nitrogen-dioxide and sapropterin

Tyrosine hydroxylase (TH) is the initial and rate-limiting enzyme in the synthesis of the neurotransmitter dopamine. TH is inhibited and nitrated at tyrosine residues in vitro by the reactive nitrogen species peroxynitrite and nitrogen dioxide (NO2) and in vivo by drugs that damage dopamine neurons. Tetrahydrobiopterin, which is the essential cofactor for TH and is concentrated in dopamine neurons, completely blocks nitration of tyrosine residues in TH caused by peroxynitrite or NO2. Various tetrahydro- and dihydro-analogs of tetrahydrobiopterin, including 6,7-dimethyl-tetrahydropterin, 6-methyl-tetrahydropterin, 6-hydroxymethyl-tetrahydropterin, tetrahydropterin, 7,8-dihydrobiopterin, 7,8-dihydroxanthopterin, and sepiapterin, also prevent nitration of tyrosines caused by the reactive nitrogen species. Biopterin and pterin, the fully oxidized forms of the pterin molecule, fail to block peroxynitrite- or NO2-induced nitration of TH. Reduced pterins prevent neither the inhibition of TH activity nor cysteine modification caused by peroxynitrite or NO2, despite blocking tyrosine nitration. However, dithiothreitol prevents and reverses these effects on TH of tetrahydrobiopterin and reactive nitrogen species. Using an enhanced green fluorescent protein-TH fusion construct as a real-time reporter of intracellular tyrosine nitration, tetrahydrobiopterin was found to prevent NO2-induced tyrosine nitration in intact cells but to leave TH activity inhibited. These results indicate that tetrahydrobiopterin prevents the tyrosine-nitrating properties of peroxynitrite and NO2. Tetrahydrobiopterin-derived radical species formed by reaction with reactive nitrogen species may account for inhibition of TH via mechanisms that do not involve tyrosine nitration.

Topics: Animals; Antioxidants; Biopterins; Cells, Cultured; Humans; Nitrogen Dioxide; Peroxynitrous Acid; Rats; Reactive Nitrogen Species; Tyrosine 3-Monooxygenase

One-electron oxidation of (6R)-5,6,7,8-tetrahydrobiopterin (H(4)B) by the azide radical generates the radical cation (H(4)B(*)(+)) which rapidly deprotonates at physiological pH to give the neutral trihydrobiopterin radical (H(3)B(*)); pK(a) (H(4)B(*)(+) <==> H(3)B(*) + H(+)) = (5.2 +/- 0.1). In the absence of ascorbate both the H(4)B(*)(+) and H(3)B(*) radicals undergo disproportionation to form quinonoid dihydrobiopterin (qH(2)B) and the parent H(4)B with rate constants k(H(4)B(*)(+) + H(4)B(*)(+)) = 6.5 x 10(3) M(-1) s(-1) and k(H(3)B(*) + H(3)B(*)) = 9.3 x 10(4) M(-1) s(-1), respectively. The H(3)B(*) radical is scavenged by ascorbate (AscH(-)) with an estimated rate constant of k(H(3)B(*) + AscH(-)) similar 1.7 x 10(5) M(-1) s(-1). At physiological pH the pterin rapidly scavenges a range of biological oxidants often associated with cellular oxidative stress and nitric oxide synthase (NOS) dysfunction including hydroxyl ((*)OH), nitrogen dioxide (NO(2)(*)), glutathione thiyl (GS(*)), and carbonate (CO(3)(*-)) radicals. Without exception these radicals react appreciably faster with H(4)B than with AscH(-) with k(*OH + H(4)B) = 8.8 x 10(9) M(-1) s(-1), k(NO(2)(*) + H(4)B) = 9.4 x 10(8) M(-1) s(-1), k(CO(3)(*-) + H(4)B) = 4.6 x 10(9) M(-1) s(-1), and k(GS(*) + H(4)B) = 1.1 x 10(9) M(-1) s(-1), respectively. The glutathione disulfide radical anion (GSSG(*-)) rapidly reduces the pterin to the tetrahydrobiopterin radical anion (H(4)B(*-)) with a rate constant of k(GSSG(*-) + H(4)B) similar 4.5 x 10(8) M(-1) s(-1). The results are discussed in the context of the general antioxidant properties of the pterin and the redox role played by H(4)B in NOS catalysis.

Topics: Ascorbic Acid; Biopterins; Carbonates; Chromatography, High Pressure Liquid; Free Radical Scavengers; Free Radicals; Glutathione Disulfide; Hydrogen-Ion Concentration; Hydroxyl Radical; Kinetics; Nitric Oxide Synthase; Nitrogen Dioxide; Oxidation-Reduction; Pulse Radiolysis; Time Factors

The nitric oxide synthases (NOS) are a class of enzymes responsible for the generation of NO via an oxygen and NADPH dependent oxidation of the amino acid arginine. These enzymes are ironheme proteins which contain FAD and FMN and, enigmatically, require tetrahydrobiopterin (BH4). NOS has recently been shown to be subject to inhibition by its product, NO. Preliminary data by us indicate that a possible role for BH4 is to prevent and/or reverse the NO-mediated inhibition of NOS. The objective of this study was to elucidate the mechanism by which BH4 protects NOS against NO inhibition. Protection of NOS from NO inhibition was observed by both BH4 and the BH4 regeneration system, dihydropteridine reductase (DHPR)/NADH. NO, rather than an oxidation product, appears to be the inhibitory species. Protection by BH4 is not likely due to a simple chemical reaction between BH4 and NO or its oxidation product, NO2. The results are consistent with a protective mechanism by which BH4 may act as a nonstoichiometric reducing agent for a redox active enzyme component, such as the ironheme, to prevent NO ligation.

Topics: Amino Acid Oxidoreductases; Animals; Biopterins; Cerebellum; NAD; Nitric Oxide; Nitric Oxide Synthase; Nitrogen Dioxide; Oxidation-Reduction; Rats