An intracellular iron chelator pleiotropically suppresses enzymatic and growth defects of superoxide dismutase-deficient Escherichia coli.

The discovery of superoxide dismutase (SOD) by McCord and Fridovich introduced biology to the field of free radical chemistry (37). The presence of this enzyme in virtually all aerobic organisms (38) forced the conclusion that superoxide (O2−) must be formed as a by-product of aerobic metabolism and, if not scavenged, must damage critical biomolecules. Left unknown were the identities of those target biomolecules. This point was controversial, since chemical studies indicated that amino acids, nucleotides, and sugars are essentially not reactive with O2− (6, 7, 15, 43). In 1986 Carlioz and Touati reported the construction of mutants of Escherichia coli that lacked both the manganese- and iron-containing isozymes of SOD (10). These mutants were capable of aerobic growth only if branched-chain, aromatic, and sulfurous amino acids were provided in the medium, and if a fermentable carbon source was available. They also displayed a high rate of spontaneous mutagenesis (14). Similar phenotypes were reported for wild-type cells during exposure to hyperbaric oxygen, presumably because of the accelerated pace of O2− formation (8). The requirement for branched-chain amino acids was traced to the inactivation by O2− of dihydroxyacid dehydratase, an enzyme in the common biosynthetic pathway (31). This enzyme utilizes a [4Fe-4S]2+ cluster as a Lewis acid to catalyze substrate dehydration. The solvent exposure of the cluster allows it to be accessible to O2−, which can univalently oxidize it. The resultant [4Fe-4S]3+ cluster is unstable and disintegrates to the [3Fe-4S]+ form, which is catalytically inactive, with loss of a ferrous iron atom to the cytosol (16, 17). Several reports following this discovery showed that other dehydratases in the cell, including the tricarboxylic acid (TCA) cycle enzymes aconitase and fumarase, contain [4Fe-4S]2+ clusters that suffer the same damage when exposed to superoxide (20, 21, 34). E. coli can repair these enzymes by a process that is presently undefined but which must entail the reduction of the cluster and replacement of the lost iron atom. The amounts of SOD and of repair enzymes in E. coli appear to be calibrated to precisely balance the rate of dehydratase damage by endogenous O2−, so that the dehydratases retain near-maximal activity during aerobic growth (22). Interestingly, the hypermutagenesis of SOD mutants arises from the iron that is released by the damaged clusters. This iron accumulates in the cytosol, where it catalyzes the oxidation of DNA (28, 30, 35). Thus, several phenotypes of SOD mutants have been traced to oxidation by O2− of iron-sulfur clusters. However, the auxotrophies for sulfur-containing and aromatic amino acids and the slow growth in rich medium are not directly attributable to cluster damage. It appears that neither amino acid biosynthetic pathway contains labile dehydratases. The sulfur requirement has been linked to the apparent leakage of sulfur from SOD mutants (5), although the specific lesion that causes this phenomenon is unknown. The aromatic amino acid biosynthetic defect has recently been ascribed to the oxidation of the intermediate 1,2-dihydroxyethyl thiamine pyrophosphate of transketolase (3). In vitro, superoxide oxidized and released this compound with progressive inactivation of the enzyme, and catabolic processes that require transketolase function were shown to be defective in SOD mutants. A resultant deficiency in erythrose-4-phosphate production could plausibly diminish aromatic amino acid biosynthesis. A pleiotropic suppressor which relieves all the auxotrophies of SOD mutants—those for sulfurous and aromatic as well as branched-chain amino acids—was isolated and characterized by Imlay and Fridovich (25, 26). It was originally hoped that this suppressor might protect multiple targets by curbing the rate of endogenous O2− production. However, the original analysis indicated that the mutation did not act to change the internal O2− concentration (26). The goal of the present study was to decipher the mechanism of suppression and thereby gain further understanding of the nature of O2− toxicity.