Bosworth 2009 Proc Natl Acad Sci U S A

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Bosworth CA, Toledo JC Jr, Zmijewski JW, Li Q, Lancaster JR (2009) Dinitrosyliron complexes and the mechanism(s) of cellular protein nitrosothiol formation from nitric oxide. Proc Natl Acad Sci U S A 106:4671-6.

» PMID: 19261856 Open Access

Bosworth CA, Toledo JC Jr, Zmijewski JW, Li Q, Lancaster JR (2009) Proc Natl Acad Sci U S A

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 O2, 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 μM) O2 concentrations and exhibits zero-order dependence on •NO concentration. Indeed, RSNO formation is not inhibited even at O2 levels <1 μM. Additionally, chelation of intracellular chelatable iron pool (CIP) reduces RSNO formation by >50%. One possible metal-dependent, O2-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 ≈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.

Keywords: Iron, Nitrosation, Reactive nitrogen species, Reactive oxygen species, Chelatable iron


Labels: MiParea: Respiration 

Stress:Oxidative stress;RONS  Organism: Fungi 

Preparation: Intact cells  Enzyme: Marker enzyme  Regulation: ADP, Inhibitor, Oxygen kinetics, Substrate, Amino acid  Coupling state: ROUTINE 

HRR: Oxygraph-2k