Recent studies have shown that nitrite is an important storage form and source of NO in biological systems. Controversy remains, however, regarding whether NO formation from nitrite occurs primarily in tissues or in blood. Questions also remain regarding the mechanism, magnitude, and contributions of several alternative pathways of nitrite-dependent NO generation in biological systems. To characterize the mechanism and magnitude of NO generation from nitrite, electron paramagnetic resonance spectroscopy, chemiluminescence NO analyzer, and immunoassays of cGMP formation were performed. The addition of nitrite triggered a large amount of NO generation in tissues such as heart and liver, but only trace NO production in blood. Carbon monoxide increased NO release from blood, suggesting that hemoglobin acts to scavenge NO not to generate it. Administration of the xanthine oxidase (XO) inhibitor oxypurinol or aldehyde oxidase (AO) inhibitor raloxifene significantly decreased NO generation from nitrite in heart or liver. NO formation rates increased dramatically with decreasing pH or with decreased oxygen tension. Isolated enzyme studies further confirm that XO and AO, but not hemoglobin, are critical nitrite reductases. Overall, NO generation from nitrite mainly occurs in tissues not in the blood, with XO and AO playing critical roles in nitrite reduction, and this process is regulated by pH, oxygen tension, nitrite, and reducing substrate concentrations.Nitric oxide is a free radical produced in biological tissues that exerts a large number of critical regulatory functions and also plays an important role in the pathogenesis of cellular injury (1). In addition to NO generation from specific NO synthases (NOSs), 3 it is now clear that nitrite can also be an important source of NO, particularly under acidic conditions (2). However, the mechanism of nitrite reduction in biological systems remains highly controversial. Furthermore, it is unknown whether NO formation from nitrite occurs primarily in tissues or in blood. Questions remain regarding the mechanism, magnitude, and contributions of the several proposed pathways of nitrite-dependent NO generation in biological systems.Recent studies reported a novel role for hemoglobin as a nitrite reductase reducing nitrite to NO and eliciting vasodilation. Based on these studies, erythrocytes were proposed to be the major intravascular site of nitrite reduction and storage (3-8). However, it is well known that hemoglobin is an extremely effective NO scavenger (9, 10). The mechanism by which NO escapes trapping by the high concentrations of hemoglobin in the blood remains a mystery with resultant controversy regarding the relative importance of nitrite reduction in blood and tissues. Therefore there is a great need to elucidate the relative importance and molecular mechanisms of NO formation from nitrite in blood and tissues.Initially nitrite was considered as a product of NO metabolism, not a source of NO, in tissues. In 1995, we observed that nitrite can be a prominent source...

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Aldehyde oxidase (AO) is a cytosolic enzyme with an important role in drug and xenobiotic metabolism. Although AO has structural similarity to bacterial nitrite reductases, it is unknown whether AO-catalyzed nitrite reduction can be an important source of NO. The mechanism, magnitude, and quantitative importance of AO-mediated nitrite reduction in tissues have not been reported. To investigate this pathway and its quantitative importance, EPR spectroscopy, chemiluminescence NO analyzer, and immunoassays of cGMP formation were performed. The kinetics and magnitude of AO-dependent NO formation were characterized. In the presence of typical aldehyde substrates or NADH, AO reduced nitrite to NO. Kinetics of AO-catalyzed nitrite reduction followed Michaelis-Menten kinetics under anaerobic conditions. Under physiological conditions, nitrite levels are far below its measured K m value in the presence of either the flavin site electron donor NADH or molybdenum site aldehyde electron donors. Under aerobic conditions with the FAD site-binding substrate, NADH, AO-mediated NO production was largely maintained, although with aldehyde substrates oxygen-dependent inhibition was seen. Oxygen tension, substrate, and pH levels were important regulators of AO-catalyzed NO generation. From kinetic data, it was determined that during ischemia hepatic, pulmonary, or myocardial AO and nitrite levels were sufficient to result in NO generation comparable to or exceeding maximal production by constitutive NO synthases. Thus, AO-catalyzed nitrite reduction can be an important source of NO generation, and its NO production will be further increased by therapeutic administration of nitrite. Nitric oxide (NO)3 exerts a large number of important regulatory biological functions and also plays an important role in the pathogenesis of cellular injury (1-5). NO synthesis was first discovered in macrophages, endothelial cells, and neuronal cells (1, 6 -8). A group of enzymes were identified, NO synthases, which metabolize arginine to citrulline with the formation of NO (9, 10). More recent studies have shown that in addition to NO generation from specific NO synthases, nitrite can be an important source of NO in biological tissues, especially under ischemic conditions (11-16). However, questions remain regarding the precise mechanisms involved in this nitrite reduction.Aldehyde oxidase (AO) (aldehyde:oxygen oxidoreductase; EC 1.2.3.1) is a cytosolic enzyme that plays an important role in the biotransformation of drugs and xenobiotics (17). AO belongs to the family of molybdenum-containing proteins with two iron-sulfur clusters, a flavin cofactor, and a molybdopterin cofactor (18,19). The similar molybdenum-containing enzyme xanthine oxidoreductase (XOR) has been shown previously to be a highly effective nitrite/nitrate reductase playing an important role in catalyzing NO generation from nitrite in mammalian tissues, especially under acidic conditions (14, 16, 20 -26).AO is present in highest levels in the liver but is also broadly distributed in o...

