Keyer K, Imlay J A
Department of Microbiology, University of Illinois, Urbana, Illinois 61801, USA.
J Biol Chem. 1997 Oct 31;272(44):27652-9. doi: 10.1074/jbc.272.44.27652.
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.
吞噬细胞产生一氧化氮和超氧化物，作为对抗病原体的氧化防御的组成部分。这两种分子在生理浓度下都不太可能杀死细胞。然而，它们的两种反应产物，过氧化氢和过氧亚硝酸盐，是强氧化剂，可穿透细胞且具有毒性。过氧化氢会造成氧化性DNA损伤，而过氧亚硝酸盐的主要毒性机制尚未确定。最近的体外研究表明，过氧亚硝酸盐能够氧化一类脱水酶的[4Fe-4S]簇（豪斯拉登，A.，和弗里多维奇，I.（1994年）《生物化学杂志》269卷，29405 - 29408页；卡斯特罗，L.，罗德里格斯，M.，和拉迪，R.（1994年）《生物化学杂志》269卷，29409 - 29415页）。我们在此证明，过氧亚硝酸盐在其致死剂量的1%时几乎完全使大肠杆菌中不稳定的脱水酶失活。过氧亚硝酸盐使簇失活的速率大大超过其氧化硫醇或自发分解的速率。这些结果表明，这些脱水酶可能是过氧亚硝酸盐在体内的主要作用靶点。簇损伤的另一个后果是100微摩尔的铁释放到细胞质中。在吞噬作用过程中，这种细胞内游离铁可能会因过氧化氢而增加致死性DNA损伤，或因额外的过氧亚硝酸盐而导致蛋白质修饰。为应对过氧亚硝酸盐的攻击，大肠杆菌使用一种未明确的清除系统迅速隔离细胞内游离铁。铁硫簇通过从其铁储存蛋白中获取铁的过程更缓慢地得到修复。这些可能是吞噬细胞与病原体斗争中的关键事件。
