Human Glutathione Transferase P1-1 and Nitric Oxide Carriers

Abstract

S-Nitrosoglutathione and the dinitrosyl-diglutathionyl iron complex are involved in the storage and transport of NO in biological systems. Their interactions with the human glutathione transferase P1-1 may reveal an additional physiological role for this enzyme. In the absence of GSH, S-nitrosoglutathione causes rapid and stable S-nitrosylation of both the Cys(47) and Cys(101) residues. Ion spray ionization-mass spectrometry ruled out the possibility of S-glutathionylation and confirms the occurrence of a poly-S-nitrosylation in GST P1-1. S-Nitrosylation of Cys(47) lowers the affinity 10-fold for GSH, but this negative effect is minimized by a half-site reactivity mechanism that protects one Cys(47)/dimer from nitrosylation. Thus, glutathione transferase P1-1, retaining most of its original activity, may act as a NO carrier protein when GSH depletion occurs in the cell. The dinitrosyl-diglutathionyl iron complex, which is formed by S-nitrosoglutathione decomposition in the presence of physiological concentrations of GSH and traces of ferrous ions, binds with extraordinary affinity to one active site of this dimeric enzyme (K(i) < 10(-12) m) and triggers negative cooperativity in the vacant subunit (K(i) = 10(-9) m). The complex bound to the enzyme is stable for hours, whereas in the free form and at low concentrations, its life time is only a few minutes. ESR and molecular modeling studies provide a reasonable explanation of this strong interaction, suggesting that Tyr(7) and enzyme-bound GSH could be involved in the coordination of the iron atom. All of the observed findings suggest that glutathione transferase P1-1, by means of an intersubunit communication, may act as a NO carrier under different cellular conditions while maintaining its well known detoxificating activity toward dangerous compounds.

S-Nitrosation of Glutathione Transferase P1-1 Is Controlled by the Conformation of a Dynamic Active Site Helix

“…S-Nitrosation stoichiometry was determined by UV-difference spectroscopy as described in Ref. 22 based on an S-NO extinction coefficient of 750 M Ϫ1 cm Ϫ1 at 330 nm. Synthesis of GSNO and CysNO-GSNO and CysNO were synthesized as described previously (25) and frozen in aliquots at Ϫ80°C.…”

“…Tryptophan Fluorescence-GSTP1-1 S-nitrosated at cysteines 47 and 101 has previously been discovered in vivo (19 -21) and produced in vitro (22,23) by reaction with GSNO. Here, we generated a series of cysteine mutants with the aim of examining the kinetics of nitrosation of GSTP1-1 in detail.…”

“…GSTP1-1 S-nitrosated at cysteines 47 and 101 has previously been discovered in vivo (19 -21) and produced in vitro (22,23). The modification is highly disruptive, significantly impairing the catalytic activity of the enzyme (22).…”

“…GSTP1-1 S-nitrosated at cysteines 47 and 101 has previously been discovered in vivo (19 -21) and produced in vitro (22,23). The modification is highly disruptive, significantly impairing the catalytic activity of the enzyme (22). Here, we establish a minimal mechanism for GSTP1-1 transnitrosation and denitrosation at both * This work was supported by the University of the Witwatersrand, South African National Research Foundation Grants 60810, 65510, and 68898 and South African Research Chairs Initiative of the Department of Science and Technology and National Research Foundation Grant 64788.…”

An association between resistant hypertension and the null GSTM1 genotype

“…It has been suggested that GST may act as a nitric oxide carrier in some circumstances. 11 Decreased GSTM1 expression in stroke-prone spontaneously hypertensive rats 10 may reduce antioxidant defences. Resulting lower renal GSTM1 levels may lead to increased superoxide formation and reduced nitric oxide bioavailability in the renal medulla and cortex.…”