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

Abstract

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 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 H2O2 led to inactivation of both m- and c-aconitase and IRP-1 activation, while extracellular had no effect. However, increased intracellular production of 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, respectively. Extracellular 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 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.