Abstract 14852: Oxidant Stress-Induced Protein Thiyl Radical Is Associated to Enhancement of Complex I S-glutathionylation in the eNOS(-/-) Murine Heart
Mitochondrial complex I catalyzes the electron transfer from NADH to ubiquinone for subsequent ATP synthesis. Complex I is one of the main superoxidegenerating sites in mitochondria. In response to oxidant stress, mitochondrial complex I is reversibly S-glutathionylated (protein S-glutathionylation, PrSSG), which can confer an inhibitory effect on mitochondrial function. To understand this mechanism, we hypothesize that PrSSG of complex I is mediated by reaction of GSH with the protein thiyl radical (PrS•) of complex I (PrS• + GS- -> PrSSG•- ; PrSSG•- + O2 -> PrSSG + O2•-). During ROS generation by isolated complex I in the presence of GSH, we detected complex I PrSSG at 51 kDa and 75 kDa subunits. Immuno-spin trapping with an anti-DMPO antibody and mass spectrometry confirmed that formation of complex I PrS• mediates PrSSG. In myocytes treated with menadione to trigger mitochondrial ROS, complex I PrSSG and PrS• were increased, supporting the hypothesis of oxidant stress induced protein thiyl radical to mediate complex I PrSSG. The hypothesis was further tested in eNOS(-/-) mouse which is known to be hypertensive and characterized by increased oxidative stress. Mitochondria isolated from eNOS(-/-) mouse heart were dysfunctional with impaired state 3 respiration (decreased by 29 %), decreased respiratory control index (from 8.5 to 3.8), and decreased enzymatic activities of electron transport chain components (by 22-37% for Complexes I-IV). Further biochemical analysis and EPR measurement indicated a defective aconitase activity (by 31%), up-regulated glutathione peroxidase/glutathione reducase activities (by 25%), and a significant increase in the superoxide generation (by 49% at state 3 conditions). These results suggested increasing ROS production and oxidant stress in the mitochondria of eNOS(-/-) mouse heart. When the complex I was immune-precipitated from the mitochondria of eNOS(-/-) myocardium, and probed with anti-GSH antibody, PrSSG was significantly enhanced, thus supporting mediation of complex I PrSSG by reactive protein thiyl radical is a unique pathway for the redox regulation of mitochondrial function.
- © 2012 by American Heart Association, Inc.