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 ( 3 NO ؊ ) 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 1 NO ؊ 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 NO͞ 3 NO ؊ and NO͞ 1 NO ؊ 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 3 NO ؊ 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, 1 NO ؊ , is physiologically inaccessible. The significance of these potentials to physiological and pathophysiological processes involving NO and O 2 under reductive conditions is discussed. N itric oxide (NO) is an endogenously generated species with a diverse array of biological functions (1). NO is one of the primary regulators of vascular tone, is involved in signal transduction in both the peripheral and central nervous system, and is an integral part of the immune response system associated with macrophage and neutrophil activation. More recently, NO has been proposed to be involved in the regulation of mitochondrial function (2, 3). Problems in NO homeostasis have been implicated in the development of a variety of diseases and disorders such as hypertension and atherosclerosis (4), diabetes (5), and many neurodegenerative diseases (6). NO is also thought to be a cytoprotective agent, capable of inhibiting radical-induced damage and oxidative stress (7). To understand the actions of NO as a physiological messenger and a cytotoxic or cytoprotective effector molecule, it is essential to understand its basic chemical interactions with biological systems and its metabolic fate.NO and its reduced derivative NO Ϫ (and͞or its conjugate acid, HNO) have very different chemical properties and display distinct and often opposite effects in cells. For example, HNO͞ NO Ϫ has been found to be toxic under conditions where NO is cytoprotective (8). HNO͞NO Ϫ reacts with O 2 to generate potent oxidizing species, capable of damaging DNA and causing cellular thiol depletion, whereas NO does neither under similar conditions (9-11). HNO has been found to be a thiophilic electrophile (12), readily capable of modifying cellular thiol functions (13,14), whereas NO reacts only indirectly with thiols. HNO͞NO Ϫ h...

The alcohol-abuse deterrent disulfiram (DSF) is shown to have a highly selective toxicity against melanoma in culture, inducing a largely apoptotic response, with much lower toxicity against several other cell lines. Melanoma cell lines derived from different stages (radial, vertical, and metastatic phase) were all sensitive to DSF treatment in vitro; melanocytes were only slightly affected. A required role of extracellular Cu is demonstrated for DSF toxicity. Low concentrations of DSF alone decreased the number of viable cells, and the addition of CuCl2 significantly enhanced the DSF-induced cell death to less than 10% of control. Significantly, the intracellular Cu concentration of melanoma cells increased rapidly upon DSF treatment. Both the intracellular Cu uptake and the toxicity induced by DSF were blocked by co-incubation with bathocuproine disulfonic acid (BCPD, 100 μM), a non-membrane-permeable Cu chelator. Chemical studies demonstrated a complicated, extracellular redox reaction between Cu(II) and DSF, which forms the complex Cu(deDTC)2 in high yield, accompanied by oxidative decomposition of small amounts of disulfiram. The Cu complex has somewhat higher activity against melanoma and is suggested to be the active agent in DSF-induced toxicity. The redox conversion of DSF was unique to Cu(II) and not engendered by the other common biological metal ions Fe(II or III), Mn(III), and Zn(II). The implications of this work are significant both in the possible treatment of melanoma as well as in limiting the known side-effects of DSF, which we propose may be diminished by cotreatment to decrease adventitious Cu.