Superoxide promotes hydroxyl-radical formation and consequent DNA damage in cells of all types. The long-standing hypothesis that it primarily does so by delivering electrons to adventitious iron on DNA was refuted by recent studies in Escherichia coli. Alternative proposals have suggested that superoxide may accelerate oxidative DNA damage by leaching iron from storage proteins or enzymic [4Fe-4S] clusters. The released iron might then deposit on the surface of the DNA, where it could catalyze the formation of DNA oxidants using other electron donors. The latter model is affirmed by the experiments described here. Whole-cell electron paramagnetic resonance demonstrated that the level of loose iron in superoxide-stressed cells greatly exceeds that of unstressed cells. Bacterial iron storage proteins were not the major source for free iron, since superoxide also increased iron levels in mutants lacking these iron storage proteins. However, overproduction of an enzyme containing a labile [4Fe-4S] cluster dramatically increased the free iron content of cells when they were growing in air. The rates of spontaneous mutagenesis and DNA damage from exogenous H 2 O 2 increased commensurately. It is striking that both growth defects and DNA damage caused by superoxide ensue from its ability to damage a subset of iron-sulfur clusters.

, which leaves the mechanism of damage promotion by O 2 ؊ unsettled. One possibility is that, through its well-established ability to leach iron from iron-sulfur clusters, O 2 ؊ increases the amount of free iron that is available to catalyze hydroxyl radical production. Experiments with iron transport mutants confirmed that increases in free-iron concentration have the effect of accelerating DNA oxidation. Thus, O 2 ؊ may be genotoxic only in doses that exceed those found in SOD-proficient cells, and in those limited circumstances it may promote DNA damage by increasing the amount of DNA-bound iron.

269, 29409 -29415).We demonstrate here that peroxynitrite at 1% of its lethal dose almost fully inactivated the labile dehydratases in Escherichia coli. The rate at which peroxynitrite inactivated the clusters substantially exceeded the rate at which it oxidized thiols or spontaneously decomposed. These results suggest that these dehydratases may be primary targets of peroxynitrite in vivo. Another consequence of the cluster damage was the release of 100 M iron into the cytosol. During phagocytosis, this intracellular free iron could increase lethal DNA damage by hydrogen peroxide or protein modification by additional peroxynitrite. In response to peroxynitrite challenges, E. coli rapidly sequestered the intracellular free iron using an undefined scavenging system. The iron-sulfur clusters were more gradually repaired by a process that drew iron from its iron-storage proteins. These are likely to be critical events in the struggle between phagocyte and pathogen.