The Reductive Power of Flavin Mononucleotide Does Not Dictate the Product Profile of the Nitroreductase, NfsA

Flavin mononucleotide (FMN)-dependent nitroreductases offer a mild and selective route to reduce nitroaromatic compounds, yet these typically fail to generate the corresponding amines. This incomplete transformation has been attributed to the two-electron redox potential of FMN. To test this hypothesis, the major nitroreductase NfsA from Escherichia coli was reconstituted with a series of FMN variants spanning midpoint potentials of -215 to -307 mV. Product profiles were examined with four substrates covering a range of electron affinities (nitrofurazone, 1,3-dinitrobenzene, 4-nitroacetophenone, and nitrobenzene), and all combinations of enzymes and substrates were found to yield only the hydroxylamine products. No amines were detected under any condition, and as a confirmation, 4-hydroxylaminacetophenone was shown to be inert to treatment with reduced nicotinamide adenine dinucleotide phosphate and NfsA reconstituted with a low-potential FMN variant (-295 mV). The inability of NsfA to generate amines is consequently not a function of the reducing potential. However, this is a determinant of the catalytic efficiency. The k_cat for nitroacetophenone turnover decreased almost 240-fold for NfsA containing an FMN variant with an E_m of -307 mV relative to that containing native FMN (-215 mV). 1,3-Dinitrobenzene experienced the smallest decrease of 52-fold in the same comparison. Redox tuning of NfsA can therefore be detrimental to catalytic efficiency and fails to generate the desired amine products. A renewed focus on active site properties is recommended for engineering new catalysts to promote nitroaromatic to arylamine conversion.