Oxoammonium cation intermediate in the nitroxide-catalyzed dismutation of superoxide.

Abstract

Dismutation of superoxide has been shown previously to be catalyzed by stable nitroxide compounds. In the present study, the mechanism of superoxide (-O°) dismutation by various five-membered ring and six-membered ring nitroxides was studied by electron paramagnetic resonance spectrometry, UV-visible spectrophotometry, cyclic voltammetry, and bulk electrolysis. Electron paramagnetic resonance signals from the carbocyclic nitroxide derivatives (piperidinyl, pyrrolidinyl, and pyrrolinyl) were unchanged when exposed to enzymatically generated -02, whereas, in the presence of°2-and reducing agents such as NADH and NADPH, the nitroxides underwent reduction to their respective hydroxylamines. The reaction of 4-hydroxy-2,2,6,6-tetramethyl-1-hydroxypiperidine (Tempol-H) with°2 was measured and, in agreement with earlier reports on related compounds, the rate was found to be too slow to be consistent with a mechanism of°2 dismutation involving the hydroxylamine as an intermediate.Voltammetric analyses of the carbocyclic nitroxide derivatives revealed a reversible one-electron redox couple at positive potentials. In contrast, oxazolidine derivatives were irreversibly oxidized. At negative potentials, all of the nitroxides studied exhibited a broad, irreversible reductive wave. The rate of°O dismutation correlated with the reversible midpoint redox potential. Bulk electrolysis at positive potentials was found to generate a metastable oxidized form of the nitroxide. The results indicate that the dismutation of°2 is catalyzed by the oxoammonium/nitroxide redox couple for carbocyclic nitroxide derivatives. In addition to the one-electron mitochondrial reduction pathway, the present results suggest the possibility that cellular bioreduction by a two-electron pathway may occur subsequent to oxidation of stable nitroxides. Furthermore, the cellular destruction of persistent spin adduct nitroxides might also be facilitated by a primary univalent oxidation.Stable nitroxide free radicals have found a wide range of applications in biology and medicine. These compounds have been used to monitor intracellular redox reactions (1), oxygen concentration (2), and pH (3), as well as electron paramagnetic resonance (EPR) microscopy of spheroids (4), as contrast agents in magnetic resonance imaging (5), and as probes in EPR imaging (6). Persistent nitroxide adducts resulting from reaction of a precursor nitrone (spin trap) with transient free radical species have been used to detect, characterize, and quantitate the production of free radicals in various in vitro and in vivo model systems (7). The cellular and in vivo pharmacology of stable nitroxides and persistent spin adduct nitroxides has been investigated in detail (8-10). Oneelectron reduction of stable nitroxides to the corresponding hydroxylamine is the primary metabolic pathway (11-15).Whereas the metabolic fate of spin adduct nitroxides is not clearly understood, oxidative degradation has been suggested (16,17).Previous studies have identified a stable five-membered nitroxi...

The chemistry and biology of nitroxide compounds

“…The mechanism of the SOD mimetic activity is now understood to involve an oxoammonium/nitroxide redox couple [8].…”

“…In the presence of such agents, an SOD-inhibitable conversion of nitroxide to the corresponding hydroxylamine was observed with a concomitant oxidation of NADH or NADPH to NAD + or NADP + as shown in equation 1d [8,9].…”

“…• OH + XH → X • + H 2 O (8) Antioxidants that scavenge the primary products of radiolysis can decrease the concentration of these highly reactive species and also the subsequent generation of secondary radicals at critical molecular targets. The cell membrane, enzymes and other proteins, and DNA are examples of BIMs that are possible critical targets in radiation-induced cytotoxicity, however DNA is thought be the crucial target which, when damaged, can result in both cell death and genetic alterations.…”

Monitoring redox-sensitive paramagnetic contrast agent by EPRI, OMRI and MRI

“…These EPRI measurements are based on the fact that the paramagnetic nitroxides are reduced to the corresponding diamagnetic one-electron reduction product (Fig. 1A), hydroxylamine in cellular incubations as well as in vivo [5][6][7][8]. It was postulated that nitroxides can accept cellular reducing equivalents, especially from the mitochondrial electron transport chain that would have otherwise gone to the reduction of molecular oxygen [9].…”

π-Stacking effects on the EPR parameters of a prototypical DNA spin label

“…rinkevic@theochem.kth. cellular bioreduction 18 are most frequently encountered in in vivo as well as in in vitro environments. These chemical processes have been extensively studied and several ways have been proposed to extend the in vivo lifetime of nitroxide spin labels.…”