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The iron-sulfur clusters of dehydratases are primary intracellular targets of copper toxicity Lee Macomber and James A. Imlay1 Department of Microbiology, University of Illinois, Urbana, IL 61801 Edited by Irwin Fridovich, Duke University Medical Center, Durham, NC, and approved March 31, 2009 (received for review December 16, 2008) Excess copper is poisonous to all forms of life, and copper over- suppressed, but no DNA damage was detected (17). Further loading is responsible for several human pathologic processes. The inspection suggested that intracellular metabolites, including primary mechanisms of toxicity are unknown. In this study, mu- glutathione, might chelate copper so that it fails to associate with tants of Escherichia coli that lack copper homeostatic systems DNA and/or undergo cycles of oxidation and reduction (17). (copA cueO cus) were used to identify intracellular targets and to How, then, does copper ‘‘toxify’’ cells? The present study used test the hypothesis that toxicity involves the action of reactive copA cueO cus mutants to identify primary routes of intracellular oxygen species. Low micromolar levels of copper were sufficient to damage. It also exploited the ability of E. coli to grow anaero- inhibit the growth of both WT and mutant strains. The addition of bically, so that the role of oxygen in copper toxicity could be branched-chain amino acids restored growth, indicating that cop- directly evaluated. per blocks their biosynthesis. Indeed, copper treatment rapidly inactivated isopropylmalate dehydratase, an iron-sulfur cluster Results enzyme in this pathway. Other enzymes in this iron-sulfur dehy- Copper Is Highly Toxic Under Environmentally Relevant Conditions. dratase family were similarly affected. Inactivation did not require Our goal was to determine the mechanism of copper toxicity oxygen, in vivo or with purified enzyme. Damage occurred con- under physiological conditions. In a simple salts medium that comitant with the displacement of iron atoms from the solvent- contained glucose as the sole carbon source, WT E. coli exhib- exposed cluster, suggesting that Cu(I) damages these proteins by ited a growth defect when Cu(II) concentrations exceeded 8 ␮M liganding to the coordinating sulfur atoms. Copper efflux by (Fig. 1A). Further, this sensitivity increased when systems that MICROBIOLOGY dedicated export systems, chelation by glutathione, and cluster control copper levels were removed. A copA cueO cusCFBA repair by assembly systems all enhance the resistance of cells to mutant showed an aerobic growth defect with as little as 0.25 ␮M this metal. Cu(II) (Fig. 1B). This extreme sensitivity contrasts with the millimolar doses that are needed to inhibit growth in complex copA ͉ Escherichia coli ͉ glutathione ͉ hydrogen peroxide ͉ Suf media (12, 14, 17). opper is universally toxic. Indian childhood cirrhosis and Copper Caused a Branched-Chain Amino Acid Auxotrophy. It has been CTyrolean infantile liver cirrhosis are human pathologic hypothesized that copper produces toxicity in cells in part by processes that result from high environmental exposure (1, 2), generating reactive oxygen species. Indeed, we found that cop- whereas Wilson disease is a copper-overloading genetic disorder per-stressed cells produced endogenous H2O2 at a rate several that stems from the dysfunction of a copper pump that normally fold higher than did untreated cells (data not shown). Excessive Ϫ keeps levels within tolerable limits (3–6). Microbes are partic- levels of superoxide (O2 ) and H2O2 can disrupt several amino ularly vulnerable to metal poisoning because they cannot control acid biosynthetic pathways (18–20); therefore, we tested whether their extracellular environment, and so studies into the mecha- copper creates similar blocks. (This experiment was complicated nisms of metal toxicity have often exploited them as model by the fact that ␣-amino acids chelate copper and thereby systems. increase the dose necessary for toxicity; to correct for this effect, Escherichia coli elaborates 3 homeostatic systems that try to alanine was added in equivalent doses to cultures from which maintain low levels of intracellular copper. CopA is a P-type amino acids of interest were withheld.) The copA cueO cusCFBA ATPase that pumps copper from the cytoplasm into the mutant could not grow aerobically in glucose/alanine medium to periplasm; it is a homologue of the ATP7B protein that is which 10 ␮M copper was added (Fig. 2A). The addition of defective in Wilson disease (7). CueO is an oxidase that is branched-chain amino acids partially restored growth, indicating believed to oxidize periplasmic Cu(I) to Cu(II), possibly to slow that this pathway is a primary target of copper toxicity. The its adventitious entry into the cytoplasm (8, 9). Both CopA and failure to fully restore growth indicates that additional growth- CueO are strongly induced when cells are