Synthesis and spectroscopic characterization of 13 C4 -labeled 4-cyano-2-oxobutyraldehyde semicarbazone: A metabolite of nitrofurazone.

Nitrofurazone usage in food-producing animals is prohibited in most countries, including the United States. Regulatory agencies regularly monitor its use in domestic, export/import animals' food products by measuring the semicarbazide (SEM) metabolite as a biomarker of nitrofurazone exposure. However, the use of SEM is controversial because it is also produced in food naturally and thus gives false positive results. A cyano-metabolite, 4-cyano-2-oxobutyraldehyde semicarbazone (COBS), is proposed as an alternate specific marker of nitrofurazone to distinguish nitrofurazone from treated or untreated animals. A synthetic method was developed to produce COBS via metallic hydrogenation of nitrofurazone. The product was isolated and characterized by one- and two-dimensional nuclear magnetic spectroscopy (NMR) experiments, Fourier-transform infrared spectroscopy (FT-IR), and mass spectrometry. The developed synthetic procedure was further extended to synthesize isotopically labeled 4-[13 C]-cyano-2-oxo- [2, 3, 4-13 C3 ]-butyraldehyde semicarbazone. Labeled COBS is useful as an internal standard for its quantification in food-producing animals. Thus, the developed method provides a possibility for its commercial synthesis to procure COBS. This is the first synthesis of the alternate specific marker metabolite of nitrofurazone for possible usage in regulatory analysis to solve a real-world problem.

Furazolidone and Nitrofurazone Metabolic Studies in Crucian Carp by Ultra-Performance Liquid Chromatography Tandem Mass Spectrometry.

In this work, the detection of the furazolidone (FZD) and nitrofurazone (NFZ) metabolites residuals in crucian carp are focused. Crucian carps of identical size were exposed to the mixed nitrofuran antibiotics under optimized bath conditions at a concentration of 50 mg/L, 26 ± 0.5°C for 24 h. Then, liquid chromatography-electrospray ionization-triple quadrupole mass spectrometry (LC-ESI-MSMS) was performed after the drug exposure experiments when the nitrofuran metabolites were enriched in organisms. During the period of 0-144 h, residue levels of the 3-amino-2-oxazolidinone (AOZ) gradually decreased with a prolonged sampling time. The changing trend in semicarbazide (SEM) with the sample collection duration is divided into two stages, and its concentration showed a trend of rising first and then falling. The metabolite concentration-time curve demonstrates that 24 h was used as a sampling time, and fish muscle was selected as tissue samples in the further quantitative study. A novel crucian carp-enrichment procedure coupled to LC-ESI-MSMS quantitative method was further explored based on much metabolite data. According to the exponential curve of the SEM-to-AOZ concentration ratio at a precisely designed FZD-to-NFZ mass ratio, the final FZD content of the veterinary NFZ antibiotics was 0.069 ± 0.005% (in terms of mass).

Nitrofurazone degradation in the self-biased bio-photoelectrochemical system: g-C3N4/CdS photocathode characterization, degradation performance, mechanism and pathways.

In this study, a self-biased bio-photoelectrochemical system (SB-BPES) was constructed using a bioanode and the g-C3N4/CdS heterojunction photocathode for nitrofurazone (NFZ) degradation under solar irradiation. The physio-chemical properties and optical performance of photocatalysts were characterized, and photo-electrochemical properties of various photocathodes were analyzed. Results showed that g-C3N4/CdS exhibited the broadest visible light absorption range (to 594 nm) and the most efficient e--h+ separation; and its corresponding photocathode showed the highest photocurrent (9.8 µA), and the lowest charge transfer resistance (5.43 ☓ 103 Ω). In the solar-illuminated SB-BPES with g-C3N4/CdS photocathode, about 80% of NFZ removal rate was achieved within 10 h. More importantly, TOC removal of 62.6% was achieved in 24 h, which was 1.8 times of that from the open circuit SB-BPES, and 4.3 folds of that from microbial degradation; also, about 1.5 times of those from SB-BPES with g-C3N4 and CdS photocathodes. Besides, reproducible current generations (∼1.0 mA) were produced. These verified that it was a self-sustained system for spontaneously pollutants degradation and electricity generation. Moreover, possible degradation mechanism and pathways were proposed according to the identified intermediates. This study provides inspiration for synchronic improving refractory organics degradation and net energy recovery.

EGCG and enzymatic cross-linking combined treatments for improving elastin stability and reducing calcification in bioprosthetic heart valves.

