Heavy Metals in the Environment - Chapter 15 pps

15 Microbial Resistance Mechanisms for Heavy Metals and Metalloids Mallika Ghosh and Barry P. Rosen Wayne State University School of Medicine, Detroit, Michigan 1. INTRODUCTION In this chapter the mechanisms of resistance to ions of the heavy metals zinc, cadmium, lead, copper, arsenic, and antimony in bacteria will be described. In addition, the pathways of arsenical resistance in the prokaryote Saccharomyces cerevisiae will be discussed. Although often grouped as heavy metals, the ions of these metals are better characterized as soft metal ions, which are those with high polarizing power (a large ratio of ionic charge to the radius of the ion), in contrast to the hard metal ions of Groups I and II such as Na ϩ and Ca 2ϩ . This distinction between hard and soft metals is important biologically. When consid- ering how these metals interact with biological molecules such as proteins, hard metals most frequently bind to proteins weakly through ionic interactions with functional groups such as carboxylates of glutamate or aspartate residues. In contrast, soft metals form much stronger, nearly covalent, bonds with functional groups such as the thiolates of cysteine residues and the imidazolium nitrogens of histidine residues. These strong interactions with proteins account for much of the biological toxicity of soft metals. All of these ions are toxic in excess, and bacterial metal resistances probably arose early in evolution owing to widespread geochemical sources of metals. Copyright © 2002 Marcel Dekker, Inc. Resistance genes to inorganic salts of soft metals, including arsenic, antimony, lead, cadmium, copper, nickel, zinc, bismuth, and mercury, are found both on extrachromosomal plasmids and in chromosomes of bacteria, archaea, and eukaryotes. For metals such as copper and zinc, which are required in low amounts but toxic in high amounts, the efflux systems are components of the homeostatic mechanisms that maintain intracellular concentrations at optimal levels. Recent reviews have been written on bacterial metal resistances (1–3), which allows this chapter to focus on a few specific mechanisms. 2. P-TYPE ATPases FOR MONOVALENT SOFT METALS: Cu(I) AND Ag(I) PUMPS P-type ATPases comprise a superfamily of enzymes that transport cations (4). Some pump cations into cells, some into organelles, others pump cations out of cells, and yet others are cation exchangers: it is not possible to predict the directionality of transport from inspection of the sequence. Every member of the superfamily has conserved sequences that include an ATP-binding domain, an aspartate residue that is the site of phosphoenzyme formation, and a phosphatase domain. There are at least five branches to the family (5). One branch includes the hard metal cation-translocating ATPases, and another the soft metal cation- translocatingATPases(Fig.1).Bothbranchescanbefurthersubdividedinto branches that comprise monovalent and divalent pumps (1). The first bacterial P-type ATPase identified was the Kdp K ϩ -translocating ATPase, a pump for uptake of monovalent ions of the hard metal potassium (6). Other hard metal bacterial P-type ATPases include the MgtA and MgtB pumps for uptake of divalent ions of the soft metal magnesium (7). The soft metal ATPases were identified more recently. They have common features not present in the hard-metal ATPases, in particular characteristic cysteine- or histidine-rich metal-binding motifs at the N-terminus and a Cys-Pro-Cys (orHis)sequenceinthesixthmembrane-spanningsegment(Fig.2).Thepresence of the CPC(H) sequence has led to the designation CPx-type ATPases (8). How- ever, given that the substrates of this class of enzymes are all soft metal ions, soft metal P-type ATPases seems an appropriate designation. The most widely recognized members of the soft metal P-type ATPases are the human copper pumps ATP7A (or MNK) (9) and ATP7B (or WIND) (10). Inheritable mutations in the genes for these pumps produce Menkes or Wilson disease, respectively. Both MNK and WND have six CXXC metal-binding sequences in their N-termini and a CPC sequence in a membrane-spanning segment (11). Peptides correspond- ing to the N-terminal metal binding domains have been shown to bind a variety of metal ions, including Cu(II), Cu(I), Ag(I), and Zn(II) (12,13). The function of the N-terminal metal binding domains is not known. Possibilities include: (1) Copyright © 2002 Marcel Dekker, Inc. F IGURE 1 The family of soft metal ion-translocating P-type ATPases. This family is growing rapidly, so only a few representative ones are shown to illustrate the nature of the lineage. The dendograms were made using the CLUSTAL4 algorithm (84) with DNASIS software from Hitachi Software Engi- neering Co., Ltd. On the left