of the shared electron. Previously, we proposed a novel mechanism for the fission of a [4Fe-4S] core into two [2Fe-2S] cores in which the minority spin localizes on one iron, thus breaking the symmetry and creating a transition state with two Fe(3+)-Fe(2+) pairs. Cleavage first through the weak Fe(2+)-S bonds lowers the activation energy. Here, we propose a test of this mechanism: break the symmetry of the cluster by changing the ligands to promote spin localization, which should enhance reactivity. The cleavage reactions for the homoligand [Fe(4)S(4)L(4)](2-) (L = SCH(3), Cl, H) and heteroligand [Fe(4)S(4)(SCH(3))(2)L(2)](2-) (L = Cl, H) clusters in the gas phase were examined via broken-symmetry density functional theory calculations. In the heteroligand clusters, the minority spin localized on the iron coordinated by the weaker electron-donor ligand, and the reaction energy and activation barrier of the cleavage were lowered, which is in accord with our proposed mechanism and consistent with photoelectron spectroscopy and collision-induced dissociation experiments. These studies suggest that proteins requiring facile fission of their [4Fe-4S] cluster in their biological function might have spin-localized [4Fe-4S] clusters.
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Project description:The reactivity of protein bound iron-sulfur clusters with nitric oxide (NO) is well documented, but little is known about the actual mechanism of cluster nitrosylation. Here, we report studies of members of the Wbl family of [4Fe-4S] containing proteins, which play key roles in regulating developmental processes in actinomycetes, including Streptomyces and Mycobacteria, and have been shown to be NO responsive. Streptomyces coelicolor WhiD and Mycobacterium tuberculosis WhiB1 react extremely rapidly with NO in a multiphasic reaction involving, remarkably, 8 NO molecules per [4Fe-4S] cluster. The reaction is 10(4)-fold faster than that observed with O(2) and is by far the most rapid iron-sulfur cluster nitrosylation reaction reported to date. An overall stoichiometry of [Fe(4)S(4)(Cys)(4)](2-) + 8NO ? 2[Fe(I)(2)(NO)(4)(Cys)(2)](0) + S(2-) + 3S(0) has been established by determination of the sulfur products and their oxidation states. Kinetic analysis leads to a four-step mechanism that accounts for the observed NO dependence. DFT calculations suggest the possibility that the nitrosylation product is a novel cluster [Fe(I)(4)(NO)(8)(Cys)(4)](0) derived by dimerization of a pair of Roussin's red ester (RRE) complexes.
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Project description:Biological [Fe-S] clusters are increasingly recognized to undergo proton-coupled electron transfer (PCET), but the site of protonation, mechanism, and role for PCET remains largely unknown. Here we explore this reactivity with synthetic model clusters. Protonation of the arylthiolate-ligated [4Fe-4S] cluster [Fe4 S4 (SAr)4 ](2-) (1, SAr=S-2,4-6-(iPr)3 C6 H2 ) leads to thiol dissociation, reversibly forming [Fe4 S4 (SAr)3 L](1-) (2) and ArSH (L=solvent, and/or conjugate base). Solutions of 2+ArSH react with the nitroxyl radical TEMPO to give [Fe4 S4 (SAr)4 ](1-) (1ox ) and TEMPOH. This reaction involves PCET coupled to thiolate association and may proceed via the unobserved protonated cluster [Fe4 S4 (SAr)3 (HSAr)](1-) (1-H). Similar reactions with this and related clusters proceed comparably. An understanding of the PCET thermochemistry of this cluster system has been developed, encompassing three different redox levels and two protonation states.
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Project description:A fundamental challenge in the redox biology of Mycobacterium tuberculosis (Mtb) is to understand the mechanisms involved in sensing redox signals such as oxygen (O2), nitric oxide (NO), and nutrient depletion, which are thought to play a crucial role in persistence. Here we show that Mtb WhiB3 responds to the dormancy signals NO and O2 through its iron-sulfur (Fe-S) cluster. To functionally assemble the WhiB3 Fe-S cluster, we identified and characterized the Mtb cysteine desulfurase (IscS; Rv3025c) and developed a native enzymatic reconstitution system for assembling Fe-S clusters in Mtb. EPR and UV-visible spectroscopy analysis of reduced WhiB3 is consistent with a one-electron reduction of EPR silent [4Fe-4S]2+ to EPR visible [4Fe-4S]+. Atmospheric O2 gradually degrades the WhiB3 [4Fe-4S]2+ cluster to generate a [3Fe-4S]+ intermediate. Furthermore, EPR analysis demonstrates that NO forms a protein-bound dinitrosyl-iron-dithiol complex with the Fe-S cluster, indicating that NO specifically targets the WhiB3 Fe-S cluster. Our data suggest that the mechanism of WhiB3 4Fe-4S cluster degradation is similar to that of fumarate nitrate regulator. Importantly, Mtb DeltawhiB3 shows enhanced growth on acetate medium, but a growth defect on media containing glucose, pyruvate, succinate, or fumarate as the sole carbon source. Our results implicate WhiB3 in metabolic switching and in sensing the physiologically relevant host signaling molecules NO and O2 through its [4Fe-4S] cluster. Taken together, our results suggest that WhiB3 is an intracellular redox sensor that integrates environmental redox signals with core intermediary metabolism.
| S-EPMC1906726 | biostudies-literature
Project description:Iron-sulfur (Fe-S) clusters are essential cofactors for enzyme activity. These Fe-S clusters are present in structurally diverse forms, including [4Fe-4S] and [3Fe-4S]. Type-identification of the Fe-S cluster is indispensable in understanding the catalytic mechanism of enzymes. However, identifying [4Fe-4S] and [3Fe-4S] clusters in particular is challenging because of their rapid transformation in response to oxidation-reduction events. In this study, we focused on the relationship between the Fe-S cluster type and the catalytic activity of a tRNA-thiolation enzyme (TtuA). We reconstituted [4Fe-4S]-TtuA, prepared [3Fe-4S]-TtuA by oxidizing [4Fe-4S]-TtuA under strictly anaerobic conditions, and then observed changes in the Fe-S clusters in the samples and the enzymatic activity in the time-course experiments. Electron paramagnetic resonance analysis revealed that [3Fe-4S]-TtuA spontaneously transforms into [4Fe-4S]-TtuA in minutes to one hour without an additional free Fe source in the solution. Although the TtuA immediately after oxidation of [4Fe-4S]-TtuA was inactive [3Fe-4S]-TtuA, its activity recovered to a significant level compared to [4Fe-4S]-TtuA after one hour, corresponding to an increase of [4Fe-4S]-TtuA in the solution. Our findings reveal that [3Fe-4S]-TtuA is highly inactive and unstable. Moreover, time-course analysis of structural changes and activity under strictly anaerobic conditions further unraveled the Fe-S cluster type used by the tRNA-thiolation enzyme.
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Project description:BtrN catalyzes the two-electron oxidation of the C3 secondary alcohol of 2-deoxy-scyllo-inosamine to the corresponding ketone and is a member of a subclass of radical S-adenosylmethionine (SAM) enzymes called radical SAM (RS) dehydrogenases. Like all RS enzymes, BtrN contains a [4Fe-4S] cluster that delivers an electron to SAM, inducing its cleavage to the common intermediate in RS reactions, the 5'-deoxyadenosyl 5'-radical. In this work, we show that BtrN contains an additional [4Fe-4S] cluster, thought to bind in contact with the substrate to facilitate loss of the second electron in the two-electron oxidation.
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