Reaction of tetrahydrobiopterin with superoxide: EPR-kinetic analysis and characterization of the pteridine radical

Abstract

It has been shown that BH₄ ameliorates endothelial dysfunction associated with conditions such as hypertension, cigarette smoking, and diabetes. This effect has been proposed to be due to a superoxide scavenging activity of BH₄. To examine this possibility we determined the rate constant for the reaction between BH₄ and superoxide using electron paramagnetic resonance (EPR) spin trapping competition experiments with 5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide (DEPMPO). We calculated a rate constant for the reaction between BH₄ and superoxide of 3.9 ± 0.2 × 10⁵ M⁻¹s⁻¹ at pH 7.4 and room temperature. This result suggests that superoxide scavenging by BH₄ is not a major reaction in vivo. HPLC product analysis showed that 7,8-BH₂ and pterin are the stable products generated from the reaction. The formation of BH₄ cation radical (BH₄^•+) was demonstrated by direct EPR only under acidic conditions. Isotopic substitution experiments demonstrated that the BH₄^•+ is mainly delocalized on the pyrazine ring of BH₄. In parallel experiments, we investigated the effect of ascorbate on 7,8-BH₂ reduction and eNOS activity. We demonstrated that ascorbate does not reduce 7,8-BH₂ to BH₄, nor does it stimulate nitric oxide release from eNOS incubated with 7,8-BH₂. In conclusion, it is likely that BH₄-dependent inhibition of superoxide formation from eNOS is the mechanism that better explains the antioxidant effects of BH₄ in the vasculature.

Introduction

There is increasing evidence in the literature suggesting that superoxide formation in the vasculature impairs endothelial-dependent vasorelaxation [1], [2]. For example, increased superoxide formation in blood vessels mediates endothelial dysfunction during early stages of cardiovascular diseases such as hypertension [3], [4], [5], coronary artery disease, atherosclerosis, and hypercholesterolemia [6], [7], [8]. Superoxide reacts with NO at a diffusion-limited rate to generate peroxynitrite [9], [10]. This intermediate is a potent oxidant that has been shown to react with plasma antioxidants [11], [12] to generate free radicals such as ascorbyl, uric acid, and the albumin-thiyl radical, consequently, diminishing antioxidant defense [11]. Thus, generation of peroxynitrite might also contribute to impaired vascular reactivity by altering vascular redox status. Clinical trials showed that supplementation with antioxidants, such ascorbic acid, ameliorate hypertension in humans and experimental animals [13], [14], [15]. However, the mechanism by which ascorbate decreases blood pressure remains unclear. As ascorbate does not increase eNOS expression [16], it was proposed that the scavenging of superoxide by ascorbate enhances the bioavailability of NO. More recently, it was proposed that ascorbate ameliorated vasoconstriction by enhancing BH₄ concentrations in endothelial cells [17].

The endothelial isoform of nitric oxide synthase (eNOS) catalyzes the stepwise oxidation of L-arginine to form L-citrulline and NO by a reaction that is strictly dependent on tetrahydrobiopterin (BH₄) [18]. Although the exact role of BH₄ in the mechanism of NO generation is not fully understood, it has been demonstrated that this cofactor induces a shift from low spin to high spin heme iron, stabilizes the homodimeric conformation of the enzyme, and acts as an allosteric effector of all isoforms [19 and references therein]. These effects, however, do not fully explain the mechanism by which BH₄ enhances NO formation by NOS. Because of its redox properties, BH₄ has been implicated in several reactions that eventually dictate the products generated by NOS. For instance, EPR spin trapping evidence indicated that BH₄ inhibits the release of superoxide from NOS [20], [21]. BH₄-free NOS generates nitroxyl (NO⁻) from L-hydroxy-L-arginine oxidation [22]. It has also been suggested that BH₄ scavenges superoxide [23], [24] and reacts with peroxynitrite [25]. However, few kinetic and/or mechanistic studies of these reactions are available in the literature.

To understand the mechanism by which BH₄ regulates NO levels and vascular tone, we examined the reaction of BH₄ with superoxide by direct electron paramagnetic resonance (EPR), EPR-spin trapping and high-performance liquid chromatography. The rate constant for the reaction was measured by the EPR-spin trapping technique with 5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide (DEPMPO) [26]. This spin trap forms a persistent DEPMPO-superoxide adduct (DEPMPO-OOH) that does not spontaneously decay to DEPMPO-hydroxyl adduct [26], [27]. These properties allow accurate quantitative EPR measurements of superoxide [26], [27]. In addition, it has been estimated that DEPMPO detects superoxide high with sensitivity [28].

Our results show that the reaction between BH₄ and superoxide proceeds with a rate constant of 3.9 ± 0.2 × 10⁵ M⁻¹s⁻¹, at pH 7.4 and room temperature. Formation of the intermediate BH₄-cation radical and 7,8-dihydrobiopterin and pterin as stable products was demonstrated by direct EPR measurements and HPLC, respectively. Our results suggest that the vasorelaxant properties of BH₄ are more likely due to specific mechanisms such as inhibition of superoxide release from eNOS than to superoxide scavenging.