Although the oxygen radical scavenging enzyme, superoxide dismutase (SOD), can reduce injury from ischemia, sepsis and inflammation, the targets of superoxide (O2-) in vivo remain unknown. Endothelium and neurons produce the free radical, nitric oxide (NO) as a secondary messenger mediating vasodilation and preventing platelet aggregation. Because NO has an unpaired electron, it reacts with O2- at or near diffusion-limited rates. We have shown that the resulting product, peroxynitrite (-OONO), has a maximal half-life of under 1 sec at pH 7.4 and 37oC before undergoing proton-catalyzed decomposition to form potent oxidants, which are most likely hydroxyl radical and nitrogen dioxide: (pKa=6.8) (t1/2<1 sec) O2- + NO => -OONO + H+ <=> HOONO => HO + NO2 We hypothesize that the simultaneous production of NO and O2- (which both can be triggered by Ca+2 entry in to cells) may occur under pathological conditions such as reperfusion of ischemic tissue, leading to the formation of -OONO which progressively injures endothelium and other cell types. The proposed hypothesis is chemically more reasonable that the iron-catalyzed Haber-Weiss reaction and is consistent with available evidence from scavenger studies in vivo. In support of this hypothesis, we have found that a burst of NO production occurs during the first few minutes of reperfusion of ischemic heart which corresponds with free radical production measured by spin trapping. We found that -OONO reacts with Cu, Zn SOD to form a stable complex, allowing us to grow crystals and to begin determining its x-ray structure. While the reaction rate with -OONO is probably too slow for intravenously administered SOD to scavenge -OONO in vivo, we will prepare site-directed mutants of SOD seeking to reduce O2- scavenging while increasing -OONO binding to serve as an experimental probe for -OONO.
The Specific Aims of this proposal are to: a) characterize the oxidative mechanisms of -OONO decomposition more fully, b) study how antioxidant enzymes react with -OONO, c) determine the toxicity of -OONO to bacteria and isolated heart and lung, and d) measure the production of NO and -OONO by isolated, perfused lung and heart subjected severe hypoxia/reoxygen-ation. We are working with isolated organs to avoid complicating reactions with blood components. These experiments will test a novel, alternative mechanism to the iron-catalyzed Haber-Weiss reaction for explaining the role of oxygen radicals in ischemic disease, and may also be relevant to sepsis and inflammation, because activated macrophages and neutrophils produce both NO and O2- and thus could form -OONO.
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