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 phys iological role of this process. By using EPR spectroscopy and enzyme activity measurements to study the mechanism in hepa tocytes, we here identify the complex as a dinitrosyl-diglutathio nyl-iron complex (DNDGIC) bound to Alpha class glutathione S-transferases (GSTs) with extraordinary high affinity (KD 10 10 M). This complex is formed spontaneously through NO mediated extraction of iron from ferritin and transferrin, in a reaction that requires only glutathione. In hepatocytes, DNDGIC may reach concentrations of 0.19 mM, apparently entirely bound to Alpha class GSTs, present in the cytosol at a concentration of about 0.3 mM. Surprisingly, about 20% of the dinitrosyl-glutathionyl-iron complex-GST is found to be asso ciated with subcellular components, mainly the nucleus, as demonstrated in the accompanying paper (Stella, L., Pallottini, V., Moreno, S., Leoni, S., De Maria, F., Turella, P., Federici, G., Fabrini, R., Dawood, K. F., Lo Bello, M., Pedersen, J. Z., and Ricci, G. (2007) J. Biol. Chem. 282, 6372– 6379). DNDGIC is a potent irreversible inhibitor of glutathione reductase, but the strong complex-GST interaction ensures full protection of glu tathione reductase activity in the cells, and in vitro experiments show that damage to the reductase only occurs when the DNDGIC concentration exceeds the binding capacity of the intracellular GST pool. Because Pi class GSTs may exert a simi lar role in other cell types, we suggest that specific sequestering of DNDGIC by GSTs is a physiological protective mechanism operating in conditions of excessive levels of nitric oxide.