Reduced Flavins Promote Oxidative DNA Damage in Non-respiringEscherichia coli by Delivering Electrons to Intracellular Free Iron

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, ␣-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.

Transcriptional Regulation of the Respiratory Genes in the CyanobacteriumSynechocystissp. PCC 6803 during the Early Response to Glucose Feeding

“…Expression of group I genes was inhibited by 6-AN, but not by electron transport inhibitors Hg 21 and CN 2 . The G6PDH inhibitor would inhibit NADPH production by G6PDH in the presence of Glc and hence would maintain a lower intracellular NADPH to NADP 1 ratio, whereas the direct (by Hg 21 ) or indirect (by CN 2 ) NADPH oxidation inhibitors would keep NADPH in a reduced state, which would maintain a higher intracellular NADPH to NADP 1 ratio (Woodmansee and Imlay, 2002). Thus, expression of group I genes is likely to be up-regulated by high cellular redox potentials.…”

Reduction of Fe(III) Ions Complexed to Physiological Ligands by Lipoyl Dehydrogenase and Other Flavoenzymes in Vitro

“…In recent studies ascorbic acid and glutathione, as well as NAD(P)H, ␣-keto acids, ferredoxin, O 2 . , and thioredoxin have been regarded as the intracellular reductants for Fe(III) (5,6,9,23). More recently, however, doubt arose whether these "classical" reductants are really responsible for the intracellular reduction of the metabolically and catalytically reactive iron, at least under certain conditions.…”

“…More recently, however, doubt arose whether these "classical" reductants are really responsible for the intracellular reduction of the metabolically and catalytically reactive iron, at least under certain conditions. It has been proposed that as yet unidentified NAD(P)H-dependent redox enzymes can effectively reduce ferric ions to the ferrous state (23). More than 1 decade ago, Shi and Dalal (27) demonstrated that lipoyl dehydrogenase, a NAD(P)H-dependent flavoenzyme that often is denominated as diaphorase (26), is able to catalyze the one electron reduction of Cr(IV) and V(V).…”

“…in mitochondrial heme synthesis) but also for the participation in deleterious radical-generating reactions. In recent years evidence has accumulated indicating that both an increased concentration (14 -22) as well as an increased re-reduction and thus redoxcycling of "reactive" iron ions (23,24) can reinforce iron-mediated damaging of biomolecules and cells.…”

Enhancement of iron toxicity in L929 cells by d-glucose: accelerated(re-)reduction

“…NAD(P)H is known to be the main cellular electron donor for redox reactions and to provide electrons for the regeneration of different cellular antioxidants, which themselves are electron donors (reducing agents), such as glutathione and ascorbate. Thus cellular NAD(P)H content is a marker for the general reducing capacity of cells [19]. To monitor a possible influence of -glucose on the NAD(P)H content, we measured the cellular autofluorescence at λ exc l 365p12.5 nm and λ em l 450-490 nm, which is predominantly caused by NADH and NADPH [20].…”