Click chemistry has played a significant role as a rapid and versatile strategy for conjugating two molecular fragments under very mild reaction conditions. Introduction of ferrocene-derived triazole systems using click chemistry has attracted enormous interest in various fields due to its potential applications in electrochemical techniques for detection and sensing. The present discussion focuses on the synthesis of ferrocene-triazole and the importance of using a CuAAC reaction for such conjugation. Applications of ferrocene-based click reactions in conjugate chemistry, asymmetric catalysis, medicinal chemistry, host-guest interactions, and materials chemistry have been highlighted.

Aldehyde oxidase, a molybdoflavoenzyme that plays an important role in aldehyde biotransformation, requires oxygen as substrate and produces reduced oxygen species. However, little information is available regarding its importance in cellular redox stress. Therefore, studies were undertaken to characterize its superoxide and hydrogen peroxide production. Aldehyde oxidase was purified to >98% purity and exhibited a single band at ∼290 kDa on native polyacrylamide gradient gel electrophoresis. Superoxide generation was measured and quantitated by cytochrome c reduction and EPR spin trapping with p-dimethyl aminocinnamaldehyde as reducing substrate. Prominent superoxide generation was observed with an initial rate of 295 nmol/min/mg. Electrochemical measurements of oxygen consumption and hydrogen peroxide formation yielded values of 650 nmol/min/mg and 355 nmol/min/mg. In view of the ubiquitous distribution of aldehydes in tissues, aldehyde oxidase can be an important basal source of superoxide that would be enhanced in disease settings where cellular aldehyde levels are increased. KeywordsAldehyde oxidase; Xanthine oxidase; Superoxide; Hydrogen peroxide; Electron paramagnetic resonance; Spin trapping; Cytochrome c reduction; Reactive oxygen species; Free radicals; Oxygen consumption Aldehyde oxidase (AO; EC 1.2.3.1) 1 is a prominent member of the molybdenum hydroxylase family of enzymes, which also includes xanthine oxidoreductase (XOR). XOR has two interconvertible forms, xanthine dehydrogenase (XDH; EC 1.1.1.204) and xanthine oxidase (XO; 1. 1.3.22). Both forms of XOR are involved in the catabolism of purines, * To whom correspondence should be addressed: Davis Heart and Lung Research Institute, 473 W. 12th Ave, Room 110G, The Ohio State University, Columbus, OH 43210. Phone: (614) . E-Mail: Jay.Zweier@osumc.edu. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Both XOR and AO are homodimeric consisting of two identical subunits with an approximate molecular mass of 145 kDa each. Each subunit consists of four discrete regions -two N-terminal domains contain distinct [2Fe-2S] centers. A linker peptide connects it to a 40 kDa FAD binding domain that positions the flavin ring in close proximity and a second linker peptide connects the FAD domain with the 85 kDa C-terminal portion of the protein that contains the molybdenum center and the substrate binding pocket [3,4]. The structure of XOR is well conserved among human, chicken, mouse and rat enzymes [5] and the amino acid sequences reveal that the molybdenum binding site is the most conserved region with 94% homo...