Abstract 16144: MitochondriaL Oxidant Stress Enhances S-Glutathionylation OF the Flavin Subcomplex of Complex I in the Post-ischemic Heart
Complex I (NQR) is a critical site of superoxide (O2•-) production and the major site for redox control in mitochondria. Previously we found that S-glutathionylation (S-glut) of NQR can occur in vitro at the FMN-binding subunit (51 kDa subunit). The S-glutathionylated domain from the 51 kDa subunit of the complex I was identified to be 200GAGAYIC206GEETALIESIEGK219, however, the functional consequences of this modification, and whether it occurs under physiological conditions remain unknown. First, to elucidate functional consequences of protein S-glut at C206, we used the peptide of 200GAGAYIC206GEETALIESIEGK219 to generate a polyclonal antibody, AbGSCA206. AbGSCA206 inhibited complex I-mediated O2•- generation by 37% as measured by EPR spin-trapping with DEPMPO. As a control, the peptide of non-glutathionylated domain (21SGDTTAPKKTSFGSLKDFDR40 of 51-kDa subunit), Ab51, did not affect complex I O2•- generation. Neither AbGSCA206 nor Ab51 inhibited electron transfer activity of complex I, suggesting that the S-glut domain is critical for O2•- generation by complex I. Second, to understand if S-glut of complex I occurs in cells or hearts subjected to mitochondrial oxidative stress, we initially treated cardiomyocytes (HL-1 cell line) with the uncoupler menadione to increase O2•- production. Immunofluorescence confocal microscopy with Ab51 and a monoclonal antibody against glutathione (AbGSH) revealed significant S-glut staining in the 51 kDa subunit of complex I. To ascertain if S-glut occurred in the intact heart subjected to oxidative stress, rats were subjected to 30 min of myocardial ischemia followed by 24 h of reperfusion. Myocardial tissue homogenates were immunoprecipitated with Ab51 and probed with AbGSH; S-glut at the 51 kDa subunit of complex I was enhanced by 60.8 ± 5.7% in post-ischemic myocardium. These results show that increasing mitochondrial oxidative stress resulting from myocardial infarction or from chemical uncoupling induces S-glut at the 51 kDa flavoprotein of the complex I. Our speculation is that S-glutathionylation of complex I is an endogenous mechanism that protects against mitochondrial damage as it would reduce the amount of ROS being generated under conditions of mitochondrial oxidative stress.
- © 2010 by American Heart Association, Inc.