is the hard metal ion ATPase subfamily, repre- sented by Pmal, the fungal H ϩ -translocation ATPase (85), NaK, the mamma- lian Na,K- ATPase (86), and SERCA1, the endoplasmic reticulum calcium pump (87). On the right is the soft metal ion ATPase subfamily. Two major branches are shown, those with histidine-rich N-termini such as the Cu(I)/ Ag(I)-translocaing CopB ATPase of E. hirae (17) and the histine-rich proteins Hra1 and Hra2 of unknown origin (20). The other branch has two groups of proteins, the monovalent copper pumps, including the human enzymes MNK (9) and WND (10) associated with Menkes and Wilson diseases, and the E. coli Cu(I)-translocating CopA ATPase (25). The second grouping are the divalent soft metal ion pumps for the metals Zn(II), Pb(II), and Cd(II), including the S. aureaus plasmid pI258 CadA ATPase (26), the ZntA pumps of E. coli (30,31), and Proteus mirabilis (88). the metal binding domains serve as sensors to activate the pump; (2) they serve as the initial binding site, transferring the metal ion to the translocation domain, which probably includes the CPC motif; (3) in eukaryotes they may be involved in trafficking of the pump to the appropriate membrane. In support of the last possibility, the metal-binding domains of either the Menkes or Wilson proteins do not appear to be essential for copper transport, but their removal appears to Copyright © 2002 Marcel Dekker, Inc. F IGURE 2 Generic model of a soft metal ion-translocating P-type ATPase. This model of a soft metal ion-translocating P-type ATPase illustrates the common features of these pumps (adapted from ref. 11). Most pump either monovalent or divalent soft metals from the cytosol, either into an intracellular com- partment or out of the cell. Some, such as E. hirae CopA, pump ions into the cell. They have an N-terminal region with one or more cytosolic metal-binding domains, most of which are cysteine-rich motifs but some are histidine- rich sequences. They have eight transmembrane segments (TMS). The cytosolic loop that connects TMS4 and TMS5 is the conserved phosphatase domain. TMS6 has the consensus CPC or CPH sequence, which could be involved in metal ion translocation. In the cytosolic loop that connects TMS6 and TMS7 regions involved in catalysis, the phosphorylation and ATP-binding domains are found. In addition, there is a conserved His-Pro sequence that is found only in soft metal ion-translocating P-type ATPases. Copyright © 2002 Marcel Dekker, Inc. affect trafficking (14,15). On the other hand, the N-terminal metal-binding domains may have a different function in bacteria, most of which lack intracellular membranes that would require trafficking signals. The first bacterial proteins identified as members of the monovalent cation- translocating ATPase subfamily were the CopA and CopB copper pumps of Enterococcus hirae (16,17). The genes for these two P-type ATPases are in an operon that also contains the gene for a transcriptional regulator, CopY, and a copper ion chaperone, CopZ. While CopA and CopB are cotranscribed, they have considerable differences both structurally and physiologically. A disruption in the copA gene renders cells copper-requiring, suggesting that CopA is required for copper import pump. In contrast, a copB disruption renders cells copper sensitive, suggesting that the 745-residue CopB protein is responsible for copper efflux from cells. That two such similar pumps transport their substrates in opposite directions illustrates the point that the directionality of transport cannot easily be deduced from inspection of the primary sequence of the proteins. Together the two pumps provide for copper homeostasis. CopB has a histidine-rich N-terminus. Of the 25 histidine residues in CopB, 16 are located in the first 100 residues, before the first transmembrane segment. It seems reasonable that this region binds copper. In the sixth putative transmembrane region is a CPH sequence that corresponds to the CPC of other soft metal P-type ATPases. CopB has been shown to be an efflux pump involved in resistance to copper in E. hirae (18). To demonstrate the direction of transport, Solioz and co-workers prepared everted membrane vesicles and showed that the vesicles accumulated 64 Cu(I) and 110 Ag(I). The affinity for metal ion was in the micromo- lar range, and for ATP the K m was approximately 10 µM. Vanadate, the classical inhibitor of P-type ATPases, inhibited CopB activity. CopB has been purified and reconstituted into proteoliposomes (19). CopB formed an acylphosphate reaction intermediate with the γ-phosphate of ATP, and formation of the phosphorylated intermediate was sensitive to vanadate. The purified protein exhibited a low level of ATPase activity that was also inhibited by vanadate. However, ATP hydrolysis