Nitric oxide is an environmental toxin. Surprisingly NO is a ubiquitous biological messenger. Nitric oxide itself is a weak oxidant, but it reacts with O2-. in the lung to produce peroxynitrite and peroxynitrous acid. HOONO, potent oxidants and toxins. The three specific aims of this proposal concern the mechanisms that explain the toxicity of HOONO. (#1) Peroxynitrite undergoes a pH-dependent rearrangement reaction to form nitrate, an important pathway for detoxification of HOONO. The mechanism of this reaction has been reported to involve radicals: HOONO to HO. + .NO2 to HONO2. However, our preliminary data suggest the mechanism involves a molecular (non-radical) rearrangement. HOONO exists in both cisoid and transoid forms, which calculations predict have acid pK constants that differ by about 1.3 pH units. The published analysis of the rate of HOONO disappearance versus pH proposes just one HOONO species and a single wave with an inflection point at the reported pK of HOONO, 6.8. However, preliminary experimental data are fit better by a curve with two waves, involving two species (presumed to be cis and trans) with pK values that differ by about 1.5 units. By obtaining sufficient data at about pH 7, we will be able to establish whether this detoxification reaction involves one or two HOONO species and occurs via a molecular rearrangement or via the hydroxyl radical. (#2) HOONO can oxidize biomolecules via an SN2 reaction of nucleophilic species Y:to give Y=O and HNO2. This is involved, for example, in the inactivation of methionine-containing proteins. The SN2 reaction will be studied using the oxidation of methionine to methionine sulfoxide. Reactions of methionine amide and ester also will be studied. (#3) Most inorganic and organic peroxyacids that oxidize sulfides to sulfoxides also epoxidize olefins. We will test whether HOONO can epoxidize cyclohexene, methyl oleate and 7,8-dihydroxy-7,8-dihydrobenzo[a]pyrene. We also will ask if the ion -OO-N=O can epoxidize alpha,beta-unsaturated carbonyl compounds via a Michael addition-elimination mechanism. All three of the specific aims will be studied both in systems where trace transition metals have been removed and in systems to which chelates of redox-active transition metal ions have been added.
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