The failures of glutaraldehyde (GLUT) cross-linked bioprosthetic heart valves (BHVs) are mainly due to degeneration and calcification. In this study, we developed a new preparation strategy for BHVs named as "HPA/EDC/EGCG" that utilized 3,4-hydroxyphenylpropionic acid (HPA)-conjugated pericardium, epigallocatechin gallate (EGCG), and horseradish peroxidase (HRP)/hydrogen peroxide (H2 O2 ) enzymatic cross-linking. HPA-pericardium conjugation was done by carbodiimide coupling reaction using 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS). Then HPA-conjugated pericardium was cross-linked by HRP/H2 O2 enzyme-catalyzed oxidation. The feeding ratios of HPA and EGCG were optimized. The consumption of amino groups, collagenase and elastase degradation in vitro, biomechanics, extracellular matrix stability, and calcification of HPA-/EDC-/EGCG-treated pericardiums were characterized. We demonstrated that HPA-/EDC-/EGCG-treated pericardiums had better elastin stabilization and less calcification. EGCG and enzymatic cross-linking treated pericardiums showed improved mechanical properties. This new EGCG and enzymatic cross-linking strategy would be a promising method to make BHVs with better elastin stability and anti-calcification property. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 1551-1559, 2019.

The health risk for seafood consumers under future ocean acidification (OA) scenarios: OA alters bioaccumulation of three pollutants in an edible bivalve species through affecting the in vivo metabolism.

The current knowledge about the effect of pCO2-driven ocean acidification on the bioaccumulation of pollutants in marine species is still scarce, as only limited types of pollutants have been investigated. Therefore, to obtain a better understanding of the effect of ocean acidification on the process of bioaccumulation and subsequent food safety, the accumulation of benzo[a]pyrene (B[a]P), chloramphenicol (CAP), and nitrofurazone (NFZ) in an edible bivalve species, Tegillarca granosa, under present and near-future ocean acidification scenarios was investigated in the present study. The health risks associated with consuming contaminated blood clams were also assessed using target hazard quotient (THQ), lifetime cancer risk (CR), or margin of exposure (MoE). To explain the alterations in bioaccumulation of these pollutants, the expressions of genes encoding corresponding key metabolic proteins were analyzed as well. The results obtained showed that ocean acidification exerted a significant effect on the accumulation of B[a]P, NFZ, and CAP in the clams. After four-week exposure to B[a]P, NFZ, or CAP contaminated seawater acidified with CO2 at pH 7.8 and 7.4, significantly greater amounts of B[a]P and lower amounts of NFZ and CAP were accumulated in the clams compared to that in the control. Although no non-carcinogenic risk of consuming B[a]P-contaminated blood clams was detected using the THQ values obtained, the CR values obtained indicated a high life-time risk in all groups. In addition, according to the MoE values obtained, the health risks in terms of consuming NFZ- and CAP-contaminated clams were significantly reduced under ocean acidification scenarios but still cannot be ignored, especially for children. The gene expression results showed that the ability of clams to eliminate B[a]P may be significantly constrained, whereas the ability to eliminate NFZ and CAP may be enhanced under ocean acidification scenarios, indicating that the changes in the accumulation of these pollutants may be due to the altered in vivo metabolism.

Targeting Leishmania amazonensis amastigotes through macrophage internalisation of a hydroxymethylnitrofurazone nanostructured polymeric system.

Dextran-coated poly (n-butyl cyanoacrylate) nanoparticles (PBCA-NPs) were prepared and were evaluated for enhanced delivery of a promising anti-Leishmania drug candidate, hydroxymethylnitrofurazone (NFOH), to phagocytic cells. Currently available chemotherapy for leishmaniasis, such as pentavalent antimonials, presents low safety and efficacy. Furthermore, widespread drug resistance in leishmaniasis is rapidly emerging. To overcome these drawbacks, the use of nanosized delivery systems can reduce systemic drug toxicity and increase the drug concentration in infected macrophages, therefore improving treatment of leishmaniasis. PBCA-NPs containing NFOH (PBCA-NFOH-NPs) were prepared by an anionic emulsion polymerisation method. The z-average and polydispersity index (PDI) were determined by photon correlation spectroscopy, the zeta potential by microelectrophoresis and the entrapment efficiency by HPLC. Cytotoxicity was determined using macrophages from BALB/c mice. Efficacy tests were performed using Leishmania amazonensis promastigotes and amastigotes. The z-average of PBCA-NFOH-NPs was 151.5 ± 61.97 nm, with a PDI of 0.104 ± 0.01, a zeta potential of -10.1 ± 6.49 mV and an entrapment efficiency of 64.47 ± 0.43%. Efficacy in amastigotes revealed IC50 values of 0.33 µM and 31.2 µM for the nanostructured and free NFOH, respectively (95-fold increase). The cytotoxicity study indicated low toxicity of the PBCA-NFOH-NPs to macrophages. The selectivity index was 370.6, which is 49-fold higher than free NFOH (7.6). Such findings indicated that improved efficacy could be due to NP internalisation following site-specific drug delivery and reactivation of immune protective reactions by the NP components. Thus, PBCA-NFOH-NPs have the potential to significantly improve the treatment of leishmaniasis, with reduced systemic side effects.

