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

One of the most important biological reactions of nitric oxide (nitrogen monoxide, (NO)-N-center dot) 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 (NO)-N-center dot. 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, (NO)-N-center dot 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 (NO)-N-center dot, and SIH added during the (NO)-N-center dot treatment freezes DNIC levels, showing that the complexes are formed from the CIP, and they are stable (resistant to SIH). DNIC formation requires free (NO)-N-center dot, because addition of oxyhemoglobin prevents formation from either (NO)-N-center dot 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 (NO)-N-center dot. In conclusion, the CIP is rapidly and quantitatively converted to paramagnetic large molecular mass DNIC from exposure to free (NO)-N-center dot but not from cellular nitrosothiol. These results have important implications for the antioxidative actions of (NO)-N-center dot and its effects on cellular iron homeostasis.