exposed to low levels limiting damage also occurred outside of this pathway. of copper (7, 10–12). Higher levels additionally activate the Cus system, which pumps copper from the periplasm back to the Copper Inactivated Iron-Sulfur Cluster Dehydratases Inside Aerobic extracellular environment (13). Mutants that lack these systems Cells. Reactive oxygen species block branched-chain biosynthesis stop growing when they are exposed to copper levels that WT because they directly damage the iron-sulfur clusters of 2 dehy- cells can tolerate (14). dratases: dihydroxy-acid dehydratase in the common branched- The mechanism by which copper poisons cells has been chain pathway and isopropylmalate isomerase (IPMI) in the elusive. A long-standing hypothesis is that copper reacts with leucine-specific branch (18, 21–23). In conformity with the endogenous H2O2 to generate hydroxyl radicals in a process analogous to the Fenton reaction: Author contributions: L.M. and J.A.I. designed research; L.M. performed research; L.M. and ϩ ϩ 3 2ϩ ϩ Ϫ ϩ ⅐ J.A.I. analyzed data; and L.M. and J.A.I. wrote the paper. Cu H2O2 Cu OH HO . [1] The authors declare no conflict of interest. When the analogous reaction is driven by iron in vivo, the This article is a PNAS Direct Submission. hydroxyl radicals are potent oxidants of DNA and cause both 1To whom correspondence should be addressed. E-mail: [email protected]. mutagenesis and lethality (15, 16). However, when copA cueO This article contains supporting information online at www.pnas.org/cgi/content/full/ cus mutants of E. coli were overloaded with copper, growth was 0812808106/DCSupplemental. www.pnas.org͞cgi͞doi͞10.1073͞pnas.0812808106 PNAS Early Edition ͉ 1of6 Downloaded by guest on October 1, 2021 Wild type copA cueO cus Wild type (No O ) copA cueO cus (No O ) A 1 0 µM B 1 A 2 B 2 0 µM 8 µM ILV ILV ILV + Cu 500 0.1 500 0.25 µM D 0.1 0.1 0.1 OD O ILV + Cu Ala 500 16 µM 500 0.5 µM Ala OD 0.01 0.01 OD 32 µM 1 µM 0.01 0.01 02 46810 0246810 Ala + Cu Hours Hours Ala + Cu 0246810 0246 810 Fig. 1. Copper is toxic to aerobic cells at micromolar doses. E. coli cultures Hours Hours were grown at 37 °C in aerobic glucose medium, and CuSO4 was added. (A) W3110 (wild type) cultures were challenged with 0 ␮M (open squares), 8 ␮M Fig. 3. Copper inhibits branched-chain biosynthesis even in the absence of (closed squares), 16 ␮M (closed circles), or 32 ␮M CuSO4 (closed diamonds). (B) oxygen. (A and B) E. coli cultures were grown anaerobically in glucose medium LEM33 (copA cueO cusCFBA) cultures were challenged with 0 ␮M (open supplemented with either 1.5 mM alanine (ala) (squares) or 0.5 mM each of squares), 0.25 ␮M (closed squares), 0.5 ␮M (closed circles), or 1 ␮M CuSO4 isoleucine (I), leucine (L), and valine (V) (circles), and they were then chal- (closed diamonds). The data are representative of 3 independent experiments. lenged with 0 ␮M (open symbols) or 2 ␮M CuSO4 (closed symbols). (A) W3110 (WT). (B) LEM33 (copA cueO cusCFBA). The data are representative of 3 independent experiments. growth data, we found that the IPMI activity of WT cells did not decrease when they were exposed to 10 ␮M Cu(II) for 8 h, whereas a similar treatment to copA cueO cusCFBA mutant cells clusters, and so it appears that all members of this enzyme class are vulnerable to copper stress. Whereas growth on glucose does reduced the activity to less than 15% (the detection limit) of not require large substrate fluxes through these enzymes, other untreated WT controls. growth conditions do: the copA cueO cusCFBA mutant failed to In principle, protracted exposure to copper might diminish grow aerobically on succinate medium even when it was supple- enzyme activity either by damaging extant enzymes or by mented with branched-chain amino acids [supporting informa- blocking new enzyme synthesis or activation. A higher dose of 16 ␮ tion (SI) Fig. S1A], a phenotype consistent with the inactivation M copper diminished the total IPMI activity of a culture of WT of fumarase A. Indeed, overexpression of fumarase A restored cells by 60% within 30 min, suggesting that the former expla- growth. nation pertains. Similarly, activity decreased in copA cueO cusCFBA mutant cells by 80% (Fig. 2B). The Mechanism of Copper Toxicity Is Independent of Oxygen. Other Furthermore, the activities of fumarase A and 6-phosphoglu- investigators have noted that copper can poison bacterial cells in conate dehydratase also declined when cells were exposed to anaerobic medium (12, 17, 24), and we observed, in fact, that copper (Fig. 2 C and D). Like IPMI and dihydroxy-acid dehy- both WT cells and the copA cueO cusCFBA mutant were much dratase, these enzymes are dehydratases that employ iron-sulfur more sensitive to growth inhibition by copper when oxygen was absent (Fig. S2). The anaerobic growth of the WT and mutant strains was effectively blocked by 1 ␮M and 125 nM copper, 1.6 A 1 ILV B respectively.

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