Modulation of Superoxide-dependent Oxidation and Hydroxylation Reactions by Nitric Oxide

Allen M Miles,D Scott Bohle,Peter A Glassbrenner,Bernhard Hansert,David A Wink,Matthew B Grisham

doi:10.1074/jbc.271.1.40

Allen M Miles, D Scott Bohle + Show 4 more

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https://doi.org/10.1074/jbc.271.1.40

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Abstract

The rapid and spontaneous interaction between superoxide (O2-.) and nitric oxide (NO) to yield the potent oxidants peroxynitrite (ONOO-) and peroxynitrous acid (ONOOH), has been suggested to represent an important pathway by which tissue may be injured during inflammation. Although several groups of investigators have demonstrated substantial oxidizing and cytotoxic activities of chemically synthesized ONOO-, there has been little information available quantifying the interaction between O2-. and NO in the absence or the presence of redox-active iron. Using the hypoxanthine (HX)/xanthine oxidase system to generate various fluxes of O2-. and H2O2 and the spontaneous decomposition of the spermine/NO adduct to produce various fluxes of NO, we found that in the absence of redox-active iron, the simultaneous production of equimolar fluxes of O2-. and NO increased the oxidation of dihydrorhodamine (DHR) from normally undetectable levels to approximately 15 microM, suggesting the formation of a potent oxidant. Superoxide dismutase, but not catalase, inhibited this oxidative reaction, suggesting that O2-. and not hydrogen peroxide (H2O2) interacts with NO to generate a potent oxidizing agent. Excess production of either radical virtually eliminated the oxidation of DHR. In the presence of 5 microM Fe+3-EDTA to insure optimum O2-.-driven Fenton chemistry, NO enhanced modestly HX/xanthine oxidase-induced oxidation of DHR. As expected, both superoxide dismutase and catalase inhibited this Fe-catalyzed oxidation reaction. Excess NO production with respect to O2-. flux produced only modest inhibition (33%) of DHR oxidation. In a separate series of studies, we found that equimolar fluxes of O2-. and NO in the absence of iron only modestly enhanced hydroxylation of benzoic acid from undetectable levels to 0.6 microM 2-hydroxybenzoate. In the presence of 5 microM Fe+3-EDTA, HX/xanthine oxidase-mediated hydroxylation of benzoic acid increased dramatically from undetectable levels to 4.5 microM of the hydroxylated product. Superoxide dismutase and catalase were both effective at inhibiting this classic O2-.-driven Fenton reaction. Interestingly, NO inhibited this iron-catalyzed hydroxylation reaction in a concentration-dependent manner such that fluxes of NO approximating those of O2-. and H2O2 virtually abolished the hydroxylation of benzoic acid. We conclude that in the absence of iron, equimolar fluxes of NO and O2-. interact to yield potent oxidants such as ONOO-/ONOOH, which oxidize organic compounds. Excess production of either radical remarkably inhibits these oxidative reactions. In the presence of low molecular weight redox-active iron complexes, NO may enhance or inhibit O2-.-dependent oxidation and hydroxylation reactions depending upon their relative fluxes.

Highlights

(ONOOH), has been suggested to represent an important pathway by which tissue may be injured during inflammation
Subsequent studies revealed that addition of ONOOϪ to a solution of nitric oxide (NO) generated by the decomposition of spermine/NO adduct (Sp/NO) transiently reduced the NO signal, suggesting an interaction between ONOOϪ and excess NO (Fig. 2)
In a similar series of experiments performed in the presence of catalase and the absence of exogenously added Feϩ3 and a constant flux of NO of 1.0 nmol/min, we found that increasing fluxes of O2. resulted in the production of rhodamine 123 (RH) in a pattern similar to that produced above such that DHR oxidation was maximal when fluxes of NO and O2. were approximately equal at 1.0 nmol/min for each (Fig. 3)

Summary

Introduction

(ONOOH), has been suggested to represent an important pathway by which tissue may be injured during inflammation. Superoxide dismutase, but not catalase, inhibited this oxidative reaction, suggesting that O2. In the presence of 5 ␮M Fe؉3-EDTA to insure optimum O2.-driven Fenton chemistry, NO enhanced modestly HX/xanthine oxidaseinduced oxidation of DHR. As expected, both superoxide dismutase and catalase inhibited this Fe-catalyzed oxidation reaction. NO in the absence of iron only modestly enhanced hydroxylation of benzoic acid from undetectable levels to 0.6 ␮M 2-hydroxybenzoate. In the presence of 5 ␮M Fe؉3-EDTA, HX/xanthine oxidasemediated hydroxylation of benzoic acid increased dramatically from undetectable levels to 4.5 ␮M of the hydroxylated product. In the presence of low molecular weight redox-active iron complexes, NO may enhance or inhibit O2.-dependent oxidation and hydroxylation reactions depending upon their relative fluxes

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