Original contributionReaction of tetrahydrobiopterin with superoxide: EPR-kinetic analysis and characterization of the pteridine radical
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.
Section snippets
Materials
Bovine Cu,Zn-SOD (5000 U/mg) and xanthine oxidase (0.1 U/mg protein) were obtained from Roche (Indianapolis, IN, USA). Hemoglobin was obtained from Calbiochem (San Diego, CA, USA). Diethylenetriaminepentaacetic acid (DTPA) was obtained from Fluka Chemika-BioChemika. 6-(R)-Tetrahydrobiopterin (6R-BH4) and 7,8-dihydrobiopterin were obtained from Alexis Biochemicals Co. (San Diego, CA, USA). Biopterin was obtained from Schircks Laboratories (Jona, Switzerland). L-[14C] Arginine was obtained from
Cytochrome c
Rates of superoxide production by xanthine (1 mM) and xanthine oxidase (0.1 U/ml) were calculated from the reduction of ferricytochrome c (50 μM). Control incubations contained SOD (10 μg/ml). Concentrations were calculated using an extinction coefficient of 21 mM−1 cm−1.
Endothelial nitric oxide synthase activity
Enzyme activity was determined either by following 14C-L-citrulline [20], [21] or hemoglobin assay. This assay used calcium chloride (0.2 mM), calmodulin (20 μg/ml), glutathione (0.1 mM), bovine serum albumin (0.1 mg/ml),
EPR-kinetic analysis of the reaction between BH4 and superoxide
Figure 1, trace A shows the DEPMPO-OOH obtained upon mixing DEPMPO (0.2 M) with xanthine/xanthine oxidase incubation mixtures, producing superoxide at a rate of 23.4 μM min−1. Lower amounts of DEPMPO-OOH were detected (Fig. 1, trace B) after adding BH4 to reaction mixtures. No spectral changes other than diminished signal intensity were observed in the presence of BH4 (Fig. 1, trace A c.f. trace B). Control experiments were performed to examine whether BH4 inhibits xanthine oxidase activity
Discussion
The direct scavenging of superoxide by BH4 has been thought to increase NO levels in endothelial cells. In order to examine this possibility, we used an EPR approach for determining the rate constant for the reaction between BH4 and superoxide. Until recently, EPR quantification of superoxide was hindered by the lack of a spin trap that generated a persistent superoxide radical adduct. This limitation has been overcome with the synthesis of DEPMPO [26], [28], EMPO [42], and 15N-EMPO [43] spin
Acknowledgements
This work was supported by AHA Grant-in-Aid 9950629N to J. V. V. Also, P. M. is supported by NIH grant GM52419 to Bettie Sue Masters, The University of Texas Health Science Center at San Antonio, San Antonio, Texas.
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