250 - Peroxiredoxin 1 Coordination to the Dinitrosyl Iron Complex of Glutathione

Abstract

Protein S-nitrosation is a reversible post-translational modification that has been associated with a variety of physiological and pathophysiological processes. The mechanisms responsible for S-nitrosation in vivo, however, remain debatable. Dinitrosyl iron complexes (DNIC), which are consistently detected in cell and tissues overproducing nitric oxide, have been proposed as possible S-nitrosating agents in vivo. On the other hand, several reports have described Peroxiredoxins (Prx) S-nitrosation in vivo. Therefore, we became interested in examining whether Prx1 nitrosation was promoted by the mono dinitrosyl iron complex of glutathione ([Fe(NO)2(GS)2]+) equilibrated with its binuclear form ([Fe2(NO)4(GS)2]4+). Apparently, the complex did not promote Prx1 nitrosation. Instead, Prx1 coordinated to the complex producing a high molecular weight complex as attested by EPR experiments at room temperature. An axial EPR signal (g┴=2.03 and g║=2.01) was observed initially, but it became rhombic (gx=2.04; gy=2.03; gz=2.01) with time. Experiments with Prx1 mutants (Prx1C52S, Prx1C83SC173S) and with Prx1 treated with a His modifying reagent showed that Cys52 is responsible for the axial signal and for about 80% of Prx1-DNIC produced, whereas a His residue is responsible for the rhombic signal. The second order rate constant for Prx1-DNIC formation was calculated as k = (6.95±0.42) M-1s-1 at pH 7.4 and 25○C. As anticipated, formation of the Prx1-DNIC complex resulted in the complete inactivation of the peroxidase activity of the enzyme. In addition, SEC experiments demonstrated that Prx1-DNICcomplex shifted the Prx1 dimer-decamer equilibrium to the dimeric state. Reaction of Prx1-DNIC with GSTP1-1, which has been previously shown to bind rapidly and avidly to DNIC, resulted in Prx1 displacement from the complex. In contrast, BSA-DNIC and Prx1-DNIC equilibrated. These results and the reported stability of the DNIC-GSTP1-1 complex argue against the possibility of cellular DNIC complexes acting as nitrosating agents.