Oxidation of tetrahydrobiopterin by biological radicals and scavenging of the trihydrobiopterin radical by ascorbate

Abstract

One-electron oxidation of (6 R)-5,6,7,8-tetrahydrobiopterin (H 4B) by the azide radical generates the radical cation (H 4B • +) which rapidly deprotonates at physiological pH to give the neutral trihydrobiopterin radical (H 3B •); p K a (H 4B • + ⇄ H 3B • + H +) = (5.2 ± 0.1). In the absence of ascorbate both the H 4B • + and H 3B • radicals undergo disproportionation to form quinonoid dihydrobiopterin (qH 2B) and the parent H 4B with rate constants k(H 4B • + + H 4B • +) = 6.5 × 10 3 M −1 s −1 and k(H 3B • + H 3B •) = 9.3 × 10 4 M −1 s −1, respectively. The H 3B • radical is scavenged by ascorbate (AscH −) with an estimated rate constant of k(H 3B • + AscH −) ∼ 1.7 × 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 4B than with AscH − with k( OH + H 4B) = 8.8 × 10 9 M −1 s −1, k(NO 2 • + H 4B) = 9.4 × 10 8 M −1 s −1, k(CO 3 •− + H 4B) = 4.6 × 10 9 M −1 s −1, and k(GS • + H 4B) = 1.1 × 10 9 M −1 s −1, respectively. The glutathione disulfide radical anion (GSSG •−) rapidly reduces the pterin to the tetrahydrobiopterin radical anion (H 4B •−) with a rate constant of k(GSSG •− + H 4B) ∼ 4.5 × 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 4B in NOS catalysis.