Abstract 16807: Superoxide Induces eNOS Protein Thiyl Radical Formation: A Novel Mechanism Regulating eNOS Function and Coupling
Imbalance of nitric oxide (NO) and superoxide (·O2-) production in the endothelium contributes to many cardiovascular diseases. Increase in production of reactive oxygen species has been associated with the cause of these diseases and damages many cellular macromolecules leading to cell death. Protein thiols are critical reducing equivalents that maintain cellular redox-state and a primary target for oxidative modification, such as sulfonation or S-glutathionylation. We demonstrate oxidant-induced formation of an eNOS protein thiyl radical from BH4-free enzyme using immunoblotting, immunostaining, mass spectrometry, and site-directed mutagenesis. Immunoblotting using an anti-DMPO antibody, demonstrated the formation of eNOS protein radicals, which were abolished by SOD and L-NAME, indicating that protein radical formation was due to ·O2- generation from the eNOS heme. With tetrahydrobiopterin reconstituted eNOS, formation of the eNOS protein radical was completely inhibited. Furthermore, in endothelial cells treated with menadione to trigger cellular ·O2- 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. Using mass spectrometric analysis, we identified Cys908 as the only residue involved in protein radical formation. Mutagenesis of this key cysteine to alanine abolishes eNOS thiyl radical formation and uncouples eNOS leading to increased ·O2- generation. In our previous study using mass spectrometric analysis, we have demonstrated that Cys 908 is involved in the oxidant-induced S-glutathionylation that induces eNOS uncoupling under oxidative stress. Thus, eNOS protein radical formation provides the groundwork for a mechanism of ·O2--directed regulation of eNOS, involving thiol oxidation; defining a unique pathway for the redox regulation of cardiovascular function.
- © 2010 by American Heart Association, Inc.