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Spectroscopic and rapid kinetic experiments were performed to detail the interaction of human glutathione S-transferases GSTA1-1, GSTM2-2, and GSTP1-1 with 6-(7-nitro-2,1,3-benzoxadiazol-4-ylthio)hexanol (NBDHEX). This compound is a representative molecule of a new class of 7-nitro-2,1,3-benzoxadiazole (NBD) derivatives (non-GSH peptidomimetic compounds) that have been designed both to give strong GST inhibition and to accumulate in tumor cells avoiding the extrusion mechanisms mediated by the multidrug resistance protein pumps. We have recently shown that submicromolar amounts of NBDHEX trigger apoptosis in several human tumor cell lines through the dissociation of the JNK⅐GSTP1-1 complex (Turella, P., Cerella, C., Filomeni, G., Bullo, A., De Maria, F., Ghibelli, L., Ciriolo, M. R., Cianfriglia, M., Mattei, M., Federici, G., Ricci, G., and Caccuri, A. M. (2005) Cancer Res. 65, 3751-3761). Results reported in the present study indicated that NBDHEX behaves like a suicide inhibitor for GSTs. It bound to the H-site and was conjugated with GSH forming a complex at the C-4 of the benzoxadiazole ring. This complex was tightly stabilized in the active site of GSTP1-1 and GSTM2-2, whereas in GSTA1-1 the release of the 6-mercapto-1-hexanol from the complex was the favored event. Docking studies demonstrated the likely localization of the complex in the GST active sites and provide a structural explanation for its strong stabilization.

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.

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Selected 7-nitro-2,1,3-benzoxadiazole derivatives have been recently found very efficient inhibitors of glutathione Stransferase (GST) P1-1, 5 an enzyme which displays antiapoptotic activity and is also involved in the cellular resistance to anticancer drugs. These new inhibitors are not tripeptide glutathione-peptidomimetic molecules and display lipophylic properties suitable for crossing the plasma membrane. In the present work, we show the strong cytotoxic activity of these compounds in the following four different cell lines: K562

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.

Presteady-state and steady-state kinetic studies performed on human glutathione transferase P1-1 (EC 2.5.1.18) with 1-chloro-2,4-dinitrobenzene as co-substrate indicate that the rate-determining step is a physical event that occurs after binding of the two substrates and before the -complex formation. It may be a structural transition involving the ternary complex. This event can be related to diffusion-controlled motions of protein portions as k cat°/ k cat linearly increases by raising the relative viscosity of the solution. Similar viscosity dependence has been observed for K m GSH , while K m CDNB is independent. No change of the enzyme structure by viscosogen has been found by circular dichroism analysis. Thus, k cat and K m GSH seem to be related to the frequency and extent of enzyme structural motions modulated by viscosity. Interestingly, the reactivity of Cys-47 which can act as a probe for the flexibility of helix 2 is also modulated by viscosity. Its viscosity dependence parallels that observed for k cat and K m GSH , thereby suggesting a possible correlation between k cat , K m GSH, and diffusioncontrolled motion of helix 2. The viscosity effect on the kinetic parameters of C47S and C47S/C101S mutants confirms the involvement of helix 2 motions in the modulation of K m GSH, whereas a similar role on k cat cannot be ascertained unequivocally. The flexibility of helix 2 modulates also the homotropic behavior of GSH in these mutants. Furthermore, fluorescence experiments support a structural motion of about 4 Å occurring between helix 2 and helix 4 when GSH binds to the G-site.Human placental glutathione transferase P1-1 (GST) 1 (EC 2.5.1.18) is a dimeric enzyme composed of two identical subunits each containing one binding site for GSH (G-site) and a second binding site for the electrophilic co-substrate (H-site). Inspection of the three-dimensional structure indicates the presence near the G-site of the irregular ␣ helix 2 (residues 37-46) which is exposed to the solvent (1). Lys-44, a part of this helix, is involved in the binding of GSH. At the end of helix 2 is Cys-47 which is probably linked by ion pair formation with Lys-54. This electrostatic interaction seems important for the correct spatial arrangement of the G-site (2); lack of this bond by replacement of Cys-47 or Lys-54 with Ser or Ala lowers the affinity for GSH and triggers a positive cooperativity toward the binding of GSH (3, 4). We therefore suggested that Cys-47 acts as a hinge which limits the extent or frequency of conformational transitions involving helix 2 (3, 4). In its absence, helix 2 would become more flexible and contact the adjacent subunit via helix 4 thereby inducing the observed cooperativity (4). Several pieces of evidence indicate that the irregular ␣ helix 2 is a flexible region even in the wild-type enzyme; it displays the highest temperature factors among all other regions of domain I (5); moreover, it offers the sole point of attack (Lys-44) for the proteolytic cleavage by trypsin (5). Finally, the strongest evide...

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 (