Identification and quantification of nitrofurazone metabolites by ultraperformance liquid chromatography-quadrupole time-of-flight high-resolution mass spectrometry with precolumn derivatization.

An ultraperformance liquid chromatography-quadrupole time-of-flight high-resolution mass spectrometry method was developed and validated for the determination of nitrofurazone metabolites. Precolumn derivatization with 2,4-dinitrophenylhydrazine and p-dimethylaminobenzaldehyde as an internal standard was used successfully to determine the biomarker 5-nitro-2-furaldehyde. In negative electrospray ionization mode, the precise molecular weights of the derivatives were 320.0372 for the biomarker and 328.1060 for the internal standard (relative error 1.08 ppm). The matrix effect was evaluated and the analytical characteristics of the method and derivatization reaction conditions were validated. For comparison purposes, spiked samples were tested by both internal and external standard methods. The results show high precision can be obtained with p-dimethylaminobenzaldehyde as an internal standard for the identification and quantification of nitrofurazone metabolites in complex biological samples. Graphical Abstract A simplified preparation strategy for biological samples.

Identification of novel members of the bacterial azoreductase family in Pseudomonas aeruginosa.

Azoreductases are a family of diverse enzymes found in many pathogenic bacteria as well as distant homologues being present in eukarya. In addition to having azoreductase activity, these enzymes are also suggested to have NAD(P)H quinone oxidoreductase (NQO) activity which leads to a proposed role in plant pathogenesis. Azoreductases have also been suggested to play a role in the mammalian pathogenesis of Pseudomonas aeruginosa. In view of the importance of P. aeruginosa as a pathogen, we therefore characterized recombinant enzymes following expression of a group of putative azoreductase genes from P. aeruginosa expressed in Escherichia coli. The enzymes include members of the arsenic-resistance protein H (ArsH), tryptophan repressor-binding protein A (WrbA), modulator of drug activity B (MdaB) and YieF families. The ArsH, MdaB and YieF family members all show azoreductase and NQO activities. In contrast, WrbA is the first enzyme to show NQO activity but does not reduce any of the 11 azo compounds tested under a wide range of conditions. These studies will allow further investigation of the possible role of these enzymes in the pathogenesis of P. aeruginosa.

A selective biomarker for confirming nitrofurazone residues in crab and shrimp using ultra-performance liquid chromatography-tandem mass spectrometry.

Reliably detecting nitrofurazone (NFZ) residues in farmed crab and shrimp was previously hindered by lack of appropriately specific analytical methodology. Parent NFZ rapidly breaks down in meat, and the commonly used side-chain metabolite, semicarbazide (SEM), is non-specific as it occurs naturally in crustacean shell often leading to 'false positive' detections in meat. Using 5-nitro-2-furaldehyde (NF) as marker metabolite, following pre-column derivatization with 2,4-dinitrophenylhydrazine (DNPH), ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) analysis in negative electrospray ionization mode enabled confirmation of NFZ residues in deliberately treated whole crab, crab meat and shrimp meat, with a limit of detection (LOD) and limit of quantification (LOQ) below 1 ng g(-1). Meanwhile, the derivatives of DNPH-NF were synthesized for the first time, purified by preparative liquid chromatography and structure characterized with nuclear magnetic resonance spectroscopy ((1)H-NMR). The purity of derivative was checked by ultra-performance liquid chromatography-tunable ultraviolet (UPLC-TUV), and the contents were beyond 99.9%. For comparison purposes, crustacean samples were analysed using both NF and SEM marker metabolites. NFZ treatment was revealed by both NF and SEM marker metabolites, but untreated crab also showed measurable levels of SEM which could potentially be misinterpreted as evidence of illegal NFZ use.

