Spectroscopic Description of the Two Nitrosyl−Iron Complexes Responsible for Fur Inhibition by Nitric Oxide

D’Autréaux, Benoît; Horner, Olivier; Oddou, Jean-Louis; Jeandey, C.; Gambarelli, Serge; Berthomieu, Catherine; Latour, Jean‐Marc; Michaud‐Soret, Isabelle

doi:10.1021/ja031671a

Cited by 89 publications

(102 citation statements)

References 65 publications

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“…However, evidence appeared suggesting that DNIC are not due to mitochondrial iron-sulfur complexes (9,50). In addition, it has been demonstrated that DNIC are relatively promiscuous regarding the two ligands other than the nitrosyls; in fact, indistinguishable EPR signals are obtained whether the additional ligands are halogens, nitrogen, oxygen, or sulfur groups (51)(52)(53)(54). The results here suggest that the iron observed in cells and tissues exposed to exogenous and endogenous ⅐ NO is derived from the CIP.…”

Section: Discussionmentioning

confidence: 63%

Nitric Oxide-induced Conversion of Cellular Chelatable Iron into Macromolecule-bound Paramagnetic Dinitrosyliron Complexes

Toledo

Bosworth

Hennon

et al. 2008

Journal of Biological Chemistry

107

139

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One of the most important biological reactions of nitric oxide (nitrogen monoxide, ⅐ NO) is its reaction with transition metals, of which iron is the major target. This is confirmed by the ubiquitous formation of EPR-detectable g ‫؍‬ 2.04 signals in cells, tissues, and animals upon exposure to both exogenous and endogenous ⅐ NO. The source of the iron for these dinitrosyliron complexes (DNIC), and its relationship to cellular iron homeostasis, is not clear. Evidence has shown that the chelatable iron pool (CIP) may be at least partially responsible for this iron, but quantitation and kinetic characterization have not been reported. In the murine cell line RAW 264.7, ⅐ NO reacts with the CIP similarly to the strong chelator salicylaldehyde isonicotinoyl hydrazone (SIH) in rapidly releasing iron from the iron-calcein complex. SIH pretreatment prevents DNIC formation from ⅐ NO, and SIH added during the ⅐ NO treatment "freezes" DNIC levels, showing that the complexes are formed from the CIP, and they are stable (resistant to SIH). DNIC formation requires free ⅐ NO, because addition of oxyhemoglobin prevents formation from either ⅐ NO donor or S-nitrosocysteine, the latter treatment resulting in 100-fold higher intracellular nitrosothiol levels. EPR measurement of the CIP using desferroxamine shows quantitative conversion of CIP into DNIC by ⅐ NO. In conclusion, the CIP is rapidly and quantitatively converted to paramagnetic large molecular mass DNIC from exposure to free ⅐ NO but not from cellular nitrosothiol. These results have important implications for the antioxidative actions of ⅐ NO and its effects on cellular iron homeostasis.Nitric oxide (nitrogen monoxide, ⅐ NO) is a multifunctional small radical molecule responsible for a remarkable array of physiological and pathophysiological phenomena (1). Although ⅐ NO gives rise to a complex array of reactive species (2) in the biological milieu, ⅐ NO itself reacts directly with only a small number of targets. These targets are either species with unpaired electrons or transition metals; the origin of this reactivity pattern lies in the ability of these targets to stabilize the unpaired electron on ⅐ NO. In the case of transition metals, this stabilization occurs because of the strong interaction of ⅐ NO orbitals and the metal d-orbitals (3). Formation of metal-nitrosyls in biological systems can give rise to several paramagnetic species, which can be observed by EPR spectroscopy, and has been utilized to glean important information regarding the biological actions of ⅐ NO (4). In particular, exposure of cells or tissues to ⅐ NO (either exogenously administered or endogenously synthesized) results in the ubiquitous appearance of a "g ϭ 2.04" axial EPR signal, which has been assigned to iron in square planar coordination with two nitrosyl ligands (denoted "dinitrosyliron complexes" (DNIC) 3 (5). The source of this iron, the nature of the other iron ligands, and the origin of their formation are not clear.The intracellular labile or chelatable iron pool (CIP) is a smal...

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Section: Discussionmentioning

confidence: 63%

Nitric Oxide-induced Conversion of Cellular Chelatable Iron into Macromolecule-bound Paramagnetic Dinitrosyliron Complexes

Toledo

Bosworth

Hennon

et al. 2008

Journal of Biological Chemistry

107

139

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“…In addition, the spectrum contains an S ϭ 1 ⁄ 2 signal with g Ќ ϭ 2.041, and g ϭ 2.018, characteristic for dinitrosyl iron complexes (DNICs) of the type Fe(NO) 2 (39,40). Similar signals have been detected with other iron proteins (41)(42)(43)(44). Quantification of the EPR signal revealed that only 1.0 -1.5% of NorA protein contributed to the DNIC.…”

Section: Purification and Spectroscopic Characterization Of Nora-mentioning

confidence: 56%

“…The best characterized to date are the protein-bound paramagnetic and EPR-silent DNICs formed upon reaction of NO with the E. coli iron uptake regulator Fur (42), which, in contrast to NorA, contains a mononuclear iron center. The optical spectrum of the paramagnetic DNIC of Fur, the major component, in contrast to NorA is characterized by a shoulder at 540 nm, and three bands at 410, 650, and 830 nm.…”

Section: Discussionmentioning

confidence: 99%

Formation of a Dinitrosyl Iron Complex by NorA, a Nitric Oxide-binding Di-iron Protein from Ralstonia eutropha H16

