Enzymatic Targets of Nitric Oxide as Detected by EPR Spectroscopy within Mammal Cells

Henry, Y.; Ducastel, Béatrice; Guissani, Annie

doi:10.1007/978-1-4613-1185-0_11

Cited by 9 publications

(20 citation statements)

References 182 publications

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“…In the 1980s the demonstration of the mammalian synthesis of inorganic nitrogen oxides (41)(42)(43), their importance in antitumor cytotoxicity by activated macrophages (44), and their detection by EPR in this setting (23,24) reignited interest. The detection of these distinctive EPR signals has now been associated with the endogenous formation of the pluripotent biological signal/messenger ⅐ NO in a multitude of pathological settings (45), including diabetes (46), organ transplant rejection (47), and sepsis (48). Because of the similarity of these signals resulting from endogenous ⅐ NO synthesis to small inorganic dinitrosyl complexes, coupled with the potent disruption of mitochondrial electron transfer by ⅐ NO and the concomitant appearance of DNIC EPR signals (49), it was first thought that cellular DNIC were derived from mitochondrial iron-sulfur clusters (23,24).…”

Section: Discussionmentioning

confidence: 99%

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: 99%

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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“…Both of these spectrometers have comparable sensitivity and reproducibility; the potential limitation of e-scan is that it does not operate at low magnetic fields; therefore, the study of high spin paramagnetic centers, such as Met-Hb or Fe III -transferrin is impossible. For more detailed information on the EPR method and, in particular, with regard to the EPR spectroscopy of biologically relevant nitrosylated compounds, the reader is referred to the monograph by Henry et al [6].…”

Section: Basic Principles Of Epr Spin Trappingmentioning

confidence: 99%

“…Additionally, heme-NO complexes can be formed as a result of reductive nitrosylation from heme and nitrite at low oxygen tension [6]. Due to the high affinity of NO for heme groups, NO has been widely used as a paramagnetic probe to characterize the oxygen binding sites of many purified heme proteins; the list of these proteins include myoglobin [31], cytochrome C [32], peroxidase [33], catalase [34], cyclooxygenase [35], P-450 monooxygenases [36] and many other enzymes.…”

Section: Tissue Heme Proteinsmentioning

confidence: 99%

Electron paramagnetic resonance (EPR) spin trapping of biological nitric oxide

Kleschyov

Wenzel

Münzel

2007

Journal of Chromatography B

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“…and specifically to iron or copper-containing proteins, forming mono-and dinitrosyl complexes (MNIC and DNIC) [43][44][45][46][47]. These reactions have usually fast binding rates and are often reversible, with dissociation rates that differ by several orders of magnitude depending on the protein and its redox state (see for instance references [15,46]).…”

Section: Metalloproteins As Spontaneous Targets Of Nitric Oxidementioning

confidence: 99%

“…Some of these nitrosylated complexes are EPR detectable and are recognized as such by use of extensive EPR catalogues. Several attempts to such compilations of EPR catalogues have been made [8,9,37,43,44]. EPR spectra recorded on whole cell cultures and organs have originally been taken as a proof of NO synthesis and accumulation following spontaneous binding to metalloproteins, mostly under pathological conditions under which inducible NO-synthase (iNOS) pathway is stimulated [11,44,45,48].…”

Section: Metalloproteins As Spontaneous Targets Of Nitric Oxidementioning

confidence: 99%

Contribution of spin-trapping EPR techniques for the measurement of NO production in biological systems

Henry¹,

Guissani²

2000

Analusis

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In this overview of NO spin-trapping methods, we have pointed out their main advantages over frozen solution EPR spectroscopy detection of nitrosylated metalloproteins: improved sensitivity, potential specificity and time-resolution. A few simple ideas concerning EPR methodology have been recalled. Examples of possible artefacts, sometimes unexpected, which can easily be encountered in any analytical methods have been described. New methodological developments have also been mentioned.

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Enzymatic Targets of Nitric Oxide as Detected by EPR Spectroscopy within Mammal Cells

Cited by 9 publications

References 182 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

Electron paramagnetic resonance (EPR) spin trapping of biological nitric oxide

Contribution of spin-trapping EPR techniques for the measurement of NO production in biological systems

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