Biomedical subjects

K Keyer

Publications and source records attributed to K Keyer.

3 recordsLinked to original sources

PubMed1997

Inactivation of dehydratase [4Fe-4S] clusters and disruption of iron homeostasis upon cell exposure to peroxynitrite.

Phagocytes produce both nitric oxide and superoxide as components of the oxidative defense against pathogens. Neither molecule is likely at physiological concentrations to kill cells. However, two of their reaction products, hydrogen peroxide and peroxynitrite, are strong oxidants, cell-permeant, and toxic. Hydrogen peroxide generates oxidative DNA damage, while the primary mechanism of toxicity of peroxynitrite has not yet been determined. Recent in vitro studies indicated that peroxynitrite is capable of oxidizing the [4Fe-4S] clusters of a family of dehydratases (Hausladen, A., and Fridovich, I. (1994) J. Biol. Chem. 269, 29405-29408; Castro, L., Rodriguez, M., and Radi, R. (1994) J. Biol. Chem. 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 microM 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.

Biological Transport↗

PubMed1996

Superoxide accelerates DNA damage by elevating free-iron levels.

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 H2O2 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.

Aerobiosis↗

PubMed1995

Superoxide and the production of oxidative DNA damage.

The conventional model of oxidative DNA damage posits a role for superoxide (O2-) as a reductant for iron, which subsequently generates a hydroxyl radical by transferring the electron to H2O2. The hydroxyl radical then attacks DNA. Indeed, mutants of Escherichia coli that lack superoxide dismutase (SOD) were 10-fold more vulnerable to DNA oxidation by H2O2 than were wild-type cells. Even the pace of DNA damage by endogenous oxidants was great enough that the SOD mutants could not tolerate air if enzymes that repair oxidative DNA lesions were inactive. However, DNA oxidation proceeds in SOD-proficient cells without the involvement of O2-, as evidenced by the failure of SOD overproduction or anaerobiosis to suppress damage by H2O2. Furthermore, the mechanism by which excess O2- causes damage was called into question when the hypersensitivity of SOD mutants to DNA damage persisted for at least 20 min after O2- had been dispelled through the imposition of anaerobiosis. That behavior contradicted the standard model, which requires that O2- be present to rereduce cellular iron during the period of exposure to H2O2. Evidently, DNA oxidation is driven by a reductant other than O2-, which leaves the mechanism of damage promotion by O2- unsettled. One possibility is that, through its well-established ability to leach iron from iron-sulfur clusters, O2- 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, O2- 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.

Aerobiosis↗