Flavin Thermodynamics Explain the Oxygen Insensitivity of Enteric Nitroreductases†

Ronald L. Koder, Chad A. Haynes, Michael E. Rodgers, David W. Rodgers, Anne-Frances Miller
2002 Biochemistry  
Bacterial nitroreductases are NAD(P)H-dependent flavoenzymes which catalyze the oxygen-insensitive reduction of nitroaromatics, quinones and riboflavin derivatives. The thermodynamic properties of the FMN cofactor of Enterobacter cloacae nitroreductase (NR) have been determined under a variety of solution conditions. The two-electron redox midpoint potential of NR is -190 mV at pH 7.0 and the pH dependence of the midpoint potential and the optical spectrum of the reduced enzyme both indicate
more » ... t the transition is from neutral oxidized flavin to anionic flavin hydroquinone. The oneelectron reduced semiquinone states of both the free enzyme and an NR-substrate analogue complex are thermodynamically and kinetically inaccessible based on optical and by electron paramagnetic resonance spectroscopies. The strong destabilization of the FMN semiquinone provides a basis for the oxygen insensitivity of NR homologues as it makes the execution of one-electron chemistry thermodynamically unfavorable. This establishes a chemical basis for the recent finding that a nitroreductase is a member of the soxRS oxidative defense regulon in E. coli (Liochev, S. I., Hausladen, A., Fridovich, I., (1999) Proc. Nat. Acad. Sci. 96(7), 3537-9). Binding affinities for the FMN cofactor in all three oxidation states have been either determined fluorometrically or calculated using thermodynamic cycles. The coupling between homodimer stability and flavin binding has been investigated using analytical ultracentrifugation, resulting in a detailed description of the thermodynamic properties of flavin binding in NR. Possible structural features responsible for these active site thermodynamics are discussed. 3 Introduction:
doi:10.1021/bi025805t pmid:12450383 fatcat:7id4ac4psjfo3k3ugacffrhg4i