Kinetic feasibility of nitroxyl reduction by physiological reductants and biological implications.

Nitroxyl (HNO), the one-electron reduced and protonated congener of nitric oxide (NO), is a chemically unique species with potentially important biological activity. Although HNO-based pharmaceuticals are currently being considered for the treatment of chronic heart failure or stroke/transplant-derived ischemia, the chemical events leading to therapeutic responses are not established. The interaction of HNO with oxidants results in the well-documented conversion to NO, but HNO is expected to be readily reduced as well. Recent thermodynamic calculations predict that reduction of HNO is biologically accessible. Herein, kinetic analysis suggests that the reactions of HNO with several mechanistically distinct reductants are also biologically feasible. Product analysis verified that the reductants had in fact been oxidized and that in several instances HNO had been converted to hydroxylamine. Moreover, a theoretical analysis suggests that in the reaction of HNO with thiol reductants, the pathway producing sulfinamide is significantly more favorable than that leading to disulfide. Additionally, simultaneous production of HNO and NO yielded a biphasic oxidative capacity.

Mechanism of aerobic decomposition of Angeli's salt (sodium trioxodinitrate) at physiological pH.

The recent determination that Angeli's salt may have clinical application as a nitrogen oxide donor for treatment of cardiovascular diseases such as heart failure has led to renewed interest in the mechanism and products of thermal decomposition of Angeli's salt under physiological conditions. In this report, several mechanisms are evaluated experimentally and by quantum mechanical calculations to determine whether HNO is in fact released from Angeli's salt in neutral, aerobic solution. The mechanism of product autoxidation is also considered.

A biochemical rationale for the discrete behavior of nitroxyl and nitric oxide in the cardiovascular system.

The redox siblings nitroxyl (HNO) and nitric oxide (NO) have often been assumed to undergo casual redox reactions in biological systems. However, several recent studies have demonstrated distinct pharmacological effects for donors of these two species. Here, infusion of the HNO donor Angeli's salt into normal dogs resulted in elevated plasma levels of calcitonin gene-related peptide, whereas neither the NO donor diethylamine/NONOate nor the nitrovasodilator nitroglycerin had an appreciable effect on basal levels. Conversely, plasma cGMP was increased by infusion of diethylamine/NONOate or nitroglycerin but was unaffected by Angeli's salt. These results suggest the existence of two mutually exclusive response pathways that involve stimulated release of discrete signaling agents from HNO and NO. In light of both the observed dichotomy of HNO and NO and the recent determination that, in contrast to the O2/O2- couple, HNO is a weak reductant, the relative reactivity of HNO with common biomolecules was determined. This analysis suggests that under biological conditions, the lifetime of HNO with respect to oxidation to NO, dimerization, or reaction with O2 is much longer than previously assumed. Rather, HNO is predicted to principally undergo addition reactions with thiols and ferric proteins. Calcitonin gene-related peptide release is suggested to occur via altered calcium channel function through binding of HNO to a ferric or thiol site. The orthogonality of HNO and NO may be due to differential reactivity toward metals and thiols and in the cardiovascular system, may ultimately be driven by respective alteration of cAMP and cGMP levels.

The reduction potential of nitric oxide (NO) and its importance to NO biochemistry.

A potential of about -0.8 (+/-0.2) V (at 1 M versus normal hydrogen electrode) for the reduction of nitric oxide (NO) to its one-electron reduced species, nitroxyl anion (3NO-) has been determined by a combination of quantum mechanical calculations, cyclic voltammetry measurements, and chemical reduction experiments. This value is in accord with some, but not the most commonly accepted, previous electrochemical measurements involving NO. Reduction of NO to 1NO- is highly unfavorable, with a predicted reduction potential of about -1.7 (+/-0.2) V at 1 M versus normal hydrogen electrode. These results represent a substantial revision of the derived and widely cited values of +0.39 V and -0.35 V for the NO/3NO- and NO/1NO- couples, respectively, and provide support for previous measurements obtained by electrochemical and photoelectrochemical means. With such highly negative reduction potentials, NO is inert to reduction compared with physiological events that reduce molecular oxygen to superoxide. From these reduction potentials, the pKa of 3NO- has been reevaluated as 11.6 (+/-3.4). Thus, nitroxyl exists almost exclusively in its protonated form, HNO, under physiological conditions. The singlet state of nitroxyl anion, 1NO-, is physiologically inaccessible. The significance of these potentials to physiological and pathophysiological processes involving NO and O2 under reductive conditions is discussed.

