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.
Highlights
The remaining iron-bound GSH ligand binds in the G-site in an almost identical manner to that observed in the structure of the GSH complex of human GST P1-1 [26]
In this paper we provide compelling evidence that GSTs can bind NO donors at the active site using as a fourth ligand, the catalytic Tyr (Tyr-7 in GST P1-1), confirming the hypotheses proposed in previous papers (9 –11)
The structure of this NO donor bound at the active site shows that 1) one GSH ligand is displaced and that the remaining GSH ligand of the complex binds in the G-site in an almost identical manner to that of the structure of GSH bound to GST; 2) iron binds in the complex in a distorted tetrahedral geometry with angles between the four iron ligands, Tyr-7, GSH, and the two nitroso groups, between 101° and 118°; 3) the protein side chain that is involved in covalent attachment with the complex is Tyr-7; 4) that other significant residues involved in the binding of the complex are Tyr-108 and Asn204, both of which form water-mediated hydrogen bonds with the oxygen of one of the nitroso groups
Summary
To 50 l of 100 mM MES buffer, pH 6.0, and 200 l of 100 mM GSH, 20 l of 100 mM GSNO was added. Dinitrosyl Diglutathionyl Iron Complex Soak—Wild-type crystals were transferred into a new drop containing 200 l of the DNDGIC solution described above, 100 mM MES buffer, pH 6.0, 22% (w/v) polyethylene glycol 8000, and 20 mM CaCl2. These crystals were soaked for several days. After multiple rounds of refinement and rebuilding the final R-factor was 18.2% (Rfree ϭ 24.4%) for all data to 2.1 Å of resolution. The stereochemistry was analyzed with the program PROCHECK [34] and gave values either similar or better than expected for structures refined at similar resolu-
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