Strube

Vries

Cramm

2007

Journal of Biological Chemistry

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In Ralstonia eutropha H16, two genes, norA and norB, form a dicistronic operon that is controlled by the NO-responsive transcriptional regulator NorR. NorB has been identified as a membrane-bound NO reductase, but the physiological function of NorA is unknown. We found that, in a NorA deletion mutant, the promoter activity of the norAB operon was increased 3-fold, indicating that NorA attenuates activation of NorR. NorA shows limited sequence similarity to the oxygen carrier hemerythrin, which contains a di-iron center. Indeed, optical and EPR spectroscopy of purified NorA revealed the presence of a di-iron center, which binds oxygen in a similar way as hemerythrin. Nitric oxide (NO)2 is an obligate intermediate of denitrification (1, 2), formed by the reduction of nitrite by nitrite reductases and subsequently converted to nitrous oxide by NO reductases. In the denitrifier Ralstonia eutropha H16, formation of the NO reductase NorB is controlled by NorR, an NObinding transcriptional activator (3, 4). The norB gene is cotranscribed with norA that encodes a protein of unknown function. Orthologs of NorA are present in many genomes of proteobacteria and firmicutes (5) and have been annotated as DnrN (Pseudomonas stutzeri), ScdA (Staphylococcus aureus), or YtfE (Escherichia coli and Salmonella enterica). The expression of DnrN, ScdA, and YtfE has been shown to be up-regulated by NO or nitrosating agents (6 -9). The non-denitrifiers S. aureus, E. coli, and S. enterica, however, do not possess the norB gene, which demonstrates that norA-like genes do not necessarily co-occur with norB. The NO-dependent expression of norA and its coexpression with norB in R. eutropha suggest a function for NorA in NO metabolism. However, it was shown previously that a nonpolar in-frame deletion of the norA gene did not affect denitrification of R. eutropha cells in terms of growth, accumulation of nitrite or nitrous oxide, or formation of dinitrogen (4). In this study we report the purification and characterization of NorA from R. eutropha. We show that NorA is an NO-binding di-iron protein that modulates the NOresponsive expression of the norAB operon in denitrifying cells of R. eutropha. EXPERIMENTAL PROCEDURESStrains and Plasmids-Strains and plasmids used in this study are listed in Table 1. E. coli JM109 was used for propagation of plasmids. E. coli S17-1 served as the donor in conjugative transfers. E. coli BL21(DE3) was used for overproduction of NorAhexahistidine fusion protein. Mutant strains of R. eutropha were constructed by a gene replacement strategy (15) using the suicide vector pLO1, and were verified by Southern blot analysis. The NorR, NorA, and NorB proteins referred to in this study are encoded by the norR1, norA1, and norB1 genes located on megaplasmid pHG1 of R. eutropha. A chromosomally encoded paralog nor gene cluster termed norR2A2B2 has not been extensively studied, but mutational analyses indicate that either of the two nor clusters is sufficient for denitrification (4).All R. eutropha strains used in this stu...

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“…2), it is possible that this complex somehow targets the same regulon as this ironbinding, Fe-uptake regulator. NO has been reported to directly nitrosylate Fur-bound iron in E. coli, thereby abolishing its ability to repress transcription under iron-replete conditions (22,23). However, the presence of NO produces the opposite effect in V. fischeri: a H-NOX-dependent increase in the repression of ironuptake genes.…”

Section: Discussionmentioning

confidence: 99%

H-NOX–mediated nitric oxide sensing modulates symbiotic colonization by Vibrio fischeri

Wang¹,

Dufour

Carlson³

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

101

140

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The bioluminescent bacterium Vibrio fischeri initiates a specific, persistent symbiosis in the light organ of the squid Euprymna scolopes. During the early stages of colonization, V. fischeri is exposed to hostderived nitric oxide (NO). Although NO can be both an antimicrobial component of innate immunity and a key signaling molecule in eukaryotes, potential roles in beneficial host-microbe associations have not been described. V. fischeri hnoX encodes a heme NO/oxygen-binding (H-NOX) protein, a member of a family of bacterial NO-and/or O 2 -binding proteins of unknown function. We hypothesized that H-NOX acts as a NO sensor that is involved in regulating symbiosis-related genes early in colonization. Whole-genome expression studies identified 20 genes that were repressed in an NO-and H-NOX-dependent fashion. Ten of these, including hemin-utilization genes, have a promoter with a putative ferric-uptake regulator (Fur) binding site. As predicted, in the presence of NO, wild-type V. fischeri grew more slowly on hemin than a hnoX deletion mutant. Host-colonization studies showed that the hnoX mutant was also 10-fold more efficient in initially colonizing the squid host than the wild type; similarly, in mixed inoculations, it outcompeted the wild-type strain by an average of 16-fold after 24 h. However, the presence of excess hemin or iron reversed this dominance. The advantage of the mutant in colonizing the iron-limited light-organ tissues is caused, at least in part, by its greater ability to acquire host-derived hemin. Our data suggest that V. fischeri normally senses a host-generated NO signal through H-NOX Vf and modulates the expression of its iron uptake capacity during the early stages of the light-organ symbiosis.symbiosis | iron uptake | transcriptional analysis | colonization

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Spectroscopic Description of the Two Nitrosyl−Iron Complexes Responsible for Fur Inhibition by Nitric Oxide

Cited by 89 publications

References 65 publications

Nitric Oxide-induced Conversion of Cellular Chelatable Iron into Macromolecule-bound Paramagnetic Dinitrosyliron Complexes

Nitric Oxide-induced Conversion of Cellular Chelatable Iron into Macromolecule-bound Paramagnetic Dinitrosyliron Complexes

Formation of a Dinitrosyl Iron Complex by NorA, a Nitric Oxide-binding Di-iron Protein from Ralstonia eutropha H16

H-NOX–mediated nitric oxide sensing modulates symbiotic colonization by Vibrio fischeri

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