was not stimulated by copper ion, although a P-type ATPase would be expected to require its metal ion substrate for activity. The sequences of two homologues of CopB, Hral and Hra2, have been reported (20). These were originally described as Escherichia coli proteins. How- ever, they are not found in the E. coli genome (21), and their derivation is unknown. Another soft metal P-type ATPase with a histidine-rich N-terminus is the Ag(I) resistance pump SilP from Salmonella typhimurium (22). Of the 25 histidines in SilP, 15 are located in the N-terminal region, including a highly charged stretch of residues with the sequence EHHHHHDHHE. However, the N-terminus of SilP exhibits little sequence similarity with the N-terminus of other soft metal P-type ATPases, including CopB, and SilP confers resistance to silver but not copper. SilP and CopB are the only two Ag(I) resistance pumps thus far Copyright © 2002 Marcel Dekker, Inc. identified, and both have histidine-rich (albeit unrelated) N-terminal metal-binding domains. This may indicate that Ag(I) recognition is via histidine residues rather than the CXXC motifs of the majority of Cu(I) pumps. The 727-residue CopA protein has a single CXXC sequence in the N-terminal region, and a CPC sequence in the putative sixth transmembrane segment (17). Sequences for other bacterial CopA homologs have been identified, and the list is growing daily. However, the physiological function of only a few of these sequences has been described, and even fewer have been investigated biochemi- cally. Two of the best characterized are the CopA pumps of Helicobacter pylori (23,24) and E. coli (25). As mentioned above, disruption of the E. hirae copA results in a copper requirement. In contrast, disruption of either of the H. pylori or E. coli pumps results in copper sensitivity. This reflects the fact that the E. hirae CopA is an uptake system, while the other two homologs are efflux pumps. Again, it is quite remarkable that the direction of ion transport can be inward for one protein and outward for others that are close homologs. The expla- nation cannot be that the proteins have the opposite orientation in the membrane because each uses ATP, which is found only in the cytosol; the catalytic domains of all three must be exposed in the cytosol. On the other hand, the topological orientation of the protein in the membrane has been determined only for the H. pylori enzyme (24). The protein has been shown to have eight transmembrane segments, with cytosolic N- and C-termini. It is reasonable to assume that all soft metal P-type ATPases will have a similar topology. CopA from E. coli is an 834-residue protein with high similarity to copper pumps such as E. hirae CopA, the human Menkes and Wilson disease proteins (25). While the CopA homologs from E. hirae and H. pylori have only a single N-terminal CXXC sequence, E. coli CopA has two, G 11 LSCGHC and G 107 MSCASC. The presence of multiple metal-binding domains in the E. coli protein may make it a better model for the human copper pumps, which have six N-terminal CXXC sequences. CopA can be predicted to have eight transmembrane segments, including C 479 PC in predicted transmembrane helix 6. In addition, there is a conserved HP motif in the soft metal P-type ATPases that corresponds to H 562 P in CopA. Regulation and resistance exhibit different metal ion specificities. The E. coli copA gene is inducible by addition of either copper or silver salts to the medium. In contrast, disruption of copA resulted in sensitivity to copper salts but not Ag(I). Thus there must be an as-yet-unidentified regulatory protein that controls copA expression, and that regulator recognizes either Ag(I) or Cu(I), while CopA recognizes only Cu(I). Everted membrane vesicles from cells expressing copA accumulated 64 Cu. ATP and DTT were both required, and vanadate inhibited transport. Even though 64 Cu(II) was added to the uptake assay, it would be reduced to Cu(I) by the strong reductant DTT. The fact that no transport of copper ion was observed without DTT strongly indicates that Cu(I) is a substrate of the pump (25). Copyright © 2002 Marcel Dekker, Inc. 3. P-TYPE ATPases FOR DIVALENT SOFT METALS: PUMPS FOR Zn(II), Pb(II), AND Cd(II) The second branch of the soft metal P-type ATPases are those for divalent soft metal ions, including Zn(II), Pb(II), and Cd(II). They can be further subgrouped into the CadA ATPases, which are found mainly in gram-positive bacteria, and ZntA ATPases, which are mainly in gram-negative bacteria (1). The first gene for a divalent soft metal P-type ATPase to be identified was cadA, a cadmium- resistance determinant on Staphylococcus aureus plasmid pI258 (26). The cadA gene encodes a 727-residue P-type ATPase that exhibits considerable sequence similarity