Dinitrosyliron complexes and the mechanism(s) of cellular protein nitrosothiol formation from nitric oxide

Abstract

Nitrosothiols (RSNO), formed from thiols and metabolites of nitric oxide (*NO), have been implicated in a diverse set of physiological and pathophysiological processes, although the exact mechanisms by which they are formed biologically are unknown. Several candidate nitrosative pathways involve the reaction of *NO with O(2), reactive oxygen species (ROS), and transition metals. We developed a strategy using extracellular ferrocyanide to determine that under our conditions intracellular protein RSNO formation occurs from reaction of *NO inside the cell, as opposed to cellular entry of nitrosative reactants from the extracellular compartment. Using this method we found that in RAW 264.7 cells RSNO formation occurs only at very low (<8 microM) O(2) concentrations and exhibits zero-order dependence on *NO concentration. Indeed, RSNO formation is not inhibited even at O(2) levels <1 microM. Additionally, chelation of intracellular chelatable iron pool (CIP) reduces RSNO formation by >50%. One possible metal-dependent, O(2)-independent nitrosative pathway is the reaction of thiols with dinitrosyliron complexes (DNIC), which are formed in cells from the reaction of *NO with the CIP. Under our conditions, DNIC formation, like RSNO formation, is inhibited by approximately 50% after chelation of labile iron. Both DNIC and RSNO are also increased during overproduction of ROS by the redox cycler 5,8-dimethoxy-1,4-naphthoquinone. Taken together, these data strongly suggest that cellular RSNO are formed from free *NO via transnitrosation from DNIC derived from the CIP. We have examined in detail the kinetics and mechanism of RSNO formation inside cells.

Fig. 1.

Formation of intracellular RSNO is not caused by the autoxidation of •NO in the extracellular space. (A) Scheme of extracellular versus intracellular nitrosative processes. External addition of FCN will prevent cellular nitrosation from entry of nitrosative species formed extracellularly from •NO autoxidation, resulting in RSNO formation from only intracellular •NO. See Results for details. (B) Cells were exposed to sper/NO (10 μM) ± FCN (1 mM) for 60 min, and RSNO level in lysates was determined as described in Methods. (C) Same as A except nitrosation of external BSA (0.075 mg/mL) was measured. n.s., not significant. *, P < 0.05 compared with control.

Fig. 2.

Time course of RSNO formation and •NO and O₂ concentrations. (A) Cells were added to PBSD containing sper/NO (10 μM) and 1 mM FCN, and O₂ concentration was monitored. (Inset) Scale expansion showing the slow decrease in O₂ at longer times. At arrow, sodium dithionite was added. Representative data from 2 experiments are shown. (B) Conditions as in A except at 0, 12, 24, 36, 48, and 60 min cells were removed and processed for RSNO content (■). In parallel experiments, aliquots were removed at the indicated time points and analyzed for •NO concentration (△). (C) Cells were incubated with sper/NO and FCN as in A for 60 min, after which oxymyoglobin (20 μM) was added to scavenge •NO. Samples were collected 0, 20, or 60 min later and processed for intracellular RSNO content.

Fig. 3.

RSNO formation during anoxia. (A) •NO and O₂ electrode traces for anaerobic experiments. Cells (2 × 10⁷ cells/mL) were allowed to respire for 5–10 min until O₂ levels reached 0 (t = 0), at which point 1 mM FCN and 5 μM sper/NO (argon-purged) were added. Cells were then incubated for 60 min. (B) RSNO determination in anaerobically-incubated cells.

Fig. 4.

Effect of ROS on cellular RSNO formation. (A) Cells preloaded with DCFH-DA were exposed to DMNQ (0 or 10 μM) for 30 min, and DCFH oxidation was determined as described in Methods. (Magnification: 20×.) (B) Cells were exposed to sper/NO and FCN as in Fig. 1 and DMNQ at the indicated concentrations, and cells were analyzed for RSNO content.

Fig. 5.

Correlation between DNIC and RSNO. Cells were exposed to 10 μM sper/NO and 1 mM FCN for 60 min, ± SIH and/or 10 μM DMNQ, and samples were analyzed for RSNO content (closed bars) or DNIC (open bars) as described in Methods. Values for RSNO are taken from Tables 1 and 2 and plotted in graphical form as percentage of control.