Nitrosylation of Human Glutathione Transferase P1-1 with Dinitrosyl Diglutathionyl Iron Complex in Vitro and in Vivo

Abstract

We have recently shown that dinitrosyl diglutathionyl iron complex, a possible in vivo nitric oxide (NO) donor, binds with extraordinary affinity to one of the active sites of human glutathione transferase (GST) P1-1 and triggers negative cooperativity in the neighboring subunit of the dimer. This strong interaction has also been observed in the human Mu, Alpha, and Theta GST classes, suggesting a common mechanism by which GSTs may act as intracellular NO carriers or scavengers. We present here the crystal structure of GST P1-1 in complex with the dinitrosyl diglutathionyl iron ligand at high resolution. In this complex the active site Tyr-7 coordinates to the iron atom through its phenolate group by displacing one of the GSH ligands. The crucial importance of this catalytic residue in binding the nitric oxide donor is demonstrated by site-directed mutagenesis of this residue with His, Cys, or Phe residues. The relative binding affinity for the complex is strongly reduced in all three mutants by about 3 orders of magnitude with respect to the wild type. Electron paramagnetic resonance spectroscopy studies on intact Escherichia coli cells expressing the recombinant GST P1-1 enzyme indicate that bacterial cells, in response to NO treatment, are able to form the dinitrosyl diglutathionyl iron complex using intracellular iron and GSH. We hypothesize the complex is stabilized in vivo through binding to GST P1-1.

Dinitrosyliron complexes and the mechanism(s) of cellular protein nitrosothiol formation from nitric oxide

“…ROS and RNS have been shown to modulate intracellular iron (32). The effect of BSO also shows that the CIP-dependent nitrosative pathway is unaffected by changes in GSH or other LMW thiol ( Table 1), indicating that GSH-DNICs (33) are not obligatory intermediates, nor are other products of the interaction between GSH and the CIP.…”

Nitric Oxide Storage and Transport in Cells Are Mediated by Glutathione S-Transferase P1-1 and Multidrug Resistance Protein 1 via Dinitrosyl Iron Complexes

“…GST P1-1 Inhibits 59 Fe Release from Cells Hyper-expressing MRP1-Although GSTs bind DNICs (14,15), their role in iron and NO metabolism in intact mammalian cells has never been assessed, particularly in the context of their relationship with MRP1. Considering that GSTs bind DNICs (14,15), MCF7-VP cells transfected with GSTA1, GSTM1, or GSTP1 (VP␣, VP, or VP) and their relevant controls (VP Vector or VP Vector*) ( Fig.…”

“…Subsequently, we discovered that the glutathione (GSH) transporter, multidrug resistance protein 1 (MRP1), mediates NO-stimulated iron and GSH efflux from cells and that this process could be inhibited by L-buthionine-(S,R)-sulfoximine (BSO) (12), an inhibitor of GSH synthesis (13). Several recent investigations have suggested that DNICs such as dinitrosyl-diglutathionyl iron complexes can bind to glutathione S-transferase (GST) isoenzymes with high affinity (K d ϭ 10 Ϫ7 -10 Ϫ10 M) (14,15). In fact, a crystal structure of the DNIC-GST P1-1 complex (PDB ID: IZGN) revealed that Tyr-7 in the active site of the enzyme coordinates to iron in the DNIC, displacing one GSH ligand (14).…”

“…Several recent investigations have suggested that DNICs such as dinitrosyl-diglutathionyl iron complexes can bind to glutathione S-transferase (GST) isoenzymes with high affinity (K d ϭ 10 Ϫ7 -10 Ϫ10 M) (14,15). In fact, a crystal structure of the DNIC-GST P1-1 complex (PDB ID: IZGN) revealed that Tyr-7 in the active site of the enzyme coordinates to iron in the DNIC, displacing one GSH ligand (14). Previous studies with toxic exogenous (e.g.…”

Mass spectrometry as a powerful tool to study therapeutic metallodrugs speciation mechanisms: Current frontiers and perspectives

“…[207,208] Casini, Lo Bello et al reported on the use of several techniques including MS to study the interaction between auranofin and GST P1-1. [209] Auranofin was found to inhibit the activity of the protein in the same IC 50 values range than a known organic inhibitor (ethacrynic acid), and the molecular mechanism of inhibition was studied by ESI-FT-ICR-MS. After 30 min incubation with P1-1, adducts corresponding to [GST P1-1+Au(PEt) 3 ] and [GST P1-1+2(Au(PEt) 3 )] were detected and were found stable overtime, indicating that at least two gold binding sites are accessible.…”