to the CopA Cu(I)-translocating ATPases. When the cadA gene was expressed in Bacillus subtilis, the CadA protein was produced as a membrane protein that could be visualized on sodium dodecyl sulfate polyacrylamide gel electrophoresis (27). CadA was shown to form a phosphorylated intermediate during the catalytic cycle (28). The phosphoenzyme intermediate was formed only in the presence of Cd(II) and γ-[ 32 P]ATP and was sensitive to hydroxylamine treatment, which is diagnostic of an acylphosphate bond. Presumably phosphorylation occurs at Asp415, which corresponds to the conserved aspartate residue in all P-type ATPases. Everted membrane vesicles prepared from B. subtilis expressing cadA accumulate 109 Cd(II) in an ATP-dependent manner (27). The topology of a CadA homolog from H. pylori has been determined. Like the H. pylori CopA, it has eight transmembrane segments (29). The zntA gene was first identified from sequencing of the E. coli genome. From its sequence, the zntA gene product could not be differentiated from copper P-type ATPases. However, disruption of zntA resulted in sensitivity of E. coli cells to Zn(II), not Cu(II) (30,31). The fact that zntA is a chromosomal gene suggests that it has a function in normal growth of E. coli, probably in zinc homeostasis. In everted membrane vesicles of E. coli, ZntA catalyzes ATP-coupled accumulation of 65 Zn(II) or 109 Cd(II) in a vanadate-sensitive reaction (30). Vesicles made from a strain with a zntA disruption did not accumulate 65 Zn(II). When zntA was expressed on a plasmid, vesicles from the disrupted strain accumulated 65 Zn(II). Although no isotope of Pb(II) is available, the data suggest that ZntA transports Pb(II). A zntA-disrupted strain was nearly three orders of magnitude more sensitive to Pb(II) than the wild-type strain (32). Moreover, 65 Zn(II) transport in vesicles was inhibited by either Pb(II) or Cd(II). Similar results were obtained when the zntA-disrupted strain expressed the S. aureus cadA gene on a plasmid. Neither conferred resistance to Cu(II) nor catalyzed uptake of 64 Cu in vesicles. Thus both ZntA and CadA are extrusion pumps for the divalent soft metal ions Zn(II), Pb(II), and Cd(II). ZntA has been purified and shown to exhibit ATPase activity at rates equivalent to those for the hard metal P-type ATPases (33). ATP hydrolysis required a divalent soft metal ion, the first metal-ion-dependent ATPase activity by a soft Copyright © 2002 Marcel Dekker, Inc. metal P-type ATPase to be clearly demonstrated. The activity was stimulated by (in order of effectiveness) Pb(II) Ͼ Cd(II) ϳ Zn(II) ϳ Hg(II). Although the free metal ions stimulated ATP hydrolysis, the rates were higher when the soft metal ions were complexed with thiolates of cysteine or glutathione. In fact, free Cd(II) or Hg(II) inhibited activity at neutral and alkaline pHs. In vivo the concentration of glutathione is in the millimolar range (34), and it is likely that soft metal ions do not exist free. These results raise the interesting possibility that the soft metal P-type ATPases recognize metal-glutathione conjugates in vivo. ZntA and CadA homologs appear to be widespread in nature, although they have not yet been found in animals. It is likely that these evolved for zinc homeostasis: zinc is required for a number of enzymes and transcription factors but is toxic in excess, so cells must have mechanisms to prevent overaccumula- tion. Although most of the zinc pumps identified to date are prokaryotic, genes for homologs have been identified in other kingdoms, for example in genome of the archeaon Methanobacterium thermoautotrophicum and in the genome of the plant Arabidopsis thaliana. It would be of interest to know whether humans have a ZntA homolog and, if so, whether there are inheritable diseases related to mutations in zinc pumps. Since these P-type ATPases are also the first Pb(II)-translocating pumps to be identified, it is possible that differential expression in Pb(II)- exposed individuals may produce variations in Pb(II) sensitivity in humans. The answers to questions such as these will become clear as genome projects are completed. 