Abstract
Oxygen-insensitive NAD(P)H:nitroreductases (NR) reduce nitroaromatics (Ar-NO2) into hydroxylamines (Ar-NHOH) through nitroso (Ar-NO) intermediates. Ar-NO may be reduced both enzymatically and directly by reduced nicotinamide adenine dinucleotide or its phosphate NAD(P)H, however, it is unclear which process is predominant in catalysis of NRs. We found that E. coli NR-A (NfsA) oxidizes 2 mol of NADPH per mol of 2,4,6-trinitrotoluene (TNT) and 4 mol of NADPH per mol of tetryl. Addition of ascorbate, which reduces Ar-NO into Ar-NHOH, changes the stoichiometry NADPH/Ar-NO2 into 1:1 (TNT) and 2:1 (tetryl), and decreases the rate of NADPH oxidation. Ascorbate does not interfere with the oxidation of NADPH during reduction of quinones by NfsA. Our analysis of ascorbate inhibition patterns and both enzymatic and non-enzymatic reduction of nitrosobenzene suggests that direct reduction of Ar-NO by NADPH rather than enzymatic reduction is the predominant mechanism during nitroaromatic reduction.
Highlights
The toxic and/or therapeutic action of nitroaromatic compounds (Ar-NO2 ) is most frequently attributed to the enzymatic reduction of their nitro group(s)
NADPH oxidation by nitrosobenzene may exceed the rate of its enzymatic oxidation (Figure 2a)
In NfsA-catalyzed NADPH oxidation by nitroaromatics, ascorbate decreases the amount of NADPH oxidized per Ar-NO2 and the reaction rate by ~2-fold (Figures 3 and 4)
Summary
The toxic and/or therapeutic action of nitroaromatic compounds (Ar-NO2 ) is most frequently attributed to the enzymatic reduction of their nitro group(s). The two-electron reduction of nitroaromatics by bacterial nitroreductases (NRs) or mammalian DT-diaphorase results in the formation of nitroso-(Ar-NO), and, subsequently, hydroxylamine (Ar-NHOH) products (Scheme 1), which alkylate DNA and other biomolecules [2]. In addition to NAD(P)H, nitrosobenzenes are rapidly by other reductants, including of NRs. thissupposition work, we found that ascorbate reducedofthe and the of Furtherascorbate evidence forInthis is provided by reports thestoichiometry direct oxidation of rate by NADPH oxidation in reactions containing nitroaromatic substrates and E. coli NR-A. 1-nitroso-2-naphthol [13], and 5-(aziridin-1-yl)-2-nitro-4-nitrosobenzamide [14].(NfsA), and did inhibit to theNAD(P)H, analogous reactions of single-electron transferring flavoenzymes and FNR. 1. The single-electron (E17) of nitroaromatic compounds andand the subsequent steady-state rate constants of their reduction by. NfsA-catalyzed reduction nitrosobenzene were from the differences in reaction rates of in nitrosobenzene may evenof exceed the rate of obtained enzymatic oxidation.
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