Mass spectroscopy and molecular modeling predict endothelial nitric oxide synthase dimer collapse by hydrogen peroxide through zinc tetrathiolate metal-binding site disruption

Abstract

Endothelial nitric oxide synthase (eNOS) is inhibited by hydrogen peroxide (H(2)O(2)), but the mechanism has not been determined. Thus, the purpose of this study was to delineate the mechanism by which H(2)O(2) inhibits eNOS activity. Using mass spectroscopy, we found that the tetrathiolate cysteine residues 94 and 99 were susceptible to oxidation by H(2)O(2). Molecular modeling predicted that these cysteic acid modifications would disrupt the van der Waals interactions and the hydrogen bonding network mediated by the tetrathiolate cysteines 94 and 99 resulting in changes in quaternary structure, zinc release, and dimer collapse. Using recombinant human eNOS (heNOS) to test the predictions of the molecular modeling we found that H(2)O(2) caused disruption of the heNOS dimer and this was accompanied by zinc release and decreased NO generation. We also found that H(2)O(2) increased the oxidation of tetrahydrobiopterin (BH(4)) to dihydrobiopterin (BH(2)), whereas preincubation of heNOS with excess BH(4) prevented the destruction of zinc tetrathiolate and dimer collapse and preserved activity. Interestingly, we found that the dimmer-stabilizing effect of BH(4) is due to its ability to act as a catalase mimetic. Further, we confirmed that, in ovine aortic endothelial cells, H(2)O(2) could also induce dimer collapse and that increasing cellular BH(4) levels could maintain eNOS in its dimeric form and NO signaling when cells were challenged with H(2)O(2). This study links the inhibitory action of H(2)O(2) on heNOS through the destruction of zinc tetrathiolate metal-binding site and dimer collapse both in vitro and in vivo.

FIG. 1.

Molecular modeling of the zinc tetrathiolate metal-binding site in heNOS. (A) The model shows that cysteines 94 and 99 from each chain are directly involved in four intramolecular van der Waals interactions that are involved in the maintenance of the zinc tetrahedral atomic coordination with the cysteines 94 and 99. The cysteines 94 and 99 also appear to be involved in a network of eight H-bonds with residues N466, G101, and L100. Together these bonds maintain the symmetrical coordination of the C94 and C99 residues. (B) The effect of oxidation of cysteines 94 and 99 leading to drastic changes in the quaternary structure of the zinc tetrathiolate coordination due to the disruption of all van der Waals interactions and alterations within the H-bond network. These alterations will disrupt zinc coordination. heNOS, human endothelial nitric oxide synthase.

FIG. 2.

H₂O₂-mediated inhibition of purified heNOS in vitro. The effect of H₂O₂ (0–500 μM) on purified heNOS protein (2 μg) dimer levels were measured using LT-PAGE and western blot analysis. (A) A representative western blot is shown demonstrating that H₂O₂ dose dependently decreases the level of active heNOS dimers. (B) The bar graph shows that the dimeric level of heNOS dimer decreased dose dependently after H₂O₂ treatment. Gel filtration analysis of purified heNOS (100 μg) exposed or not to 100 μM of H₂O₂ was also carried out to confirm the LT-PAGE data. heNOS is a mixture of dimer and monomer (80:20 ratio); however, H₂O₂ treatment induces full monomerization. (C) Representative gel filtration chromatograms from three independent experiments. (D) The effect of H₂O₂ (100 μM) on the ability of heNOS to metabolize [³H]-l-arginine to [³H]-l-citrulline was also determined. The presence of H₂O₂ in the reaction mixture results in a 60% inhibition in NOS activity. Values are mean ± SD from three experiments. *p < 0.05 versus untreated, ^†p < 0.05 versus previous concentration of H₂O₂. H₂O₂, hydrogen peroxide; LT-PAGE, low-temperature polyacrylamide gel electrophoresis; SD, standard deviation.

FIG. 3.