4. THE ArsAB ATPase: AN As(III)/Sb(III) EFFLUX PUMP In E. coli high-level resistance to As(V), As(III), and Sb(III) is conferred by the arsRDABC operon of plasmid R773 (35). The arsC gene encodes an arsenate reductase that converts As(V) to As(III). As(III) and Sb(III) are the substrates of the ArsAB efflux pump, which is an As(III)/Sb(III)-translocating ATPase. The 429-residue ArsB subunit is an integral membrane protein located in the inner membrane of E. coli. The results from construction of a series of gene fusions between arsB and phoA, lacZ,orblaM demonstrate that ArsB has 12 membrane- spanning segments, with the N- and C-termini in the cytosol (36). ArsB can function even in the absence of the ArsA subunit to catalyze translocation of the arsenite anion, with energy supplied in the form of a membrane potential (37) (Fig.3A).TheArsABcomplexisabletocoupleATPhydrolysistothetransport of arsenite, making it a more efficient efflux system than ArsB alone (Fig. 3B). As a semimetal or metalloid, arsenic can have nonmetallic properties, existing in solution as the oxyanions arsenate and arsenite. Alternatively, As(III) and Sb(III) have properties similar to those of soft metals, interacting with high affinity with thiolates. ArsB has only a single cysteine residue that is not required for Copyright © 2002 Marcel Dekker, Inc. F IGURE 3 The ArsAB pump. The E. coli Ars arsenite translocator has two modes of energetics (37). (A) ArsB is an inner-membrane protein that can function as a secondary ∆ω-coupled uniporter. (B) The ArsAB pump is a com- plex of the ArsA ATPase and ArsB membrane carrier and functions exclu- sively as a primary ATP-driven pump. ArsA has two halves, A1 and A2, which are homologous to each other and are connected by a 25-residue linker peptide. Hydrolysis of ATP by ArsA is coupled to arsenite or antimonite translocation by ArsB. ArsB catalysis; thus ArsB does not interact with As(III) as soft metals but must recognize and transport the nonmetallic, oxyanionic forms of the metalloids (38). In contrast, the 583 residue ArsA protein, which is the catalytic subunit of the pump, is allosterically activated by the soft metals As(III) or Sb(III) (39). In the absence of its partner, ArsB, ArsA can be expressed and purified as a soluble protein. ArsA has two halves, A1 and A2, connected by a 25-residue linker. A1 and A2 are homologous to each other, clearly the result of an ancient gene dupli- cation and fusion. Both A1 and A2 have a consensus nucleotide-binding domain (NBD), both of which are required for activity (40,41). As(III) or Sb(III) specifically stimulates ATP hydrolysis (39). It is clear that they do so as soft metals, with the thiolates of Cys113, Cys172, and Cys422 serving as ligands to the metals (42). Those three cysteine residues are located in different regions of the primary sequence, which implies that the protein folds in such a way that the cysteines are brought in proximity to each other, allowing them to coordinate with the metalloid (43). From X-ray diffraction data of crystals of As(III) or Sb(III) complexed to small molecule dithiols, the lengths of an As- Copyright © 2002 Marcel Dekker, Inc. F IGURE 4 Proposed geometry of the ArsA metalloid-binding domain. The metalloid-binding site ArsA is proposed to be a trigonal pyramidal structure containing the three-coordinately liganded sulfur thiolates of Cys113, Cys172, and Cys422, with either As(III) or Sb(III) at the apex. The bond angles and distances are predicted from crystallographic analysis of small arsenic or antimony thiol compounds. S bond and Sb-S bond have been shown to be 2.23 and 2.45 A ˚ , respectively, with S-As-S and S-Sb-S angles of 92.7° and 84.8°, respectively (44). Extrapolat- ing from the shape of these small molecules, a model of the metal-binding site in ArsA has been proposed (Fig. 4) (43). Filling of the allosteric site with metal results in an increase in the rate of ATP hydrolysis at the NBD. This is associated with conformational changes that occur upon metalloid binding (45). A1 and A2 are tethered by a 25-residue flexible linker peptide (46), but otherwise the NBDs do not interact in the absence of metalloid (47). When As(III) or Sb(III) coordi- nates with Cys113 and Cys172 in A1 and Cys422 in A2, this draws the two halves of the protein closer, bringing the two nucleotide-binding sites into contact, which increasestherateofATPhydrolysis(48,49)(Fig.5). How activation occurs is an open question, as is whether both NBDs are catalytic. Both sites bind nucleotides, even when the other is inactivated by muta- genesis (50). The two sites do not appear to be equivalent, either in sequence or in binding properties. In the absence of metalloid activator, the A2 site binds the ATP analog 5′-p-fluorosulfonylbenzoyladenosine (FSBA) but not ATP, leading to the hypothesis that the A1 NBD is a high-affinity binding site but that the A2 site can bind ATP only when the enzyme is activated (47). Although mutations in either NBD eliminate the high, activated rate, ArsAs with inactive A2 sites still hydrolyze ATP at the basal, unactivated rate (48). In contrast, A1 substitu- tions result in complete inactivation. These results suggest that the A1 site exhibits independent unisite catalysis in the absence of As(III) or Sb(III); participation of the A2 site requires metalloid binding, which produces multisite catalysis (48). Copyright © 2002 Marcel Dekker, Inc. [...]