Fig. 4.
Grx2 is present as a dissociable dimeric iron-sulfur cluster complex. (A) Grx2 exists in an inactive dimeric form where the catalytic cysteine (Cys76) of each of the two monomeric Grx2 molecules is ligated reversibly to an iron moiety of the [Fe2S2] cluster. Two GSH molecules also serve as ligands. Diminution of GSH in the solution causes partial dissociation of the complex, and treatment with sodium dithionite (DT) results in full dissociation, resulting in release of two active Grx2 monomers. Superoxide radicals also are predicted to dissociate this complex, leading to activation of Grx2 under oxidative stress conditions, suggesting that the iron–sulfur cluster-bound Grx2 may function as a redox sensor in vivo[13,57]; (B) The Grx2 complex can be stabilized by mitochondrial lysis in the presence of GSH, or fully activated by sodium dithionite treatment. Following t-Bid treatment of mitochondria isolated from elderly F334 rats to release Grx1, lysates of t-Bid-treated mitochondria were analyzed for Grx2 activity (nmol/min/mg of mitochondrial protein). The mitochondrial lysates were adjusted to include either GSH (1 mM, white bars) or sodium dithionite (DT, 10 mM, black bars). The corresponding values were compared to the predicted Grx2 activity, calculated on the basis of the Grx2 content and its specific activity [31] (diagonal bar). Data represent mean±SEM, n=3; (C) and (D) Quantitative evaluation of the contribution of Grx2 to the deglutathionylation activities of (C) SSM and (D) IFM from the hearts of young adult and elderly F344 rats. Isolated heart SSM and IFM from young adult and elderly rats were lysed in RIPA buffer supplemented with either GSH (1 mM, white bar) or sodium dithionite (DT, 10 mM, black bar). The total deglutathionylation activity was then determined and the contribution of Grx2 in each case was calculated by subtracting the amount of activity attributable to Grx1 according to its known content and corresponding activity measured in previous separate experiments. Results for Grx2 are reported as mean (nmol/min/mg mitochondrial protein)±SEM (n=5 for young adult animals and n=8 for elderly animals). The values for observed Grx2 activities are compared to the predicted Grx2 activities (diagonally hatched bar), calculated from the previously determined Grx2 contents and the GRx2 specific activity.