Generation of nitroxyl by heme protein-mediated peroxidation of hydroxylamine but not N-hydroxy-L-arginine

Abstract

The chemical reactivity, toxicology and pharmacological responses to nitroxyl (HNO) are often distinctly different from those of nitric oxide (NO). The discovery that HNO donors may have pharmacological utility for treatment of cardiovascular disorders such as heart failure and ischemia reperfusion has led to increased speculation of potential endogenous pathways for HNO biosynthesis. Here, the ability of heme proteins to utilize H 2 O 2 to oxidize hydroxylamine (NH 2 OH) or N-hydroxy-L-arginine (NOHA) to HNO was examined. Formation of HNO was evaluated with a recently developed selective assay in which the reaction products in the presence of reduced glutathione (GSH) were quantified by HPLC. Release of HNO from the heme pocket was indicated by formation of sulfinamide (GS(O)NH 2 ), while the yields of nitrite and nitrate signified the degree of intramolecular recombination of HNO with the heme. Formation of GS(O)NH 2 was observed upon oxidation of NH 2 OH, whereas NOHA, the primary intermediate in oxidation of L-arginine by NO synthase, was apparently resistant to oxidation by the heme proteins utilized. In the presence of NH 2 OH, the highest yields of GS(O)NH 2 were observed with proteins in which the heme was coordinated to a histidine (horseradish peroxidase, lactoperoxidase, myeloperoxidase, myoglobin and hemoglobin) in contrast to a tyrosine (catalase) or cysteine (cytochrome P450). That peroxidation of NH 2 OH by horseradish peroxidase produced free HNO, which was able to affect intracellular targets, was verified by conversion of 4,5-diaminofluorescein to the corresponding fluorophore within intact cells.

The Specificity of Nitroxyl Chemistry Is Unique Among Nitrogen Oxides in Biological Systems

“…Both NH 2 OH and hydroxyurea can be oxidized by peroxidases (33,69) [ Fig. 5 for NH 2 OH; a related two step process is suggested for hydroxyurea (69)].…”

“…Whether HNO will be capable of escaping the heme pocket seems to be protein dependent. A survey of heme proteins (33) showed that proteins such as Mb and myeloperoxidase, which have proximal histidine residues, can generate free HNO, as indicated by formation of sulfinamide. A lower affinity of the ferric heme for HNO may also correlate to a lower affinity for NO upon reductive nitrosylation.…”

“…HNO has been assumed to be an intermediate of SAHA oxidation by a peroxidase mechanism (139). Addition of HRP/H 2 O 2 /NH 2 OH to cells loaded with DAF resulted in an increase in fluorescence (33), indicating that HNO produced by this reaction can migrate into cells (42). Interestingly, HNO pharmacology has been shown to be effective with cardiovascular conditions in which inflammation will produce H 2 O 2 .…”

A Novel Role for HNO in Local and Spreading Vasodilatation in Rat Mesenteric Resistance Arteries

“…However, more recent evidence suggests that HNO may also be active endogenously (22,33,35,38), and therefore likely to be of physiological significance. Indeed a number of potential biosynthetic pathways for HNO generation have been identified, including a) direct oxidation of the nitric oxide synthase (NOS) intermediates hydroxylamine (8) and Nhydroxy-L-arginine (19); b) nitrosothiol decomposition (44); c) direct reduction of NO by either mitochondrial cytochrome c, ubiquinol, and=or xanthine oxidase (20,36), and d) by NOS in the absence of the cofactor tetrahydrobiopterin (BH 4 ) (1).…”

Nitrosyl hydride (HNO) replaces dioxygen in nitroxygenase activity of manganese quercetin dioxygenase

“…But the significance of "free" HNO in nature is as yet uncertain (22). Although several biological pathways for HNO formation have been suggested, e.g., via nitrosothiol decomposition (23,24) or enzymatic malfunction (25)(26)(27), no true physiological role for endogenous HNO has been accepted. Following our previous characterization of the trapping of free HNO by various O 2 -binding globins (11), we hypothesized that HNO might similarly interact with O 2 -dependent oxygenases; we herein report the unprecedented substitution of HNO for O 2 in the activity of Mn-QDO, resulting in the incorporation of the nitroxyl-derived N atom into the product, which we denote as nitroxygenase activity.…”