Nitric oxide and peroxynitrite dependent aconitase inactivation and iron regulatory protein-1 activation in mammalian fibroblasts.

OXIDO NITRICO

BIOQUIMICA

BIBLIOGRAFIA NACIONAL QUIMICA

1998

The reaction of reactive oxygen and nitrogen species with the [4Fe–4S]2+cluster of mitochondrial (m-) and cytosolic (c-) aconitases leads to loss of catalytic activity and, in the case of the c-aconitase, triggers total cluster disruption to yield the iron-regulatory protein-1 (IRP-1). Herein we have studied the relative contribution and interplay of reactive oxygen species (O [equation] and H2O2), nitric oxide (•NO), and peroxynitrite in the modulation of m- and c-aconitase and IRP-1 activities in V79-M8 mammalian fibroblasts, identifying key variables that control the various reactivities at the cellular level. Extracellular production of H2O2led to inactivation of both m- and c-aconitase and IRP-1 activation, while extracellular [equation] had no effect. However, increased intracellular production of [equation] caused a loss in m- and c-aconitase activity and IRP-1 activation. Nitric oxide released from NOC-12 had a more complex effect on aconitase and IRP-1 activities. Mitochondrial aconitase was more sensitive than c-aconitase to•NO-mediated inactivation and minimal activation of IRP-1 was observed during a 30-min exposure to the•NO donor. The action of•NO was down- or upregulated by the presence of extra- or intracelular [equation], respectively. Extracellular [equation] decreased the•NO-mediated inactivation of aconitases, due to the preferential extracellular decomposition and the lower diffusivity of peroxynitrite compared to•NO. On the other hand,•NO exposure concomitant with enhanced intracellular [equation] fluxes lead to intracellular peroxynitrite formation as evidenced by Western blot analysis of nitrated proteins, which increased the effects observed with•NO alone. Peroxynitrite-mediated aconitase inactivation, IRP-1 activation, and cellular protein nitration were more pronounced in cells with low GSH content such as V79-M8 glutathione-depleted cells as well as in pGSOD4 cells which contain 32% of the GSH of the parental strain. Mechanistically, our results imply that the differential actions of the studied reactive species toward cellular aconitases depend on at least three critical factors: (i) their reaction rates with aconitases, (ii) the cellular compartment where they are formed, and (iii) the intracellular status of glutathione.

Archives of Biochemistry and Biophysics v. 359, no. 2, 1998. -- p. 215-224

Elsevier

1998

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doi:10.1006/abbi.1998.0898

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