The impact of nitric oxide toxicity on the evolution of the glutathione transferase superfamily: a proposal for an evolutionary driving force

Abstract

Glutathione transferases (GSTs) are protection enzymes capable of conjugating glutathione (GSH) to toxic compounds. During evolution an important catalytic cysteine residue involved in GSH activation was replaced by serine or, more recently, by tyrosine. The utility of these replacements represents an enigma because they yield no improvements in the affinity toward GSH or in its reactivity. Here we show that these changes better protect the cell from nitric oxide (NO) insults. In fact the dinitrosyl·diglutathionyl·iron complex (DNDGIC), which is formed spontaneously when NO enters the cell, is highly toxic when free in solution but completely harmless when bound to GSTs. By examining 42 different GSTs we discovered that only the more recently evolved Tyr-based GSTs display enough affinity for DNDGIC (KD < 10(-9) M) to sequester the complex efficiently. Ser-based GSTs and Cys-based GSTs show affinities 10(2)-10(4) times lower, not sufficient for this purpose. The NO sensitivity of bacteria that express only Cys-based GSTs could be related to the low or null affinity of their GSTs for DNDGIC. GSTs with the highest affinity (Tyr-based GSTs) are also over-represented in the perinuclear region of mammalian cells, possibly for nucleus protection. On the basis of these results we propose that GST evolution in higher organisms could be linked to the defense against NO.

Keywords: Dinitrosyl Iron Complex; Enzyme Inhibitors; Enzyme Structure; Enzymes; Evolution; Glutathione Transferase; Nitric Oxide.

FIGURE 1.

Formula of DNDGIC and structures of GSH bound to GSTs. A, chemical structure of DNDGIC. B, superimposition of GSH in the crystallographic structures of GSTs (for the 20 Tyr-GSTs, 14 Ser-GSTs, and 8 Cys-GSTs listed in Table 2). Catalytic residues are also shown. The only divergent crystal structures are hGSTZ1-1 (a Ser-GST) and hGSTO1-1 (a Cys-GST).

FIGURE 2.

Orientation of the catalytic residue and kinetic constants for the three GSTs subfamilies. A, representative structures for Tyr-GSTs (PDB code 1K3L) (red), Ser-GSTs (PDB code 3LJR) (green), and Cys-GSTs (PDB code 1EEM) (blue). The segment of protein backbone containing the catalytic residue is shown in a ribbon representation, whereas GSH is reported as sticks. The Tyr residue is located on the β1 strand, whereas Ser and Cys residues are on the β1α1 loop. B, experimental K_m values for GSH binding to Tyr-GSTs (red circle), Ser-GSTs (green square), and Cys-GSTs (blue triangle); starting from the left: data refer to GSTs in the same sequence as reported in Table 1 (excluding the five mutants). C, experimental K_D values for DNGIC/GST interaction. Tyr-GSTs (red circle), PfGST (red open circle), Ser-GSTs (green square), Cys-GSTs (blue triangle). For a few GSTs lacking detectable amounts of bound DNGIC, only a limiting lower value of K_D is reported. Red arrow indicates that the true K_D could be even higher (red arrow ↓) or lower (red arrow ↑). Starting from left: data refer to GSTs in the same sequence as that reported in Table 1 (excluding the five mutants).

FIGURE 3.

EPR experiments. EPR spectra at 22–25 °C of DNGIC bound to representative GSTs belonging to Tyr-GSTs (hGSTA1-1 and hGSTM2-2), Ser-GSTs (GSTD1-3, GSTD4-4), and Cys-GSTs (hGSTO1-1 and OaGST). Experiments were performed as described under “Experimental Procedures.”

FIGURE 4.

Experimental and theoretical studies of DNGIC binding to GSTs. A, DNGIC·GST complex structure as obtained from DFT calculations (green) superimposed on the crystallographic model (red) (PDB code 1ZGN). B, structures of DNGIC with Tyr (red), Ser (green), and Cys (blue) as fourth ligands, obtained from DFT calculations. C, free energy of the GST/DNGIC interaction obtained from docking calculations. Tyr-GSTs (red circle), Ser-GSTs (green square), and Cys-GSTs (blue triangle). D, free energies of GST/DNGIC interaction as in C were corrected for the energy of the coordination bond involving the Tyr residue (−4.1 kcal/mol for a Tyr with a pK_a = 9.8 as in GSTP1-1) and the Cys residue (−3.3 kcal/mol for a Cys with pK_a = 7.3 as in GSTB1-1). Coordination energy due to Tyr or Cys residues with different pK_a have been calculated as described under “Experimental Procedures.” Symbols are as described in C. Black symbols are experimental K_D values, whereas the corresponding theoretical K_D values are reported with the same vertical alignment. Starting from left, data refer to GSTs in the same sequence of that reported in Table 1 (excluding the five mutants), red open circle and black open circle are the theoretical and experimental energy values for the atypical PfGST. Red arrow indicates that the true ΔG⁰ value could be even more (red arrow ↓) or less (red arrow ↑) negative (higher or lower affinity, respectively).

FIGURE 5.

The role of protein charge. A, dependence of the affinity of GSTs for DNDGIC as a function of pI values for Tyr-GSTs (red circle), PfGST (red open circle) (r² = 0.92; p < 0.001) excluding the anomalous PfGST. B, correlation between pI and pK_a values of the catalytic residues of Tyr-GSTs. Linear regression shows positive correlation (r² = 0.78, p = 0.009). Tyr-GSTs, red circle; PfGST, red open circle. Ser- and Cys-GSTs did not show correlations. C, coulombic potential on the dimer surface of selected Tyr-GSTs facing the two G-sites (models on left) and opposite to the G-sites (models on right). Glutathione is represented as yellow spheres. The protein surface is colored based on the electrostatic potential values, as red (negative values), blue (positive values), or white (near zero values) in the range −5 to +5 kcal/(mol·e). D, “traffic light” graph. Predictive dependence of GR inhibition (%) in the presence of a stoichiometric amount of GSTs (site at high affinity) and DNDGIC, as a function of experimental K_D for DNDGIC. On the basis of the K_D, the unbound DNDGIC and then the percentage of GR inhibition (K_i = 3 μm) (continuous line) (9, 10) were calculated. Good protection is assumed for inhibition less than 10%. Low or no protection is assumed for inhibition >30%. Tyr-GST (red circle), PfGST (black open circle), Ser-GST (green square), and Cys-GST (blue triangle). Arrow indicates that K_D measured by EPR experiments could be even lower (←) or higher (→). See also Table 1.