... monitored during the individual steps of the catalytic cycle This has allowed modeling of the reaction cycle (52) The results suggest that conformational change is the rate-limiting step in the overall reaction in the unactivated state, and that binding of the allosteric activator in some way overcomes this rate-limiting step This is reminiscent of the E1-to-E2 transition in P-type ATPases, where the conformational... report binding and hydrolysis of nucleotides (51,52) In both the A1 and A2 halves of the protein there is a 12-residue consensus sequence DTAPTGHTIRLL (the DTAP domain) that undergoes considerable conformational change during catalysis (51) Using intrinsic tryptophan fluorescence from ArsAs in which single typtophan residues had been introduced at specific locations, the movement of domains in ArsA could... catalysis In the absence of metalloid activator, the A1 and A2 halves of ArsA are independent of each other, with only the A1 NBD exhibiting a basal rate of ATP hydrolysis (48) Binding of Sb(III) or As(III) to Cys113, Cys172, and Cys422 brings the halves together This results in interaction of the A1 and A2 NBDs to interact and promotion of catalysis Conformational changes in the conserved DTAP domains report... stable and inducible resistance to arsenite and antimonite has also been reported in Chinese hamster cell lines (64) Although the responsible gene(s) and protein(s) have not been identified, resistance is associated with increased efflux of arsenite from the cells (65) As mentioned above, arsenical and antimonial drugs such as Pentostam, an Sb(V)-containing drug, are still the first line in the treatment... As(GS) 3 (83), the process is probably too slow to be involved in biological formation of the conjugate In conclusion, although the pathways of resistance in prokaryotes and eukaryotes are similar in overall design, most of the prokaryotic proteins are unrelated evolutionarily to their eukaryotic analogs (Fig 6) In both E coli and yeast, the first step in resistance is detoxification of arsenate In prokaryotes... source of reducing potential (89,90) In E coli, the resulting arsenite is extruded from the cells by the Ars efflux system, which, depending on the subunit composition, can be either the ArsB uniporter or the ArsAB ATPase (37) In S cerevisiae, there are two types of arsenite transporters Acr3p is a plasma membrane arsenite efflux protein that probably functions as a uniporter coupled to the membrane potential... As-thiol conjugates into intracellular compartments (71) There are multiple pathways for arsenite resistance in Leishmania While amplification of pgpA leads to increased resistance, in cell lines selected for arsenite resistance, disruption of pgpA does not eliminate resistance (72) This is consistent with the existence of an independent resistance mechanism, and in the plasma membrane of Leishmania there... mechanism Other components in the pathway for arsenical resistance in humans are not known Human enzymes that catalyze arsenate reduction and conjugation to GSH have not been identified, nor has an Acr3p homolog been found It will be of interest to determine whether the pathways of metalloid detoxification in humans are similar to those in yeast 6 CONCLUSIONS The intracellular concentrations of heavy metals. .. reductases, while in eukaryotic yeast Acr2p catalyzes this reaction Arsenite, the product of the reductase reaction, is then ex- FIGURE 6 Pathways of arsenical detoxification in prokaryotes and eukaryotes In both E coli and S cerevisiae, the first step in resistance is reduction of arsenate to arsenite by the bacterial ArsC or yeast Acr2p enzymes In both organisms, glutathione and glutaredoxin serve as the source... conformational change from one state to the other is a major feature of the mechanism 5 ARSENICAL RESISTANCE IN EUKARYOTES Although this chapter has focused on soft metal resistance in bacteria, the prevalence of arsenic in the environment ensures that arsenical resistance mechanisms are universal and ubiquitous (1,35,53) Arsenic is a well-documented human carcinogen, and the adverse effects from exposure . hard-metal ATPases, in particular characteristic cysteine- or histidine-rich metal-binding motifs at the N-terminus and a Cys-Pro-Cys (orHis)sequenceinthesixthmembrane-spanningsegment(Fig.2).Thepresence of. CXXC metal-binding sequences in their N-termini and a CPC sequence in a membrane-spanning segment (11). Peptides correspond- ing to the N-terminal metal binding domains have been shown to bind a. (3) in eukaryotes they may be involved in trafficking of the pump to the appropriate membrane. In support of the last possibility, the metal-binding domains of either the Menkes or Wilson proteins do

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