We report here that, like nonheme iron, protein-bound intracellular heme iron is also a target for destruction by endogenously produced nitric oxide (NO). In isolated rat hepatocytes NO synthesis results in substantial (approximately 60%) and comparable loss of catalase and cytochrome P450 as well as total microsomal heme, and decreased heme synthetic (delta-aminolevulinate synthetase and ferrochelatase) and increased degradative (heme oxygenase) enzymatic activities. The effect is reversible, and intact cytochrome P450 apoproteins are still present, as judged by heme reconstitution of isolated microsomes. The effects on delta-aminolevulinate synthetase and heme oxygenase are likely to be secondary to heme liberation, while the effects on ferrochelatase appear to be a direct effect of NO, perhaps destruction of its nonheme iron-sulfur center.

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...

Pretreatment of rat hepatocytes with low-dose nitrogen oxide (addition of SNAP in vitro or induction of nitric oxide synthase in vitro or in vivo) imparts resistance to killing and decrease in aconitase and mitochondrial electron transfer from a second exposure to a higher dose of SNAP. Induction of this resistance is prevented by cycloheximide, indicating upregulation of protective protein(s). Ferritin levels are increased as are nonheme iron-NO EPR signals. Tin-protoporphyrin (SnPP) prevents protection, suggesting involvement of hsp32 (heme oxygenase) and/or guanylyl cyclase (GC). Cross-resistance to H2Oz killing is also observed, which is also prevented by cycloheximide and SnPP. Thus, hepatocytes possess inducible protective mechanisms against nitrogen oxide and reactive oxygen toxicity.

Porphyria cutanea tarda (PCT), the most common form of porphyria in humans, is due to reduced activity of uroporphyrinogen decarboxylase (URO-D) in the liver. Previous studies have demonstrated that protein levels of URO-D do not change when catalytic activity is reduced, suggesting that an inhibitor of URO-D is generated in hepatocytes. Here, we describe the identification and characterization of an inhibitor of URO-D in liver cytosolic extracts from two murine models of PCT: wild-type mice treated with iron, ␦-aminolevulinic acid, and polychlorinated biphenyls; and mice with one null allele of Uro-d and two null alleles of the hemochromatosis gene (Uro-d ؉/؊ , Hfe ؊/؊ ) that develop PCT with no treatments. In both models, we identified an inhibitor of recombinant human URO-D (rhURO-D). The inhibitor was characterized by solid-phase extraction, chromatography, UV-visible spectroscopy, and mass spectroscopy and proved to be uroporphomethene, a compound in which one bridge carbon in the uroporphyrinogen macrocycle is oxidized. We synthesized uroporphomethene by photooxidation of enzymatically generated uroporphyrinogen I or III. Both uroporphomethenes inhibited rhURO-D, but the III isomer porphomethene was a more potent inhibitor. Finally, we detected an inhibitor of rhURO-D in cytosolic extracts of liver biopsy samples of patients with PCT. These studies define the mechanism underlying clinical expression of the PCT phenotype, namely oxidation of uroporphyrinogen to uroporphomethene, a competitive inhibitor of URO-D. The oxidation reaction is iron-dependent.heme ͉ porphyrins ͉ iron ͉ oxidation

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