Mechanism and kinetics of inducible nitric oxide synthase auto-S-nitrosation and inactivation

Abstract

Nitric oxide (NO), the product of the nitric oxide synthase (NOS) reaction, was previously shown to result in S-nitrosation of the NOS Zn(2+)-tetrathiolate and inactivation of the enzyme. To probe the potential physiological significance of NOS S-nitrosation, we determined the inactivation time scale of the inducible NOS isoform (iNOS) and found it directly correlates with an increase in the level of iNOS S-nitrosation. A kinetic model of NOS inactivation in which arginine is treated as a suicide substrate was developed. In this model, NO synthesized at the heme cofactor is partitioned between release into solution (NO release pathway) and NOS S-nitrosation followed by NOS inactivation (inactivation pathway). Experimentally determined progress curves of NO formation were fit to the model. The NO release pathway was perturbed through addition of the NO traps oxymyoglobin (MbO(2)) and β2 H-NOX, which yielded partition ratios between NO release and inactivation of ~100 at 4 μM MbO(2) and ~22000 at saturating trap concentrations. The results suggest that a portion of the NO synthesized at the heme cofactor reacts with the Zn(2+)-tetrathiolate without being released into solution. Perturbation of the inactivation pathway through addition of the reducing agent GSH or TCEP resulted in a concentration-dependent decrease in the level of iNOS S-nitrosation that directly correlated with protection from iNOS inactivation. iNOS inactivation was most responsive to physiological concentrations of GSH with an apparent K(m) value of 13 mM. NOS turnover that leads to NOS S-nitrosation might be a mechanism for controlling NOS activity, and NOS S-nitrosation could play a role in the physiological generation of nitrosothiols.

Figure 1

iNOS S-nitrosation and inactivation during enzymatic turnover. (A) iNOS S-nitrosation during enzymatic turnover. Each reaction contained 4 μM iNOS, 5 mM Arg, 2 mM NADPH, 50 mM NaCl, and 50 mM HEPES pH 7.5. iNOS activity was initiated by NADPH addition and 100 μL aliquots were taken at the indicated timepoints and quenched with an equal volume of 250 mM HEPES at pH 7.4 containing 2 mM EDTA, 0.2 mM neocuproine, 60 mM N-ethylmaleimide, and 10% SDS. Relative levels of iNOS S-nitrosation were determined by the biotin switch method (see Experimental Procedures). (B) iNOS inactivation during enzymatic turnover. Two separate reactions were initiated containing 90 nM iNOS, 15 mM Arg, 150 mM NaCl, and 100 mM HEPES pH 7.5. To one reaction iNOS activity was initiated at time zero by NADPH (600 μM) addition (black bars), to the other reaction NADPH was only added immediately prior to rate determination (grey bars). At the given timepoints, 100 μL aliquots of each reaction were added to 200 μL of 45 μM MbO₂ in 100 mM HEPES pH 7.5 to obtain final concentrations of 30 nM iNOS, 5 mM Arg, 50 mM NaCl, 30 μM MbO₂, and 100 mM HEPES pH 7.5.

Figure 2

Kinetic model of iNOS inactivation. (A) Complete kinetic model of iNOS auto-inactivation by S-nitrosation and dimer dissociation where the kinetic readout is reaction of NO with MbO₂. Gray boxes indicate the NO formation, inactivation, and NO release/detection pathways. E represents the enzyme (i.e. iNOS), R represents arginine, E•R represents arginine bound within the iNOS active-site, E•NO represents nitric oxide sequestered within iNOS but not necessarily bound to the heme iron, E-SNO represents iNOS S-nitrosated at the Zn²⁺-tetrathiolate cysteines, and E_i represents inactivated iNOS. (B) A simplified kinetic model in which the inactivation and NO release/detection pathways are represented by net rate constants. (C) General kinetic model of a suicide substrate where S represents the substrate and P represents the product.

Figure 3

Progress curve of NO formation exhibits inactivation over time. The reaction contained 15 nM iNOS, 5 mM Arg, 200 μM NADPH, 50 mM NaCl, 4 μM MbO₂, and 100 mM HEPES pH 7.5 in 300 μL total volume in a 96-well microplate. NO formation was determined by the increase in absorbance at 401 nm observed upon reaction of NO with MbO₂ to form metMb and nitrite. Data was fitted to equation 3 and the resulting fit is shown as a solid black line. The dashed line represents the linear fit to the first five minutes of the progress curve.

Figure 4

Protection of iNOS inactivation by reductants. Progress curves of iNOS catalyzed NO formation in the presence of increasing concentrations of (A) GSH and (B) TCEP. Individual reactions contained 15 nM iNOS, 5 mM Arg, 200 μM NADPH, 50 mM NaCl, 4 μM MbO₂, 100 mM HEPES pH 7.5, and varying concentrations of either GSH or TCEP in 300 μL total volumes in a 96-well microplate. NO formation was measured via the change in absorbance at 401 nm upon reaction of MbO₂ with NO. (C) Plot of [NO]_∞ versus GSH (circles, solid line) or TCEP (squares, dashed line) concentration. Data was fitted using equations 2, 5, and 6. (D) Plot of inactivation rate (λ) versus GSH (circles, solid line) or TCEP (squares, dashed line) concentration. Data was fitted using equations 2, 4, and 6.

Figure 5

Protection of iNOS auto-S-nitrosation by reductants. (A) The anti-biotin blot shows a dose-dependent decrease in iNOS S-nitrosation upon addition of GSH and TCEP. (B) Quantitation of anti-biotin blots with increasing concentrations of GSH (black bars) and TCEP (grey bars). Levels of iNOS S-nitrosation were normalized to the level observed with no added reductant. Error bars represent standard deviation from the mean. Each reaction contained 2 μM iNOS, 5 mM Arg, 2 mM NADPH, 50 mM NaCl, and varying concentrations of either GSH or TCEP in HEN buffer (250 mM HEPES, 2 mM EDTA, 0.2 mM neocuproine, pH 7.5) in 100 μL total volume. iNOS activity was initiated by NADPH addition and quenched after 20 min in HEN buffer with 60 mM N-ethylmaleimide and 10% SDS. Relative levels of iNOS S-nitrosation were determined by the biotin switch method (see Experimental Procedures).

Figure 6

Plots of iNOS inactivation rate (λ) versus (A) MbO₂ concentration and (B) β2 H-NOX concentration. Individual reactions contained 15 nM iNOS, 5 mM Arg, 200 μM NADPH, 50 mM NaCl, 100 mM HEPES pH 7.5, and varying concentrations of either MbO₂ or β2 H-NOX in 300 μL total volumes in a 96-well microplate. NO formation was measured as described under Experimental Procedures. The resulting progress curves were fitted to equation 3 to determine the inactivation rate (λ). The plots of inactivation rate (λ) versus NO trap concentration were fitted using equations 1 and 4. The inset in (A) was fitted to equation 8.

Figure 7

(A) Potential tunnel for NO diffusion from the heme to the Zn²⁺-tetrathiolate within iNOS. The tunnel within a structure of the iNOS heme domain (pdb 1DWV) (85) was generated using CAVER (86). (B) Summary model of iNOS auto-inactivation through S-nitrosation of the Zn²⁺-tetrathiolate and protection from inactivation by reductants. The illustrated scheme is superimposed on the kinetic model that was developed by treating the substrate arginine as a mechanism-based inhibitor. Rescue by cellular reductants (GSH) is also shown as well as trapping of released NO by MbO₂.