H₂O₂ induces release of zinc from heNOS. Purified heNOS (1 mg) was treated with and without H₂O₂ (100 μM) in the presence or absence of BH₄ (100 μM) for 30 min at 37°C (in the dark) in a cuvette containing PAR (150 μM). A cuvette containing only heNOS was used to autozero the spectrophotometer. Absorbance scan spectra were recorded between 300 and 600 nm. Shown is a representative recording from three independent experiments. H₂O₂ causes a release of zinc from the heNOS dimer that was prevented by the presence of excess BH₄. BH₄, tetrahydrobiopterin; PAR, 4-(2-pyridyl (azo) resorcinol).

FIG. 4.

H₂O₂ exposure oxidizes bound BH₄ in recombinant heNOS. DTT-free pure heNOS (100 μg) was incubated with BH₄ (50 μM), unbound BH₄ was removed, and heNOS was treated with H₂O₂ (10 μM) at 37°C for 30 min. The levels of total BH₄ and BH₂ bound to the protein were determined. Results were expressed as fmol/μg protein ± SD. n = 3. *p < 0.05 versus untreated.

FIG. 5.

Increasing BH₄ levels decreases the H₂O₂-mediated heNOS dimer collapse. (A) Representative gel filtration chromatograms from three independent experiments. (B) The presence of BH₄ prevents dimer collapse induced by H₂O₂. BH₄ also attenuates the H₂O₂-mediated inhibition of [³H]-l-arginine to [³H]-l-citrulline conversion by heNOS. Values are mean ± SD from three experiments. *p < 0.05 versus untreated, ^†p < 0.05 versus no additional BH₄.

FIG. 6.

BH₄ acts as a catalase mimetic. A 10 mM H₂O₂ in 50 mM potassium phosphate buffer (pH 7.8) was incubated in the presence or absence of BH₄ (14 μM) and the rate of conversion of H₂O₂ to H₂O was determined spectrophotometrically at 240 nm (for 30 s). Data are represented as relative changes from spontaneous H₂O₂ to H₂O conversion; n = 3. *p < 0.05 versus no BH₄.

FIG. 7.

H₂O₂ exposure disrupts the eNOS dimer in ovine PAECs. PAECs were exposed to H₂O₂ (100 μM) for 15 min. Protein extracts were then prepared and separated on 7.5% polyacrylamide gels using LT-PAGE, electrophoretically transferred to Hybond membranes, and analyzed using a specific antiserum raised against eNOS. (A) A representative western blot is shown. (B) The densitometric values demonstrate that H₂O₂ decreases the level of eNOS dimers in PAECs and decreases the NO generation (C) in response to laminar shear stress (5 min, 20 dyn/cm²). (D) BH₄ levels (expressed as pmol/mg protein) are also decreased in cells exposed to H₂O₂. Values are mean ± SD; n = 3–6. *p < 0.05 versus untreated. ^†p < 0.05 versus no H₂O₂. PAECs, pulmonary arterial endothelial cells.

FIG. 8.

Increasing BH₄ levels decrease H₂O₂-mediated reduction in eNOS activity in ovine PAECs. PAECs were treated or not with the BH₄ precursor sepiapterin (100 μM) for 18 h and then exposed to H₂O₂ (100 μM) for 15 min. Protein extracts were then prepared and separated on 7.5% polyacrylamide gels via LT-PAGE, then electrophoretically transferred to Hybond membranes, and analyzed using a specific antiserum raised against eNOS. (A) A representative western blot is shown. (B) The densitometric values demonstrate that BH₄ reduces the H₂O₂-mediated decrease in eNOS dimers. Cells were also treated or not with the BH₄ precursor sepiapterin (100 μM) for 18 h in the presence or absence of N-acetylserotonin (NAS) (10 μM). Cells were then treated with H₂O₂ (100 μM) for 15 min and then exposed to laminar shear stress (20 dyn/cm²) for 5 min. Results are presented as a percentage of NO_x release from sheared cells not exposed to H₂O₂ or sepiapterin. Addition of sepiapterin increases NO production by PAECs both in the presence and absence of H₂O₂ (C). Data are mean ± SD; n = 6. *p < 0.05 versus sheared alone, ^†p < 0.05 versus H₂O₂ alone.