Kinetics of the reactions of nitrogen dioxide with glutathione, cysteine, and uric acid at physiological pH.

Nitrogen dioxide (NO(2)(*)) is a key biological oxidant. It can be derived from peroxynitrite via the interaction of nitric oxide with superoxide, from nitrite with peroxidases, or from autoxidation of nitric oxide. In this study, submicromolar concentrations of NO(2)(*) were generated in < 1 micros using pulse radiolysis, and the kinetics of scavenging NO(2)(*) by glutathione, cysteine, or uric acid were monitored by spectrophotometry. The formation of the urate radical was observed directly, while the production of the oxidizing radical obtained on reaction of NO(2)(*) with the thiols (the thiyl radical) was monitored via oxidation of 2,2'-azino-bis-(3-ethylthiazoline-6-sulfonic acid). At pH 7.4, rate constants for reaction of NO(2)(*) with glutathione, cysteine, and urate were estimated as approximately 2 x 10(7), 5 x 10(7), and 2 x 10(7) M(-1) s(-1), respectively. The variation of these rate constants with pH indicated that thiolate reacted much faster than undissociated thiol. The dissociation of urate also accelerated reaction with NO(2)(*) at pH > 8. The thiyl radical from GSH reacted with urate with a rate constant of approximately 3 x 10(7) M(-1) s(-1). The implications of these values are: (i) the lifetime of NO(2)(*) in cytosol is < 10 micros; (ii) thiols are the dominant 'sink' for NO(2)(*) in cells/tissue, whereas urate is also a major scavenger in plasma; (iii) the diffusion distance of NO(2)(*) is approximately 0.2 microm in the cytoplasm and < 0.8 microm in plasma; (iv) urate protects GSH against depletion on oxidative challenge from NO(2)(*); and (v) reactions between NO(2)(*) and thiols/urate severely limit the likelihood of reaction of NO(2)(*) with NO* to form N(2)O(3) in the cytoplasm.

The reaction of superoxide radicals with S-nitrosoglutathione and the products of its reductive heterolysis.

Generation of superoxide radicals (0.01-0.1 microm s(-1)) by radiolysis of aqueous solutions containing S-nitrosoglutathione (45-160 microm, pH 3.8-7.3) resulted in loss of this solute at rates varying with solute concentration, radical generation rate, and pH. The results were quantitatively consistent with the loss being attributed to competition between reaction of superoxide with S-nitrosoglutathione (rate constant 300 +/- 100 m(-1) s(-1)) and the pH-dependent disproportionation of superoxide/hydroperoxyl. This rate constant is much lower than previous estimates and seven orders of magnitude lower than the rate constants between superoxide and superoxide dismutase or superoxide and nitric oxide. This indicates that interaction between superoxide and S-nitrosoglutathione is unlikely to be biologically important, contrary to previous suggestions that reaction could serve to prevent the rapid reaction between superoxide and nitric oxide. Reductive homolysis of S-nitrosoglutathione by the carbon dioxide radical anion, a model for biological reductants such as disulfide radical anions, occurred with a rate constant of 7.4 x 10(8) m(-1) s(-1) and produced nitric oxide stoichiometrically. Thiyl radicals were not produced, indicating the alternative homolysis route to generate nitroxyl did not occur.