Bacterial nitroreductases are NAD(P)H-dependent flavoenzymes which catalyze the oxygen-insensitive reduction of nitroaromatics, quinones, and riboflavin derivatives. Despite their broad substrate specificity, their reactivity is very specific for two-electron, not one-electron, chemistry. We now describe the thermodynamic properties of the flavin mononucleotide cofactor of Enterobacter cloacae nitroreductase (NR), determined under a variety of solution conditions. The two-electron redox midpoint potential of NR is -190 mV at pH 7.0, and both the pH dependence of the midpoint potential and the optical spectrum of the reduced enzyme indicate that the transition is from neutral oxidized flavin to anionic flavin hydroquinone. The one-electron-reduced semiquinone states of both the free enzyme and an NR-substrate analogue complex are strongly suppressed based on optical spectroscopy and electron paramagnetic resonance measurements. This can explain the oxygen insensitivity of NR and its homologues, as it makes the execution of one-electron chemistry thermodynamically unfavorable. Therefore, we have established a chemical basis for the recent finding that a nitroreductase is a member of the soxRS oxidative defense regulon in Escherichia coli [Liochev, S. I., Hausladen, A., Fridovich, I. (1999) Proc. Natl. Acad. Sci. U.S.A. 96 (7), 3537-3539]. We also report binding affinities for the FMN cofactor in all three oxidation states either determined fluorometrically or calculated using thermodynamic cycles. Thus, we provide a detailed picture of the thermodynamics underlying the unusual activity of NR.