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 (H(2)O(2)) and nitric oxide (NO). We previously found that NO inhibits catalases in Escherichia coli, stabilizing H(2)O(2) around treated cells and promoting catastrophic chromosome fragmentation via continuous Fenton reactions generating hydroxyl radicals. Indeed, H(2)O(2)-alone treatment kills catalase-deficient (katEG) mutants similar to H(2)O(2)+NO treatment. However, the Fenton reaction, in addition to H(2)O(2), requires Fe(II), which H(2)O(2) excess instantly converts into Fenton-inert Fe(III). For continuous Fenton when H(2)O(2) 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 H(2)O(2)-alone and H(2)O(2)+NO treatments. Moreover, the quadruple katEG ndh nuo mutant lacking both catalases and efficient respiration was rapidly killed by H(2)O(2)-alone, but this killing was delayed by NO, rather than potentiated by it. Taken together, we conclude that NO boosts the levels of both H(2)O(2) and Fe(II) Fenton reactants, making continuous hydroxyl-radical production feasible and resulting in irreparable oxidative damage to the chromosome.