Abstract 15435: Oxidative Stress Induced Enos Thiol Oxidation: a Novel Mechanism Regulating Enos and Cardiovascular Function
Previously, we have demonstrated that eNOS S-glutathionylation uncouples eNOS, leading to increase superoxide (•O2-) generation from the purified enzyme, oxidant-induced endothelium, and hypertensive vessels. An increase in production of reactive oxygen species (ROS) resulting in a decrease in nitric oxide bioavailability in the endothelium contributes to many cardiovascular diseases and these ROS can oxidize cellular macromolecules. Recently, we have also demonstrated using immunoblotting, immunostaining, and mass spectrometry that eNOS oxidant-induced protein thiyl radical formation at Cys908 of the reductase domain occurs with BH4-depleted enzyme or following exposure to exogenous •O2-. Mutagenesis of this key cysteine to alanine abolished eNOS thiyl radical formation and uncoupled eNOS leading to increased •O2- generation. However, critical questions remain regarding the importance of this cysteine on eNOS structure and function, and the mechanism leading to eNOS S-glutathionylation. The mutation of Cys908 to Ala slightly alters the binding of FAD and FMN to the enzyme. The ratio of FAD/FMN (0.98 ± 0.02 of WT, and 0.80 ± 0.02 of Cys908A mutant) was determined using HPLC. Moreover, this mutation greatly decreases the NOS activity and cytochrome c reductase activity of the C908A (473.8 ± 13.4 nmol/mg/min of WT, and 198.9 ± 12.5 nmol/mg/min of C908A), indicating that the mutation affects electron efflux within the reductase. In the presence of glutathione, the formation of eNOS thiyl radical from the uncoupled eNOS was shown to react with glutathione to form eNOS S-glutathionylation as demonstrated by immunoblotting. This eNOS S-glutathionylation is inhibited in the presence of the radical trap DMPO, suggesting that Cys908 is involved in S-glutathionylation through thiyl radical intermediates. In the absence of glutathione, this thiyl radical reacted with vicinal cysteine to form inter-disulfide bonds leading to an increase in eNOS dimer as shown by non-reducing SDS-PAGE separation and immunoblotting analysis. Thus, eNOS protein radical formation at Cys908 provides the basis for a mechanism of •O2--directed regulation of eNOS, involving thiol oxidation; defining a unique pathway for the redox regulation of cardiovascular function.
- © 2011 by American Heart Association, Inc.