Nitric oxide precipitates catastrophic chromosome fragmentation by bolstering both hydrogen peroxide and Fe(II) Fenton reactants in E. coli

Immune cells kill invading microbes by producing reactive oxygen and nitrogen species, primarily hydrogen peroxide (H₂O₂) and nitric oxide (NO). We previously found that NO inhibits catalases in Escherichia coli, stabilizing H₂O₂ around treated cells and promoting catastrophic chromosome fragmentation via continuous Fenton reactions generating hydroxyl radicals. Indeed, H₂O₂-alone treatment kills catalase-deficient (katEG) mutants similar to H₂O₂+NO treatment. However, the Fenton reaction, in addition to H₂O₂, requires Fe(II), which H₂O₂ excess instantly converts into Fenton-inert Fe(III). For continuous Fenton when H₂O₂ is stable, a supply of reduced iron becomes necessary. We show here that this supply is ensured by Fe(II) recruitment from ferritins and Fe(III) reduction by flavin reductase. Our observations also concur with NO-mediated respiration inhibition that drives Fe(III) reduction. We modeled this NO-mediated inhibition via inactivation of ndh and nuo respiratory enzymes responsible for the step of NADH oxidation, which results in increased NADH pools driving flavin reduction. We found that, like the katEG mutant, the ndh nuo double mutant is similarly sensitive to H₂O₂-alone and H₂O₂+NO treatments. Moreover, the quadruple katEG ndh nuo mutant lacking both catalases and efficient respiration was rapidly killed by H₂O₂-alone, but this killing was delayed by NO, rather than potentiated by it. Taken together, we conclude that NO boosts the levels of both H₂O₂ and Fe(II) Fenton reactants, making continuous hydroxyl-radical production feasible and resulting in irreparable oxidative damage to the chromosome.