A simple and inexpensive method to control oxygen concentrations within physiological and neoplastic ranges.

Traditional methods for regulating oxygen concentration ([O2]) in in vitro experiments over the range found in normal and tumor tissues require the use of expensive equipment to generate controlled gas atmospheres or the purchase of a range of gas cylinders with certified O2 percentages. Here we describe a simple and inexpensive enzymatic method for generating low, precise steady-state [O2] levels that are stable for several hours. This method is particularly applicable to the in vitro study of some classes of hypoxia-targeted antitumor prodrugs and bioreductively activated agents.

An unusually cold active nitroreductase for prodrug activations.

A set of PCR primers based on the genome sequence were used to clone a gene encoding a hypothetical nitroreductases (named as Ssap-NtrB) from uropathogenic staphylococcus, Staphylococcus saprophyticus strain ATCC 15305, an oxygen insensitive flavoenzyme. Activity studies of the translation product revealed that the nitroreductase catalyses two electron reduction of a nitroaromatic drug of nitrofurazone (NFZ), cancer prodrugs of CB1954 and SN23862 at optimum temperature of 20 °C together with retaining its maximum activity considerably at 3 °C. The required electrons for such reduction could be supplied by either NADH or NADPH with a small preference for the latter. The gene was engineered for heterologous expression in Escherichia coli, and conditions were found in which the enzyme was produced in a mostly soluble form. The recombinant enzyme was purified to homogeneity and physical, spectral and catalytical properties were determined. The findings lead us to propose that Ssap-NtrB represents a novel nitro reductase with an unusual cold active property, which has not been described previously for prodrug activating enzymes of nitroreductases.

Characterization of the interaction between nitrofurazone and human serum albumin by spectroscopic and molecular modeling methods.

The interaction of nitrofurazone (NF) and human serum albumin (HSA) has been studied by fluorescence spectroscopy, FT-IR spectroscopy and molecular modeling methods. The results showed that the fluorescence of HSA was quenched by NF in a static quenching mechanism. Thermodynamic parameters revealed that hydrogen bonds and van der Waals force played the major role during the interaction. The calculated binding distance (r) indicated that the non-radioactive energy transfer came into being in the interaction between NF and HSA. HSA had a single class of binding site at Sudlow' site I in subdomain IIA for NF, which was verified by the displacement experiment. The molecular modeling study further confirmed the specific binding sites of NF on HSA, such as the interaction between N11 and N14 of NF with Lue 283 and Ser 287 predominately through hydrogen bonds. Three-dimensional fluorescence spectra indicated that the polarity around the tryptophan residues decreased and the conformation of HSA changed after adding NF. FT-IR spectra showed that NF could induce the polypeptides of HSA unfolding because it changed α-helix and ß-sheet into ß-turn and random structure of HSA.

A prodrug approach to improve the physico-chemical properties and decrease the genotoxicity of nitro compounds.

In therapeutics research, the nitro compounds are part of an important group of drugs with multiple pharmacological activities. However, in drug design, the inclusion of a nitro group in a molecule changes the physico-chemical and electronic properties and is associated with increased mutagenicity and carcinogenicity. In addition, several studies have related the relationship between the antimicrobial and/or anti-protozoal activity and the mutagenic effect to reduction of the nitro group. This work reviews the toxicity of nitro compounds and shows how the use of prodrugs can increase the biological activity and decrease the genotoxicity of nitro compounds, without any modification in nitro reduction behavior, but rather by physico-chemical improvement. Examples are given of metronidazole and nitrofurazone prodrugs.

Cyano metabolite as a biomarker of nitrofurazone in channel catfish.

The use of nitrofuran drugs in food-producing animals continues to attract international concern as a food safety issue. Methods for monitoring nitrofuran residues have been directed to the intact side chain of tissue-bound metabolites. Semicarbazide, the side chain of nitrofurazone (NFZ), can enter food products from non-NFZ sources, suggesting the need for an alternative biomarker for confirmatory purposes. We characterized a cyano derivative as a major metabolite of NFZ in channel catfish (Ictalurus punctatus). The depletion of cyano metabolite was examined in the muscle of channel catfish after oral dosing (10 mg of NFZ/kg of body weight). Parent NFZ was rapidly eliminated in muscle, with a half-life of 6.3 h. The cyano metabolite was detected for up to 2 weeks, with an elimination half-life of 81 h. The cyano metabolite represents an alternative biomarker for confirming the use of NFZ in channel catfish.

