The mechanism of oxygen and nitric oxide toxicity in Escherichia coli

Oxidative DNA damage requires only H₂O₂, free iron, and an as-yet unidentified electron donor that reduces ferric iron to the ferrous state. In this study I established that respiratory blocks accelerate the rate of DNA damage because they increased the availability of the electron donor. The goal of this work was to identify that donor. Cells were protected from cyanide-stimulated DNA damage if they lacked flavin reductase, an enzyme that transfers electrons from NADH to free FAD. Flavins that were reduced by purified flavin reductase rapidly transferred electrons to free iron and drove a DNA-damaging Fenton system in vitro. Thus the rate of oxidative DNA damage can be limited by the rate at which electron donors reduce free iron, and reduced flavins become the predominant donors in E. coli when respiration is blocked. It remains unclear whether flavins or other reductants drive Fenton chemistry in respiring cells.; The presence of nitric oxide (NO) greatly accelerates the rate at which hydrogen peroxide (H₂O₂) kills E. coli. The goal of this study was to determine the mechanism of this synergism. The filamentation of the dead cells, and their protection by cell-permeable iron chelators, indicated that NO/H₂O₂ killed cells by damaging their DNA through the Fenton reaction. NO also blocked respiration, an event which previous studies have shown can stimulate oxidative DNA damage. The resultant accumulation of NADH accelerates the reduction of free flavins by flavin reductase, and these reduced flavins drive Fenton chemistry by tranferring electrons to free iron. The possibility that H₂O₂ and NO synergize when macrophages attack captive bacteria is discussed.