Heme-coordinating inhibitors of neuronal nitric oxide synthase. Iron-thioether coordination is stabilized by hydrophobic contacts without increased inhibitor potency

Abstract

The heme-thioether ligand interaction often occurs between heme iron and native methionine ligands, but thioether-based heme-coordinating (type II) inhibitors are uncommon due to the difficulty in stabilizing the Fe-S bond. Here, a thioether-based inhibitor (3) of neuronal nitric oxide synthase (nNOS) was designed, and its binding was characterized by spectrophotometry and crystallography. A crystal structure of inhibitor 3 coordinated to heme iron was obtained, representing, to our knowledge, the first crystal structure of a thioether inhibitor complexed to any heme enzyme. A series of related potential inhibitors (4-8) also were evaluated. Compounds 4-8 were all found to be type I (non-heme-coordinating) inhibitors of ferric nNOS, but 4 and 6-8 were found to switch to type II upon heme reduction to the ferrous state, reflecting the higher affinity of thioethers for ferrous heme than for ferric heme. Contrary to what has been widely thought, thioether-heme ligation was found not to increase inhibitor potency, illustrating the intrinsic weakness of the thioether-ferric heme linkage. Subtle changes in the alkyl groups attached to the thioether sulfur caused drastic changes in the binding conformation, indicating that hydrophobic contacts play a crucial role in stabilizing the thioether-heme coordination.

Figure 1

Ferric and ferrous difference spectra for 3 and 4. PANEL A: Titration of ferric nNOS-BH₄ with 3. PANEL B: Titration of ferrous nNOS-BH₄ with 3. PANEL C: Titration of ferric nNOS-imidazole with 4 (the concentration of imidazole was 300 μM). PANEL D: Titration of ferrous nNOS-BH₄ with 4. In each chart, the absorption spectrum of either heme domain nNOS-BH₄ (Panels A, B, and D) or heme domain nNOS-imidazole (Panel C) was set as the baseline. Aliquots of inhibitor solution were added sequentially; each curve represents the difference spectrum after addition of a particular aliquot. Spectra were normalized to zero absorbance at 420 (Panel A), 427 (B, D), and 415 nm (C), respectively. In each chart, distinct ΔA_max and ΔA_min values were observed, indicating conversion of the heme iron from high spin to low spin (Panels A, B, and D) or from low spin to high spin (Panel C). The insets are plots of (ΔA_max - ΔA_min) versus inhibitor concentration. The value (ΔA_max - ΔA_min) increased logarithmically with increasing concentration, as expected for binding isotherms. Hanes-Woolf plots were constructed for (inhibitor concentration)/(ΔA_max - ΔA_min) versus inhibitor concentration; the negative x-intercepts of the linear regressions were taken as the spectral constants (K_s). Based on the data shown above and data from replicate experiments, K_s values of 34 ± 2 (Panel A), 15.5 ± 0.1 (B), 15 ± 3 (C), and 8 ± 1 μM (D) were calculated. For ferric 4-nNOS, the K_s value was calculated from the apparent K_s value as described. Ferrous difference spectra were obtained under anaerobic conditions, and nNOS was reduced using 2-10 mM sodium dithionite. All titrations were performed in 100 mM Hepes buffer at pH 7.5. The concentration of nNOS heme domain was 3.75 μM in the ferric titrations and 2.4 μM in the ferrous titrations. For each titration, the total volume change was < 2%. The data shown are representative of at least two replicate experiments.

Figure 2

Absolute absorption spectra in the ferric and ferrous oxidation states for Panel A) 3 -nNOS; B) 4 -nNOS; C) 8 -nNOS; and D) H₄B -nNOS with no inhibitor present as a control. In each panel, the solid line represents the ferric spectrum, and the dotted line represents the ferrous spectrum.

Figure 3

Active site binding conformations for 1 (panel D), 3 (A), 4 (B), and 6 (C) based on the refined X-ray crystal structures. Note that for 3 and 6 alternate conformations were observed, shown with two different colors. For 1 and 4, a single conformation was observed. The hydrogen bonds are depicted with dashed lines. The mean Fe-S distance from the two subunits for 3, 4, and 6 is labeled. The 2Fo-Fc electron density map contoured at 1 σ is also displayed around the bound inhibitor and the heme iron.

Scheme 1