Steady-state and stopped-flow kinetic studies of three Escherichia coli NfsB mutants with enhanced activity for the prodrug CB1954.

The enzyme nitroreductase, NfsB, from Escherichia coli has entered clinical trials for cancer gene therapy with the prodrug CB1954 [5-(aziridin-1-yl)-2,4-dinitrobenzamide]. However, CB1954 is a poor substrate for the enzyme. Previously we made several NfsB mutants that show better activity with CB1954 in a cell-killing assay in E. coli. Here we compare the kinetic parameters of wild-type NfsB with CB1954 to those of the most active single, double, and triple mutants isolated to date. For wild-type NfsB the global kinetic parameters for both k(cat) and K(m) for CB1954 are about 20-fold higher than previously estimated; however, the measured specificity constant, k(cat)/K(m) is the same. All of the mutants are more active with CB1954 than the wild-type enzyme, the most active mutant showing about 100-fold improved specificity constant with CB1954 over the wild-type protein with little effect on k(cat). This enhancement in specificity constants for the mutants is not seen with the antibiotic nitrofurazone as substrate, leading to reversed nitroaromatic substrate selectivity for the double and triple mutants. However, similar enhancements in specificity constants are found with the quinone menadione. Stopped-flow kinetic studies suggest that the rate-determining step of the reaction is likely to be the release of products. The most active mutant is also selective for the 4-nitro group of CB1954, rather than the 2-nitro group, giving the more cytotoxic reduction product. The double and triple mutants should be much more effective enzymes for use with CB1954 in prodrug-activation gene therapy.

Interaction between nitroheterocyclic compounds with beta-cyclodextrins: phase solubility and HPLC studies.

Chagas disease is a serious health problem for Latin America. Nitrofurazone (NF) and Hidroxymethylnitrofurazone (NFOH) are active against Trypanosoma cruzi. The effect of beta-cyclodextrin (beta-CD) and dimethyl-beta-cyclodextrin (DM-beta-CD) complexation on the UV absorption and retention time of nitrofurazone (NF) and its hydroxymethylated analog (NFOH) were studied in solution. The retention behavior was analyzed on a reversed phase C18 column and the mobile phase used was acetonitrile-water (20/80 v/v), in which cyclodextrins (beta-CD or DM-beta-CD) were incorporated as a mobile phase additive. The decrease in the retention times of NF (or NFOH) with increasing concentration of HP-beta-CD enables the determination of the complex stability constants by HPLC. A phase-solubility study was performed, according to the method reported by Higuchi and Connors, to evaluate the changes of NF/NFOH in the complexation state, and the diagrams obtained suggested that it forms complexes with a stoichiometry of 1:1. This is an important study for the characterization of potential formulations to be used as therapeutic options for the treatment of Chagas disease.

Kinetic and structural characterisation of Escherichia coli nitroreductase mutants showing improved efficacy for the prodrug substrate CB1954.

Escherichia coli nitroreductase (NTR) is a flavoprotein that reduces a variety of quinone and nitroaromatic substrates. Among these substrates is the prodrug 5-[aziridin-1-yl]-2,4-dinitrobenzamide (CB1954) that is activated by NTR to form two products, one of which is highly cytotoxic. NTR in combination with CB1954 has entered clinical trials for virus-directed enzyme-prodrug therapy of cancer. Enhancing the catalytic efficiency of NTR for CB1954 is likely to improve the therapeutic potential of this system. We previously identified a number of mutants at six positions around the active site of NTR that showed enhanced sensitisation to CB1954 in an E. coli cell-killing assay. In this study we have purified improved mutants at each of these positions and determined their steady-state kinetic parameters for CB1954 and for the antibiotic nitrofurazone. We have also made a double mutant, combining two of the most beneficial single mutations. All the mutants show enhanced specificity constants for CB1954, and, apart from N71S, the enhancement is selective for CB1954 over nitrofurazone. One mutant, T41L, also shows an increase in selectivity for reducing the 4-nitro group of CB1954 rather than the 2-nitro group. We have determined the three-dimensional structures of selected mutants bound to the substrate analogue nicotinic acid, using X-ray crystallography. The N71S mutation affects interactions of the FMN cofactor, while mutations at T41 and F124 affect the interactions with nicotinic acid. The structure of double mutant N71S/F124K combines the effects of the two individual single mutations, but it gives a greater selective enhancement of activity with CB1954 over nitrofurazone than either of these, and the highest specificity constant for CB1954 of all the mutations studied.

