Lability and liability of endogenous copper pools.

Although the genome of Escherichia coli K-12 encodes just a few copper-containing enzymes, this transition metal is critical for the bacterium to derive energy from oxygen reduction and to protect itself from injury (1). For example, the CuB site within cytochrome bo oxidase facilitates the flow of electrons from ubiquinol to oxygen during aerobic respiration. A putative copper-binding site with cupric reductase activity has also been identified within NADH dehydrogenase II, but this site may involve an adventitious activity rather than a bona fide role for the enzyme in copper homeostasis (2). More certain is the role of copper within TynA, a periplasmic monoamine oxidase that allows the cell to use phenylethylamine as its sole carbon and energy source (3). The homodimeric enzyme contains two copper atoms that facilitate autocatalytic generation of the enzyme’s cofactor (4, 5). Damaging reactive oxygen intermediates, such as those generated by the oxidative burst of professional phagocytes, are detoxified in the periplasm by Cu,Zn superoxide dismutase (6). Periplasmic Cu(I) is oxidized to the less toxic Cu(II) by CueO, a multicopper oxidase (7). Despite the utility of copper in enzymatic reaction centers, at least two factors may have selected against its usage. The first involves the limited bioavailability of copper necessitating competition between commensal E. coli, other microflora residing within the intestinal lumen, and the mammalian host, for which copper is an essential micronutrient. Indeed, agricultural settings are likely to be among the few ecological niches where the bacterium may encounter an abundance of copper, because copper salts have been used as both a feed additive and fungicide (8). Even within the laboratory, the trace amounts of copper present in culture media are suboptimal for growth, and the concentration of copper within the bacterium exceeds that of its environment (9, 10). Under such copper starvation conditions, this cellular copper is thought to be tightly sequestered within enzymatic reaction centers (11). However, that view may require revision in light of the accompanying article by Fung et al., which suggests that the cell maintains a labile pool of copper ions that flux in response to nutritional status and environmental conditions (12). As demonstrated by Fung et al., the maintenance of an endogenous pool of copper is not without costs; like most transition metals (essential and nonessential), copper can be toxic in vivo. Thus, toxicity is the second factor that may limit copper’s biological use. Despite early hypotheses about copper toxicity that focused on the ability of Cu(I) to generate reactive oxygen species (ROS), it has become clear that much of the copper toxicity in E. coli is actually due to disruption of iron metabolism via non-ROS mechanisms (13, 14). In fact, the toxicity of copper increases under anaerobic conditions, when there is no oxygen available for ROS generation (13). The most sensitive target of copper toxicity appears to be iron-sulfur (Fe-S) cluster metabolism, both at the stage of Fe-S cluster biogenesis as well as via disruption of mature Fe-S cluster-containing metalloenzymes (13, 15). Furthermore, the most toxic oxidation state of copper appears to be Cu(I), which likely predominates in the reducing environment of the cell, especially under anaerobic conditions (16). Much of the Cu(I) toxicity likely stems from its thiolphilic nature, which allows it to directly displace other metal ions, such as iron, that are bound less tightly to thiolate or sulfide ligands, as predicted by the IrvingWilliams series. To combat copper toxicity, E. coli utilizes an efflux strategy to remove excess copper from the cytoplasm via CopA, a P-type ATPase efflux pump. Full functionality of the CopA system depends on a periplasmic multicopper oxidase, CueO, that may oxidize Cu(I) as a substrate in addition to using copper ions as cofactors (7). The expression of both CopA and CueO is regulated by the Cu(I)-dependent activator CueR (17). Supplementing CopA is CusCBA, a tripartite RND transport system that spans the cell envelope and clears Cu(I) from the cytoplasm or, with the assistance of CusF, from the periplasmic compartment (18). In contrast to CopA, the CusCFBA system relies on the proton motive force to drive copper efflux, and transcription of cusCFBA is dependent upon a two-component regulatory system consisting of CusR and CusS (19). The signal cascade is initiated by Cu(I), which is presumably detected by a periplasmic sensor domain of CusS (20). The signal is then transmitted across the cytoplasmic membrane via CusS-dependent phosphorylation of the response regulator CusR, followed by activation of the cusC promoter, PcusC. Although the addition of exogenous copper ions is known to activate the CusRS system, Fung et al. observed activation of PcusC in anaerobic cultures deprived of amino acids that was independent of exogenous copper. They attributed this effect to an endogenous pool of Cu(I), because it was suppressed by the membrane-permeable Cu(I) chelator neocuproine but not by bathocuproine, a membrane-impermeable derivative. In addition, the sulfur-containing amino acids methionine and cysteine were shown to suppress the anaerobic activation of PcusC, suggesting a potential role for these amino acids in the maintenance of endogenous pools of copper ions. Intriguingly, Fung et al. also found that anaerobic, amino aciddeprived conditions compromised the growth of copA and/or cusC mutants on fumarate. Through a series of logical experiments, the authors were able to demonstrate that the growth defect was due to inactivation of fumarate reductase (Frd), a critical