Abstract 152: Site-Specific Protein S-glutathiolation Modulates the Enzymatic Function of Mitochondrial Complex I by Increasing Electron Transfer Activity and Decreasing Electron Leakage for Superoxide Production
The generation of reactive oxygen species in mitochondria acts as a redox signal in triggering cellular events such as apoptosis, proliferation, and senescence. Overproduction of superoxide (O2· −) and O2· −-derived oxidants change the redox status of the mitochondrial GSH pool. Mitochondria contains high concentrations of protein thiols (20–25 nmol exposed thiols/mg protein) and GSH (5–10 mM or 3–5 nmol thios/mg protein), which control the redox state of mitochondria. An early response of protein thiols to mitochondrial oxidative stress is to reversibly form GSH-protein mixed disulfide via protein S-glutathiolation. Physiologically this redox modification has been implicated to play an antioxidant role and an adaptive response to combat oxidative injury in cells. Complex I is the major host of regulatory protein thiols in mitochondria. The Complex I isolated from heart mitochondria after exposure to oxidized GSH, GSSG (1–3 mM), resulted in specific S-glutathiolation at the 51 kDa and 75 kDa subunits as detected by anti-GSH monoclonal antibody. To investigate the molecular mechanism of S-glutathiolation of Complex I, we employed the techniques of mass spectrometry and EPR spin trapping for analysis. The gel bands of 51 kDa and 75 kDa polypeptides were cut, and digested with trypsin, and then subjected to the analysis of tandem mass spectrometry (LC/MS/MS). Sequence coverage of 87% for 51 kDa and 71% for 75 kDa were obtained. LC/MS/MS analysis of tryptic digests revealed that two specific cysteines (C206 and C187) of the 51 kDa subunit and one specific cysteine (C367) of the 75 kDa subunit were involved in redox modifications with GS binding. The electron transfer activity (ETA) of S-glutathiolated Complex I (GS-Complex I) in catalyzing NADH oxidation by ubiquinone-1 (Q1) was significantly enhanced (8–25% increase). However, O2· − generation (SGA) mediated by GS-Complex I suffered a mild loss (10–32% inhibition) as measured by EPR spin trapping with 5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide (DEPMPO). In conclusion, site-specific protein S-glutathiolation of Complex I at 51 kDa and 75 kDa subunits modulates its enzymatic function by increasing the electron transfer efficiency and decreasing the electron leakage for O2·− production.