The ubiquitous cellular labile iron pool (LIP) is often associated with the production of the highly reactive hydroxyl radical, which forms through a redox reaction with hydrogen peroxide. Peroxynitrite is a biologically relevant peroxide produced by the recombination of nitric oxide and superoxide. It is a strong oxidant that may be involved in multiple pathological conditions, but whether and how it interacts with the LIP are unclear. Here, using fluorescence spectroscopy, we investigated the interaction between the LIP and peroxynitrite by monitoring peroxynitrite-dependent accumulation of nitrosated and oxidized fluorescent intracellular indicators. We found that, in murine macrophages, removal of the LIP with membrane-permeable iron chelators sustainably accelerates the peroxynitrite-dependent oxidation and nitrosation of these indicators. These observations could not be reproduced in cell-free assays, indicating that the chelator-enhancing effect on peroxynitrite-dependent modifications of the indicators depended on cell constituents, presumably including LIP, that react with these chelators. Moreover, neither free nor ferrous-complexed chelators stimulated intracellular or extracellular oxidative and nitrosative chemistries. On the basis of these results, LIP appears to be a relevant and competitive cellular target of peroxynitrite or its derived oxidants, and thereby it reduces oxidative processes, an observation that may change the conventional notion that the LIP is simply a cellular source of pro-oxidant iron.

In this study, we examined the mechanisms and kinetic profiles of intracellular nitrosative processes using diaminofluorescein (DAF-2) as a target in RAW 264.7 cells. The intracellular formation of the fluorescent, nitrosated product diaminofluorescein triazol (DAFT) from both endogenous and exogenous nitric oxide (NO) was prevented by deoxygenation and by cell membrane-permeable superoxide (O2(-)) scavengers but not by extracellular bovine Cu,Zn-SOD. In addition, the DAFT formation rate decreased in the presence of cell membrane-permeable Mn porphyrins that are known to scavenge peroxynitrite (ONOO(-)) but was enhanced by HCO3(-)/CO2. Together, these results indicate that nitrosative processes in RAW 264.7 cells depend on endogenous intracellular O2(-) and are stimulated by ONOO(-)/CO2-derived radical oxidants. The N2O3 scavenger sodium azide (NaN3) only partially attenuated the DAFT formation rate and only with high NO (>120 nM), suggesting that DAFT formation occurs by nitrosation (azide-susceptible DAFT formation) and predominantly by oxidative nitrosylation (azide-resistant DAFT formation). Interestingly, the DAFT formation rate increased linearly with NO concentrations of up to 120-140 nM but thereafter underwent a sharp transition and became insensitive to NO. This behavior indicates the sudden exhaustion of an endogenous cell substrate that reacts rapidly with NO and induces nitrosative processes, consistent with the involvement of intracellular O2(-). On the other hand, intracellular DAFT formation stimulated by a fixed flux of xanthine oxidase-derived extracellular O2(-) that also occurs by nitrosation and oxidative nitrosylation increased, peaked, and then decreased with increasing NO, as previously observed. Thus, our findings complementarily show that intra- and extracellular O2(-)-dependent nitrosative processes occurring by the same chemical mechanisms do not necessarily depend on NO concentration and exhibit different unusual kinetic profiles with NO dynamics, depending on the biological compartment in which NO and O2(-) interact.

The ubiquitous cellular labile iron pool (LIP) is often associated with the production of the highly reactive species hydroxyl radical, which forms through redox reaction with hydrogen peroxide. Several studies indicate that LIP‐dependent hydroxyl radical production causes cellular damage through oxidation of biomolecules. Although LIP's interaction with hydrogen peroxide and its effects are well established, the interaction of LIP and peroxynitrite, and it's effects are not yet understood. Does peroxynitrite, a potent oxidant species known to interact with transition metals, act as an oxidant species in cells just like hydrogen peroxide? In murine macrophage cells we observed that removal of LIP with membrane‐permeable iron chelators attenuates peroxynitrite‐dependent oxidation and nitrosation of fluorescent probes in both normal and oxidative stress conditions which is in stark contrast to H2O2‐dependent oxidation. These results could not be reproduced in cell‐free systems indicating that the chelator‐enhancing effect on peroxynitrite‐dependent modifications of the probes depend on cells constituents, presumably including LIP, that reacts with these chelators. It is also observed that neither free nor ferrous‐complexed chelators stimulated intracellular or extracellular nitrosative and oxidative processes. These results indicates that LIP is a competitive cellular target for peroxynitrite and its derived oxidants, and thereby reduces oxidative processes, which may change the current view of LIP as a cellular source of pro‐oxidant iron.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.