Multi-residue monitoring for the simultaneous determination of five nitrofurans (furazolidone, furaltadone, nitrofurazone, nitrofurantoine, nifursol) in poultry muscle tissue through the detection of their five major metabolites (AOZ, AMOZ, SEM, AHD, DNSAH) by liquid chromatography coupled to electrospray tandem mass spectrometry--in-house validation in line with Commission Decision 657/2002/EC.

Following the ban of four nitrofurans in the mid-90s (furazolidone, furaltadone, nitrofurantoine, nitrofurazone), the nifursol, a veterinary drug from the nitrofuran class of antibacterials which has been used prophylactically as feed additive for treating turkeys against histomoniasis (blackhead disease) was also declared in Annex IV of the European Union Directive no. 90/2377/EC in 2002 according to the Regulation no. 1756/2002/EC. As for the four other nitrofurans, nifursol disappears from tissues within a few days after treatment of food-producing animals. But toxic metabolites are still present for longer periods (several weeks or even months). The major metabolite that can readily be monitored in the tissues following nifursol abuse is the 3,5-dinitro-salicylic acid hydrazine (DNSAH). This article displays some improvements and the revalidation of the analytical method by liquid chromatography coupled to electrospray tandem mass spectrometry (LC-esiMS/MS) already in use in our laboratory for monitoring nitrofuran metabolites but also including the nifursol metabolite at the confirmatory minimum required performance level (MRPL) of 1 microg kg(-1). The validation is applied both to artificially and to naturally incurred turkey muscle.

Depletion of four nitrofuran antibiotics and their tissue-bound metabolites in porcine tissues and determination using LC-MS/MS and HPLC-UV.

Depletion of the nitrofuran antibiotics furazolidone, furaltadone, nitrofurantoin and nitrofurazone and their tissue-bound metabolites AOZ, AMOZ, AHD and SEM from pig muscle, liver and kidney tissues is described. Groups of pigs were given feed medicated with one of the nitrofuran drugs at a therapeutic concentration (400?mg?kg(-1)) for ten days. Animals were slaughtered at intervals and tissue samples collected for analysis for six weeks following withdrawal of medicated feed. These samples were analysed both for parent nitrofurans (using LC-MS/MS and HPLC-UV), and for tissue-bound metabolites (using LC-MS/MS). The parent drugs were detectable only sporadically and only in pigs subjected to no withdrawal period whatsoever. This confirms the instability of the four major nitrofuran antibiotics in edible tissues. In contrast, the metabolites accumulated to high concentrations in tissues (ppm levels) and had depletion half lives of between 5.5 and 15.5 days. The metabolites of all four drugs were still readily detectable in tissues six weeks after cessation of treatment. This emphasizes the benefits of monitoring for the stable metabolites of the nitrofurans.

Structural and mechanistic studies of Escherichia coli nitroreductase with the antibiotic nitrofurazone. Reversed binding orientations in different redox states of the enzyme.

The antibiotics nitrofurazone and nitrofurantoin are used in the treatment of genitourinary infections and as topical antibacterial agents. Their action is dependent upon activation by bacterial nitroreductase flavoproteins, including the Escherichia coli nitroreductase (NTR). Here we show that the products of reduction of these antibiotics by NTR are the hydroxylamine derivatives. We show that the reduction of nitrosoaromatics is enzyme-catalyzed, with a specificity constant approximately 10,000-fold greater than that of the starting nitro compounds. This suggests that the reduction of nitro groups proceeds through two successive, enzyme-mediated reactions and explains why the nitroso intermediates are not observed. The global reaction rate for nitrofurazone determined in this study is over 10-fold higher than that previously reported, suggesting that the enzyme is much more active toward nitroaromatics than previously estimated. Surprisingly, in the crystal structure of the oxidized NTR-nitrofurazone complex, nitrofurazone is oriented with its amide group, rather than the nitro group to be reduced, positioned over the reactive N5 of the FMN cofactor. Free acetate, which acts as a competitive inhibitor with respect to NADH, binds in a similar orientation. We infer that the orientation of bound nitrofurazone depends upon the redox state of the enzyme. We propose that the charge distribution on the FMN rings, which alters upon reduction, is an important determinant of substrate binding and reactivity in flavoproteins with broad substrate specificity.