Abstract
When cells are exposed to external H(2)O(2), the H(2)O(2) rapidly diffuses inside and oxidizes ferrous iron, thereby forming hydroxyl radicals that damage DNA. Thus the process of oxidative DNA damage requires only H(2)O(2), free iron, and an as-yet unidentified electron donor that reduces ferric iron to the ferrous state. Previous work showed that H(2)O(2) kills Escherichia coli especially rapidly when respiration is inhibited either by cyanide or by genetic defects in respiratory enzymes. In this study we established that these respiratory blocks accelerate the rate of DNA damage. The respiratory blocks did not substantially affect the amounts of intracellular free iron or H(2)O(2), indicating that that they accelerated damage because they increased the availability of the electron donor. The goal of this work was to identify that donor. As expected, the respiratory inhibitors caused a large increase in the amount of intracellular NADH. However, NADH itself was a poor reductant of free iron in vitro. This suggests that in non-respiring cells electrons are transferred from NADH to another carrier that directly reduces the iron. Genetic manipulations of the amounts of intracellular glutathione, NADPH, alpha-ketoacids, ferredoxin, and thioredoxin indicated that none of these was the direct electron donor. However, cells were protected from cyanide-stimulated DNA damage if they lacked flavin reductase, an enzyme that transfers electrons from NADH to free FAD. The K(m) value of this enzyme for NADH is much higher than the usual intracellular NADH concentration, which explains why its flux increased when NADH levels rose during respiratory inhibition. 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.
Highlights
(O2.) and stronger hydrogen oxidants peroxide (H2O2) [1,2,3]. than oxygen, and they can efficiently oxidize iron-sulfur clusters and protein thiols with which oxygen reacts slowly or not at all [4, 5] (Reaction 1)
Most Intracellular “Free Iron” Is in the Ferrous Form—The vulnerability of E. coli to Fenton chemistry suggests that at least some of its free iron is in the ferrous form
Free FADH2 Is an Efficient Reductant of Free Iron—These results indicate that the rate of oxidative DNA damage depends upon how quickly free iron is reduced, and they show that free FADH2 can be an efficient reductant
Summary
(O2.) and stronger hydrogen oxidants peroxide (H2O2) [1,2,3]. than oxygen, and they can efficiently oxidize iron-sulfur clusters and protein thiols with which oxygen reacts slowly or not at all [4, 5] (Reaction 1). Oxidative DNA damage is ascribed to a fourth oxygen species, the hydroxyl radical (HO1⁄7). The hydroxyl radical is powerful enough to react at diffusion-limited rates with either the base or sugar residues of DNA, leading to base modification and/or strand breakage (Reaction 4, below) [10, 11]. The pertinent metal appears to be ferrous iron. The evidence for this conclusion is that cell-permeable iron chelators fully protect intracellular DNA from exogenous H2O2 [12, 13]. The appearance of oxygen in the atmosphere allowed some organisms to develop more efficient metabolic schemes, but it raised the possibility that inadvertent chemical oxidations could disrupt metabolism and damage biomolecules. Molecular oxygen is a triplet species that is constrained to accept electrons one at a time, and its univalent redox potential is low REACTION 3, Fenton reaction
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