Glutathione Transferases Sequester Toxic Dinitrosyl-Iron Complexes in Cells

Abstract

It is now well established that exposure of cells and tissues to nitric oxide leads to the formation of a dinitrosyl-iron complex bound to intracellular proteins, but little is known about how the complex is formed, the identity of the proteins, and the physiological role of this process. By using EPR spectroscopy and enzyme activity measurements to study the mechanism in hepatocytes, we here identify the complex as a dinitrosyl-diglutathionyl-iron complex (DNDGIC) bound to Alpha class glutathione S-transferases (

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

“…These results at 293 K were consistent with the formation of a specific DNIC-GST P1-1 protein complex (g ϭ 2.04) (14,27) and support the EPR studies done at 77 K that suggest the formation of a chemically distinct DNIC in the presence of GST P1-1 (Fig. 5A, panel iii, cf.…”

“…5A, panel x, marked by arrows) in the signals after incubation with GSNO. These findings were consistent with the formation of a specific DNIC-GST P1-1 protein complex (g ϭ 2.04) (14,27).…”

“…Further, GST enzymes bind DNICs with very high affinity and could act as a storage form of NO for regulating intracellular processes (14,27). Most of these previous studies examining DNIC-GST complexes have been performed on purified proteins (14, 27), and none of these have examined the cooperative role of GSTs and MRP1 on intracellular iron trafficking and release in mammalian cells.…”

“…Most of these previous studies examining DNIC-GST complexes have been performed on purified proteins (14, 27), and none of these have examined the cooperative role of GSTs and MRP1 on intracellular iron trafficking and release in mammalian cells. However, EPR studies by Pedersen et al (27) using cultured rat hepatocytes and liver homogenates showed that DNICs may bind to ␣ class GSTs. This is inconsistent with our current work, where GST A1-1 hyper-expression did not influence NO-mediated 59 Fe release (Fig.…”

“…4, EPR (7,12) at 77 K was used to assess the formation of cellular DNICs after exposure to GSNO (0.5 mM) for 3 h at 37°C. As a control, we first examined the EPR signal of a synthetically prepared (15) low M r DNIC, containing GSH (dinitrosyl-diglutathionyl iron complex), which gives the characteristic spectrum of this complex with g ϭ 2.04 (15,27) (Fig. 5A, panel i).…”