129 - Cardiolipin Interactions with Cytochrome C increase Tyrosine Nitration Yields and Site-Specificity

Abstract

Cytochrome c is a small protein that functions as carrier in the mitochondrial respiratory chain and pro-apoptotic mediator in the cytosol. This protein can also act as a peroxidase when exposed to high doses of hydrogen peroxide; when interacting with cardiolipin, a phospholipid from the inner mitochondrial membrane or after its oxidative post-translational modification by tyrosine nitration. Since mitochondria are a key place of intra cellular-generated peroxynitrite, the nitration of cytochrome c becomes a biologically-relevant event. In this work, we studied the effect of cardiolipin on cytochrome c tyrosine nitration and free radical formation when exposed to peroxynitrite, as well as the effect of a single residue, Y67, in the overall nitration. The effect of cardiolipin in cytochrome c exposed to peroxynitrite was studied by EPR analysis, showing an increase of free radicals when the protein was in presence of cardiolipin-containing liposomes. This increase of free radical formation was translated into an increase of tyrosine nitration, determined by western blot and proteomic analyses, in particular Y67. Also, studies were performed in order to establish the effect of nitration in the binding constant of cytochrome c with cardiolipin. Our results showed an increase of the binding constant when peroxynitrite was present. In order to determine the importance of Y67 in the reaction of peroxynitrite with cytochrome c in presence of cardiolipin, a mutant lacking this residue was used (Y67F). Notably, all differences observed using WT cyt c were abolished when using the Y67F mutant, suggesting that this residue, very close to the heme moiety of the protein, was responsible for the differences in reactivity and nitration observed. Also, when measuring the binding constant in presence of the Y67F mutant, no difference was observed upon addition of peroxynitrite to the reaction mixture. Our results support that a structural change induced by cardiolipin in cytochrome c ( i.e. weakening of the 6th coordination bond at the heme) increases tyrosine nitration yields, particularly in the heme-adjacent tyrosine 67, suggesting an iron-catalyzed event.