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 Zn2+-tetrathiolate and inactivation of the enzyme. To probe the potential physiological significance of NOS S-nitrosation, the inactivation timescale of the inducible NOS isoform (iNOS) was determined and found to directly correlate with an increase in 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 (MbO2) and β2 H-NOX, which yielded partition ratios between NO release and inactivation of ~100 at 4 μM MbO2 and ~22,000 at saturating trap concentrations. The results suggest that a portion of the NO synthesized at the heme cofactor reacts with the Zn2+-tetrathiolate without being released into solution. Perturbation of the inactivation pathway through addition of the reducing agents GSH or TCEP resulted in a concentration-dependent decrease in iNOS S-nitrosation that directly correlated with protection from iNOS inactivation. iNOS inactivation was most responsive to physiological concentrations of GSH with an apparent Km value of 13 mM. NOS turnover that leads to NOS S-nitrosation might be a mechanism to control NOS activity, and NOS S-nitrosation could play a role in the physiological generation of nitrosothiols.

Mechanism of Sirt1 NAD+-dependent Protein Deacetylase Inhibition by Cysteine S-Nitrosation

“…For instance, if genomic damage exceeds the ability of the cell to repair its DNA, then inhibition of Sirt1 deacetylase activity by S-nitrosation would increase p53 acetylation and promote apoptosis (45). Interestingly, we recently observed a similar dependence on physiological GSH concentrations for iNOS inhibition by S-nitrosation of the iNOS Zn 2ϩ -tetrathiolate (46). In summary, we have demonstrated that Sirt1 is directly modified and inhibited by cysteine S-nitrosation.…”

S-nitrosylation: integrator of cardiovascular performance and oxygen delivery

“…The abundance of SNO-GRK2 and SNO-β-arrestin2 is diminished in eNOS -/-mice and enhanced in GSNOR -/-mice (28, 34); eNOS and GSNOR thus promote S-nitrosylation and denitrosylation of these proteins, respectively, through the intermediacy of GSNO. eNOS also binds dynamin (46), but whether S-nitrosylation is mediated directly by a transnitrosylase activity of NOS (48,49) or via GSNO is not known. GSNOR -/-mice further exhibit increases in cardiac output under basal conditions, reflecting marked peripheral vasodilation (29) as well perhaps as pronounced myocardial angiogenesis that results from stimulatory S-nitrosylation of HIF-1α under normoxic conditions (35).…”

Nitrosothiols in the Immune System: Signaling and Protection

“…Thus, it is feasible that negative feedback onto S-nitrosylation-dependent NF-jB inactivation should be taken into account. In this mechanism, inactivation of IKK/NF-jB by S-nitrosylation could downregulate iNOS expression and the associated S-nitrosylation activity, an effect reinforced by the fact that iNOS itself is susceptible to inactivation by S-nitrosylation (117,163). As a result, NF-jB activation may be equilibrated in cells, and the levels of NF-jB-induced cytokines and endothelial adhesion molecules (vascular cell adhesion protein 1, intercellular adhesion molecule 1, and E-and P-selectins) sustained for leukocyte extravasation and function.…”