Thiol dependence of nitric oxide synthase.
ABSTRACT Nitric oxide synthases (NOS) require NADPH and tetrahydrobiopterin (H4biopterin) to convert L-arginine to L-citrulline. The additional requirement and effects of thiols during purification and activity assays of NOS are unclear; for example, glutathione (GSH) has been reported to stimulate or, in the presence of catalase, to inhibit enzyme activity. We therefore studied the effects of different thiols, thiol reagents, antioxidants, and H4biopterin-regenerating systems on purified porcine cerebellum NOS. GSH in the presence of catalase did not inhibit NOS. In contrast, GSH and, to a lesser degree, several other thiols consistently stimulated total L-arginine turnover up to 4-fold. In the presence of GSH, Vmax of NOS was increased, the usually observed loss of activity during the 15 min assay was less dramatic, and the apparent S0.5 value for H4biopterin decreased. Stabilization of NOS activity by GSH was augmented by protein disulfide isomerase (PDI), indicating that, at least in part, GSH acted by reductive protection of NOS protein thiols. Consistent with this, four different protein thiol reagents abolished NOS activity. In other experiments, specific allosteric binding was excluded as a potential mechanism of GSH regulation of NOS. In addition, GSH may affect NOS kinetics by recycling or preventing the autoxidation of H4biopterin. In support of this, the non-thiol reductant ascorbate and dihydropteridine reductase mimicked the effects of GSH on NOS kinetics, but not on NOS stability. Thus, NOS activity depends on both H4biopterin and the reduced state of essential protein thiols.
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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.Biochemistry 02/2012; 51(5):1028-40. · 3.38 Impact Factor
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ABSTRACT: Significance. Despite many advances in blood substitute research, the development of materials that are effective in maintaining blood volume and oxygen delivery remains a priority for emergency care and trauma. Clinical trials on hemoglobin (Hb) based oxygen carriers (HBOCs) have not provided information on the mechanism of toxicity, although all commercial formulations have safety concerns. Specifically, it is important to reconcile the different hypotheses of Hb toxicity, such as nitric oxide (NO) depletion and oxidative reactions, to provide a coherent molecular basis for designing a safe HBOC. Recent Advances. Different sized HBOCs often exhibit differences in the degree of HBOC-induced vasoactivity. This has been attributed to differences in the degree of NO scavenging and in the extent of Hb extravasation. Additionally, it is appears that Hb can undergo reactions that compensate for NO scavenging by generating bioactive forms of NO. Critical Issues. Engineering modifications to enhance bioactive NO production can result in diminished oxygen delivery by virtue of increased oxygen affinity. This strategy can prevent the HBOC from fulfilling the intended goal on preserving oxygenation; however, the NO production effects will increase perfusion and oxygen transport. Future Directions. Hb modifications influence NO scavenging and the capacity of certain HBOCs to compensate for NO scavenging through nitrite-mediated reactions that generate bioactive NO. Based on the current understanding of these NO-related factors, possible synthetic strategies are presented that address how HBOC formulations can be prepared that: i) effectively deliver oxygen; ii) maintain tissue perfusion; and iii) limit/reverse underlying inflammation within the vasculature.Antioxidants & Redox Signaling 12/2012; · 8.20 Impact Factor
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ABSTRACT: Ascorbate (Asc) has been shown to increase nitric oxide (NO) bioavailability and thereby improve endothelial function in patients showing signs of endothelial dysfunction. Tetrahydrobiopterin (BH4) is a co-factor of endothelial nitric oxide synthase (eNOS) which may easily become oxidized to the inactive form dihydrobiopterin (BH2). Asc may increase NO bioavailability by a number of mechanisms involving BH4 and eNOS. Asc increases BH4 bioavailability by either reducing oxidized BH4 or preventing BH4 from becoming oxidized in the first place. Asc could also increase NO bioavailability in a BH4-independent manner by increasing eNOS activity by changing its phosphorylation and S-nitrosylation status or by upregulating eNOS expression. In this review, we discuss the putative mechanisms by which Asc may increase NO bioavailability through its interactions with BH4 and eNOS.Nitric Oxide 12/2013; · 3.27 Impact Factor