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.

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Ferredoxin (Fd) is the major iron-containing protein in photosynthetic organisms and is central to reductive metabolism in the chloroplast. The Chlamydomonas reinhardtii genome encodes six plant type [Fe 2 S 2 ] ferredoxins, products of PETF, FDX2-FDX6. We performed the functional analysis of these ferredoxins by localizing Fd, Fdx2, Fdx3, and Fdx6 to the chloroplast by using isoform-specific antibodies and monitoring the pattern of gene expression by iron and copper nutrition, nitrogen source, and hydrogen peroxide stress. In addition, we also measured the midpoint redox potentials of Fd and Fdx2 and determined the kinetic parameters of their reactions with several ferredoxin-interacting proteins, namely nitrite reductase, Fd:NADP ؉ oxidoreductase, and Fd:thioredoxin reductase. We found that each of the FDX genes is differently regulated in response to changes in nutrient supply. Moreover, we show that Fdx2 (E m ‫؍‬ ؊321 mV), whose expression is regulated by nitrate, is a more efficient electron donor to nitrite reductase relative to Fd. Overall, the results suggest that each ferredoxin isoform has substrate specificity and that the presence of multiple ferredoxin isoforms allows for the allocation of reducing power to specific metabolic pathways in the chloroplast under various growth conditions.Ferredoxins are small (ϳ11,000-kDa), soluble, iron-sulfur cluster-containing proteins with strongly negative redox potentials (Ϫ350 to Ϫ450 mV) that function as electron donors at reductive steps in various metabolic pathways (1-3). In photosynthetic organisms, the well studied ferredoxin (Fd 4

totic phenotype in the transformed cell. This conceptual frameThe human melanocyte is continuously exposed to intrinsic and work offers testable steps to determine the role of redox extrinsic sources of reactive biochemical species, but is finely tuned via the intrinsic anti-oxidant and radical properties of alterations in the carcinogenic evolution, prevention and treatmelanin to suppress the build-up of an altered redox phenoment of melanoma and other diseases of the melanocyte. type. We propose that this control is lost during melanomageKey words: Transcription factor, NF-kB, AP-1, Reactive nesis and inappropriate redox-sensitive transcriptional factor oxygen species, Glutathione, Superoxide anion activations occur which result in enhancement of an anti-apopand glutathione (GSH), melanin as an anti-oxidant and cellular pro-oxidant, response of melanin to ultraviolet (UV) light, anti-oxidant levels and melanomagenesis, redox regulation of transcription factors, and a conceptual framework for the pathogenesis of melanoma based on altered redox control.In response to UV light an inflammatory response is generated involving the production of massive amounts of various cytokines and growth factors by keratinocytes, a vigorous inflammatory/immunologic host response, and in some cases angiogenesis. Each one of these responses generates or stimulates the production of reactive oxygen or nitrogen species. Additionally, UV light interacts directly with biochemical constituents of the melanocyte to generate intracellular reactive oxygen species (ROS), hydrogen peroxide and/or superoxide anion. The sum total of these alterations is that melanocytes are subjected to an panoply of redox changes with secondary effects on melanin synthesis and a variety of signaling cascades.We suggest that alterations in these processes are fundamentally involved in the pathogenesis of melanoma and perhaps other pathologic states. These observations encum-

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The nitroxyl, a one-electron reduced form of nitric oxide, has been suggested to play an important role in many biological systems which involve heme proteins. 1,2 Like nitric oxide, the nitroxyl has been linked to processes such as vasodilation 3 and cytotoxicity. 4 It has been proposed to be the released product of arginine oxidation by inducible P450 NoS . 5 Nitroxyl-heme adducts are postulated as intermediates in mechanisms of several types of nitric oxide reductases. 6,7 In a previous paper, we described the reversible electrochemical reduction of nitrosyl myoglobin (NO-Mb) to a long-lived nitroxyl adduct. 8 In this work, we describe the characterization and unusual stability of nitroxyl myoglobin in aqueous solution, and clearly identify it as an HNOadduct.Voltammetry of NO-Mb adduct at high pH and in the absence of exogenous NO gave evidence of a reversible formation of a one-electron reduced product at -0.63 V vs NHE, eq 1. The potential of this reduction is at the edge of those known in biological systems, but in the range of certain highly reduced ferredoxin and siroheme proteins which are accessible in aqueous medium. 9,10 The product was postulated as a nitroxyl anion, NO -, adduct in analogy to several such adducts formed from reductions of Fe porphyrin nitrosyl in organic solvents. 11,12 The lifetime of the nitroxyl adduct, as indicated by voltammetric reversibility, was dramatically increased at high pH in these measurements.The chemical reduction of solution-based NO-Mb, 1, to HNO-Mb, 2, was achieved using an excess of Cr II reagents (as the tacn or edta complexes), and could be followed by the shift in the Soret absorbance from 421 to 423 nm, Figure 1. 13 Electrochemical reduction of 1, using N-methyl-4,4′-bipyridine iodide as a mediator (E 1/2 NHE ) -800 mV), also produces the spectral changes we attribute to 2. 14 The reduced protein thus formed can be separated by size-exclusion chromatography to yield purified samples for analysis.

Melanin protects the skin and eyes from the harmful effects of UV irradiation, protects neural cells from toxic insults, and is required for sound conduction in the inner ear. Aberrant regulation of melanogenesis underlies skin disorders (melasma and vitiligo), neurologic disorders (Parkinson's disease), auditory disorders (Waardenburg's syndrome), and opthalmologic disorders (age related macular degeneration). Much of the core synthetic machinery driving melanin production has been identified; however, the spectrum of gene products participating in melanogenesis in different physiological niches is poorly understood. Functional genomics based on RNA-mediated interference (RNAi) provides the opportunity to derive unbiased comprehensive collections of pharmaceutically tractable single gene targets supporting melanin production. In this study, we have combined a high-throughput, cell-based, one-well/one-gene screening platform with a genome-wide arrayed synthetic library of chemically synthesized, small interfering RNAs to identify novel biological pathways that govern melanin biogenesis in human melanocytes. Ninety-two novel genes that support pigment production were identified with a low false discovery rate. Secondary validation and preliminary mechanistic studies identified a large panel of targets that converge on tyrosinase expression and stability. Small molecule inhibition of a family of gene products in this class was sufficient to impair chronic tyrosinase expression in pigmented melanoma cells and UV-induced tyrosinase expression in primary melanocytes. Isolation of molecular machinery known to support autophagosome biosynthesis from this screen, together with in vitro and in vivo validation, exposed a close functional relationship between melanogenesis and autophagy. In summary, these studies illustrate the power of RNAi-based functional genomics to identify novel genes, pathways, and pharmacologic agents that impact a biological phenotype and operate outside of preconceived mechanistic relationships.