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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.

SUBMITTER: Bosworth CA

PROVIDER: S-EPMC2660757 | biostudies-literature | 2009 Mar

REPOSITORIES: biostudies-literature

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

Bosworth Charles A CA Toledo José C JC Zmijewski Jaroslaw W JW Li Qian Q Lancaster Jack R JR

Proceedings of the National Academy of Sciences of the United States of America 20090304 12

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 ...[more]

PMID: 19261856

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Project description:An increase in production of reactive oxygen species resulting in a decrease in nitric oxide bioavailability in the endothelium contributes to many cardiovascular diseases, and these reactive oxygen species can oxidize cellular macromolecules. Protein thiols are critical reducing equivalents that maintain cellular redox state and are primary targets for oxidative modification. We demonstrate endothelial NOS (eNOS) oxidant-induced protein thiyl radical formation from tetrahydrobiopterin-free enzyme or following exposure to exogenous superoxide using immunoblotting, immunostaining, and mass spectrometry. Spin trapping with 5,5-dimethyl-1-pyrroline N-oxide (DMPO) followed by immunoblotting using an anti-DMPO antibody demonstrated the formation of eNOS protein radicals, which were abolished by superoxide dismutase and L-NAME, indicating that protein radical formation was due to superoxide generation from the eNOS heme. With tetrahydrobiopterin-reconstituted eNOS, eNOS protein radical formation was completely inhibited. Using mass spectrometric and mutagenesis analysis, we identified Cys-908 as the residue involved in protein radical formation. Mutagenesis of this key cysteine to alanine abolished eNOS thiyl radical formation and uncoupled eNOS, leading to increased superoxide generation. Protein thiyl radical formation leads to oxidation or modification of cysteine with either disulfide bond formation or S-glutathionylation, which induces eNOS uncoupling. Furthermore, in endothelial cells treated with menadione to trigger cellular superoxide generation, eNOS protein radical formation, as visualized with confocal microscopy, was increased, and these results were confirmed by immunoprecipitation with anti-eNOS antibody, followed by immunoblotting with an anti-DMPO antibody. Thus, eNOS protein radical formation provides the basis for a mechanism of superoxide-directed regulation of eNOS, involving thiol oxidation, defining a unique pathway for the redox regulation of cardiovascular function.

Dataset Information

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

Publications

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

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