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 ϳ10,000-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.

Metallothionein genes are transcriptionally regulated by a number of inducers including heavy metals. Previous mutational analyses of the mouse metallothionein-I gene (mMTI) promoter have delineated a heavy-metal regulatory region between -60 and -42 relative to the transcription start site. A synthetic copy of a 12-base-pair (bp) conserved sequence located within this region was subsequently shown to confer heavy-metal regulation on a heterologous gene. However, specific disruption of this metal regulatory element (MRE) within a wild-type mMTI promoter reduced but did not eliminate the heavy-metal response. The additional metal regulatory activity was localized to an upstream region containing four sequences homologous to the identified MRE. Similar sequences were also found in multiple copies in metallothionein genes from other species. Here we test synthetic copies of all five mMTI MRE homologues for metal regulatory activity. At least four of these sequences are able to confer heavy-metal regulation on a heterologous promoter.