Flavin Thermodynamics Explain the Oxygen Insensitivity of Enteric Nitroreductases

Abstract

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.

Microbial community structure and function of Nitrobenzene reduction biocathode in response to carbon source switchover

“…9B). The measured midpoint potential of the FMN cofactor of Enterobacter cloacae nitroreductase was À0.19 V (Koder et al, 2002), consistent with our observation that NB reduction with the gBC was found to start at around À0.20 V (Fig. 2B).…”

“…Diverse functional genes related to nitroaromatics reduction compounds using NAD(P)H as electrons donor (Koder et al, 2002). In this study, 32 nitroreductase genes were detected that came from 19 genera and 3 uncultured proteobacteria (a and g) among 5 classes of Actinobacteria, Bacilli, a-proteobacteria, b-proteobacteria and g-proteobacteria.…”

Structural Basis of 5-Nitroimidazole Antibiotic Resistance

“…Thus, the first step of the antibiotic reduction becomes R-NO 2 3 RNϭO (Fig. 7, Step ❷), similar to other bacterial nitro reductases (38). Importantly, this 2-electron reduction avoids formation of the toxic nitro radical anion ( Fig.…”

Ligand-induced Conformational Changes in the Capping Subdomain of a Bacterial Old Yellow Enzyme Homologue and Conserved Sequence Fingerprints Provide New Insights into Substrate Binding

“…Interestingly, all of the other structurally characterized OYEs to date have a leucine instead of methionine at position 25, and they all exhibit planar FMNs in the oxidized state. It has been proposed that a bent flavin would destabilize the formation of semiquinones and thus one-electron reactions of the flavin (39). It is interesting to note that semiquinone species were observed during the photoreduction of OPR1 (13) and OYE (40), but not of YqjM (41) and SYE1 (31).…”