Woodmansee Anh N, Imlay James A
Department of Microbiology, University of Illinois, Urbana, IL 61801, USA.
J Biol Chem. 2002 Sep 13;277(37):34055-66. doi: 10.1074/jbc.M203977200. Epub 2002 Jun 21.
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.
当细胞暴露于外部过氧化氢（H₂O₂）时，H₂O₂会迅速扩散到细胞内部并氧化亚铁离子，从而形成损伤DNA的羟基自由基。因此，氧化性DNA损伤过程仅需要H₂O₂、游离铁以及一种尚未确定的电子供体，该电子供体可将三价铁还原为二价铁状态。先前的研究表明，当呼吸作用被氰化物或呼吸酶的基因缺陷抑制时，H₂O₂杀死大肠杆菌的速度特别快。在本研究中，我们确定这些呼吸阻断会加速DNA损伤的速率。呼吸阻断对细胞内游离铁或H₂O₂的量没有实质性影响，这表明它们加速损伤是因为增加了电子供体的可用性。这项工作的目标是确定该供体。正如预期的那样，呼吸抑制剂导致细胞内NADH的量大幅增加。然而，NADH本身在体外是游离铁的不良还原剂。这表明在不进行呼吸的细胞中，电子从NADH转移到另一种直接还原铁的载体上。对细胞内谷胱甘肽、NADPH、α-酮酸、铁氧化还原蛋白和硫氧还蛋白的量进行基因操作表明，这些都不是直接的电子供体。然而，如果细胞缺乏黄素还原酶（一种将电子从NADH转移到游离FAD的酶），则可免受氰化物刺激的DNA损伤。该酶对NADH的米氏常数（Kₘ）远高于通常的细胞内NADH浓度，这解释了为什么在呼吸抑制期间NADH水平升高时其通量会增加。被纯化的黄素还原酶还原的黄素能迅速将电子转移给游离铁，并在体外驱动一个破坏DNA的芬顿体系。因此，氧化性DNA损伤的速率可能受电子供体还原游离铁的速率限制，并且当呼吸受阻时，还原型黄素成为大肠杆菌中的主要供体。目前尚不清楚黄素或其他还原剂是否在进行呼吸的细胞中驱动芬顿化学反应。
