Nitroaromatic compounds (ArNO₂) maintain their importance in relation to industrial processes, environmental pollution, and pharmaceutical application. The manifestation of toxicity/therapeutic action of nitroaromatics may involve their single- or two-electron reduction performed by various flavoenzymes and/or their physiological redox partners, metalloproteins. The pivotal and still incompletely resolved questions in this area are the identification and characterization of the specific enzymes that are involved in the bioreduction of ArNO₂ and the establishment of their contribution to cytotoxic/therapeutic action of nitroaromatics. This review addresses the following topics: (i) the intrinsic redox properties of ArNO₂, in particular, the energetics of their single- and two-electron reduction in aqueous medium; (ii) the mechanisms and structure-activity relationships of reduction in ArNO₂ by flavoenzymes of different groups, dehydrogenases-electrontransferases (NADPH:cytochrome P-450 reductase, ferredoxin:NADP(H) oxidoreductase and their analogs), mammalian NAD(P)H:quinone oxidoreductase, bacterial nitroreductases, and disulfide reductases of different origin (glutathione, trypanothione, and thioredoxin reductases, lipoamide dehydrogenase), and (iii) the relationships between the enzymatic reactivity of compounds and their activity in mammalian cells, bacteria, and parasites.

Ferredoxin:NADP⁺ oxidoreductase from Plasmodium falciparum (PfFNR) catalyzes the NADPH-dependent reduction of ferredoxin (PfFd), which provides redox equivalents for the biosynthesis of isoprenoids and fatty acids in the apicoplast. Like other flavin-dependent electrontransferases, PfFNR is a potential source of free radicals of quinones and other redox cycling compounds. We report here a kinetic study of the reduction of quinones, nitroaromatic compounds and aromatic N-oxides by PfFNR. We show that all these groups of compounds are reduced in a single-electron pathway, their reactivity increasing with the increase in their single-electron reduction midpoint potential (E¹₇). The reactivity of nitroaromatics is lower than that of quinones and aromatic N-oxides, which is in line with the differences in their electron self-exchange rate constants. Quinone reduction proceeds via a ping-pong mechanism. During the reoxidation of reduced FAD by quinones, the oxidation of FADH^. to FAD is the possible rate-limiting step. The calculated electron transfer distances in the reaction of PfFNR with various electron acceptors are similar to those of Anabaena FNR, thus demonstrating their similar “intrinsic” reactivity. Ferredoxin stimulated quinone- and nitro-reductase reactions of PfFNR, evidently providing an additional reduction pathway via reduced PfFd. Based on the available data, PfFNR and possibly PfFd may play a central role in the reductive activation of quinones, nitroaromatics and aromatic N-oxides in P. falciparum, contributing to their antiplasmodial action.

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.

With the aim to clarify the mechanism(s) of action of nitroaromatic compounds against the malaria parasite Plasmodium falciparum, we examined the single-electron reduction by P. falciparum ferredoxin:NADP⁺ oxidoreductase (PfFNR) of a series of nitrofurans and nitrobenzenes (n = 23), and their ability to inhibit P. falciparum glutathione reductase (PfGR). The reactivity of nitroaromatics in PfFNR-catalyzed reactions increased with their single-electron reduction midpoint potential (E¹₇). Nitroaromatic compounds acted as non- or uncompetitive inhibitors towards PfGR with respect to NADPH and glutathione substrates. Using multiparameter regression analysis, we found that the in vitro activity of these compounds against P. falciparum strain FcB1 increased with their E¹₇ values, octanol/water distribution coefficients at pH 7.0 (log D), and their activity as PfGR inhibitors. Our data demonstrate that both factors, the ease of reductive activation and the inhibition of PfGR, are important in the antiplasmodial in vitro activity of nitroaromatics. To the best of our knowledge, this is the first quantitative demonstration of this kind of relationship. No correlation between antiplasmodial activity and ability to inhibit human erythrocyte GR was detected in tested nitroaromatics. Our data suggest that the efficacy of prooxidant antiparasitic agents may be achieved through their combined action, namely inhibition of antioxidant NADPH:disulfide reductases, and the rapid reduction by single-electron transferring dehydrogenases-electrontransferases.

Derivatives of tirapazamine and other heteroaromatic N-oxides (ArN→O) exhibit promising antibacterial, antiprotozoal, and tumoricidal activities. Their action is typically attributed to bioreductive activation and free radical generation. In this work, we aimed to clarify the mechanism(s) of aerobic mammalian cell cytotoxicity of ArN→O performing the parallel studies of their reactions with NADPH:cytochrome P-450 reductase (P-450R), adrenodoxin reductase/adrenodoxin (ADR/ADX), and NAD(P)H:quinone oxidoreductase (NQO1); we found that in P-450R and ADR/ADX-catalyzed single-electron reduction, the reactivity of ArN→O (n = 9) increased with their single-electron reduction midpoint potential (E¹₇), and correlated with the reactivity of quinones. NQO1 reduced ArN→O at low rates with concomitant superoxide production. The cytotoxicity of ArN→O in murine hepatoma MH22a and human colon adenocarcinoma HCT-116 cells increased with their E¹₇, being systematically higher than that of quinones. The cytotoxicity of both groups of compounds was prooxidant. Inhibitor of NQO1, dicoumarol, and inhibitors of cytochromes P-450 α-naphthoflavone, isoniazid and miconazole statistically significantly (p < 0.02) decreased the toxicity of ArN→O, and potentiated the cytotoxicity of quinones. One may conclude that in spite of similar enzymatic redox cycling rates, the cytotoxicity of ArN→O is higher than that of quinones. This is partly attributed to ArN→O activation by NQO1 and cytochromes P-450. A possible additional factor in the aerobic cytotoxicity of ArN→O is their reductive activation in oxygen-poor cell compartments, leading to the formation of DNA-damaging species similar to those forming under hypoxia.