Characterization of the catalytic subunit of an anion pump

The ArsA protein, the 63-kDa catalytic subunit of an oxyanion-translocating ATPase, was purified by successive chromatography using Q-Sepharose, red agarose, and phenyl-Sepharose to a specific activity in excess of 1 mumol of ATP hydrolyzed per min per mg of protein. ATPase activity was dependent on the presence of the oxyanionic substrates. Inhibitors of other classes of ion-translocating ATPases had no effect on ArsA ATPase activity, including N,N'-dicyclohexyl-carbodiimide, azide, vanadate, and nitrate. The apparent Km for ATP was determined to be 0.13 mM. The optimal pH range for ATP hydrolysis was 7.5 to 7.8. ATPase activity required Mg2+ at a molar ratio of 2 ATP:1 Mg2+. Limited proteolysis by trypsin was used to study conformational changes produced upon binding of substrates to the ArsA protein. In the absence of substrates, the ArsA protein was rapidly cleaved by trypsin to a major product of 30 kDa. ATP was partially protected from trypsin digestion, while the anionic substrate antimonite alone had no effect on proteolysis. Combination of the two substrates nearly completely protected the ArsA protein from proteolysis. Proteolytic cleavage correlated with loss of anion-stimulated ATPase activity and substrate protection from cleavage correlated with retention of activity. These results demonstrate that ATP and antimonite together produce a conformational change which is different from that of the ArsA protein in the presence of either substrate alone and suggest interaction between the oxyanion and ATP binding sites.     

Role of conserved aspartates in the ArsA ATPase

The ArsA ATPase is the catalytic subunit of the arsenite-translocating ArsAB pump that is responsible for resistance to arsenicals and antimonials in Escherichia coli. ATPase activity is activated by either arsenite or antimonite. ArsA is composed of two homologous halves A1 and A2, each containing a nucleotide binding domain, and a single metalloid binding or activation domain is located at the interface of the two halves of the protein. The metalloid binding domain is connected to the two nucleotide binding domains through two DTAPTGH sequences, one in A1 and the other in A2. The DTAPTGH sequences are proposed to be involved in information communication between the metal and catalytic sites. The roles of Asp142 in A1 D 142TAPTGH sequence, and Asp447 in A2 D 447TAPTGH sequence was investigated after altering the aspartates individually to alanine, asparagine, and glutamate by site-directed mutagenesis. Asp142 mutants were sensitive to As(III) to varying degrees, whereas the Asp447 mutants showed the same resistance phenotype as the wild type. Each altered protein exhibited varying levels of both basal and metalloid-stimulated activity, indicating that neither Asp142 nor Asp447 is essential for catalysis. Biochemical characterization of the altered proteins imply that Asp142 is involved in Mg (2+) binding and also plays a role in signal transduction between the catalytic and activation domains. In contrast, Asp447 is not nearly as critical for Mg (2+) binding as Asp142 but appears to be in communication between the metal and catalytic sites. Taken together, the results indicate that Asp142 and Asp447, located on the A1 and A2 halves of the protein, have different roles in ArsA catalysis, consistent with our proposal that these two halves are functionally nonequivalent.     

The linker peptide of the ArsA ATPase

Plasmid R773 encodes an As(III)/Sb(III)-translocating ATPase that confers resistance to those metalloids in Escherichia coli. The catalytic subunit of the pump, the ArsA ATPase, consists of homologous N- and C-terminal nucleotide-binding domains connected by a 25-residue linker. The role of this linker sequence was examined by deletion of five, 10, 15 or 23 residues or insertion of five glycine residues. Cells expressing arsA with the 5-residue insertion had wild-type arsenite resistance. Resistance of cells expressing modified arsA genes with deletions was dependent on the linker length. Cells with five or 10 deleted residues exhibited slightly reduced resistance. Deletion of 15 or 23 residues resulted in further decreases in resistance. Each altered ArsA was purified. The enzyme with the 5-residue insertion had the same affinity for ATP and Sb(III) as the wild-type enzyme. Enzymes with 5-, 10-, 15- or 23-residue deletions exhibited decreased affinity for both Sb(III) and ATP. The enzyme with a 23-residue deletion exhibited only basal ATPase activity and was unable to be allosterically activated by Sb(III). These results suggest that the linker has evolved to a length optimal for bringing the two halves of the protein into proper contact with each other, facilitating catalysis.     

Cys-113 and Cys-422 form a high affinity metalloid binding site in the ArsA ATPase

The arsRDABC operon of Escherichia coli plasmid R773 encodes the ArsAB extrusion pump for the trivalent metalloids As(III) and Sb(III). ArsA, the catalytic subunit has two homologous halves, A1 and A2. Each half has a consensus signal transduction domain that physically connects the nucleotide-binding domain to the metalloid-binding domain. The relation between metalloid binding by ArsA and transport through ArsB is unclear. In this study, direct metalloid binding to ArsA was examined. The results show that ArsA binds a single Sb(III) with high affinity only in the presence of Mg(2+)-nucleotide. Mutation of the codons for Cys-113 and Cys-422 eliminated Sb(III) binding to purified ArsA. C113A/C422A ArsA has basal ATPase activity similar to that of the wild type but lacks metalloid-stimulated activity. Accumulation of metalloid was assayed in intact cells, where reduced uptake results from active extrusion by the ArsAB pump. Cells expressing the arsA(C113A/C422A)B genes had an intermediate level of metalloid resistance and accumulation between those expressing only arsB alone and those expressing wild type arsAB genes. The results indicate that, whereas metalloid stimulation of ArsA activity enhances the ability of the pump to reduce the intracellular concentration of metalloid, high affinity binding of metalloid by ArsA is not obligatory for transport or resistance. Yet, in mixed populations of cells bearing either arsAB or arsA(C113A/C422A)B growing in subtoxic concentrations of arsenite, cells bearing wild type arsAB replaced cells with mutant arsA(C113A/C422A)B in less than 1 week, showing that the metalloid binding site confers an evolutionary advantage.     

Unisite and multisite catalysis in the ArsA ATPase

The ars operon of plasmid R773 encodes an As(III)/Sb(III) extrusion pump. The catalytic subunit, the ArsA ATPase, has two homologous halves, A1 and A2, each with a consensus nucleotide-binding sequence. ATP hydrolysis is slow in the absence of metalloid and is accelerated by metalloid binding. ArsA M446W has a single tryptophan adjacent to the A2 nucleotide-binding site. Tryptophan fluorescence increased upon addition of ATP, ADP, or a nonhydrolyzable ATP analogue. Mg(2+) and Sb(III) produced rapid quenching of fluorescence with ADP, no quenching with a nonhydrolyzable analogue, and slow quenching with ATP. The results suggest that slow quenching with ATP reflects hydrolysis of ATP to ADP in the A2 nucleotide-binding site. In an A2 nucleotide-binding site mutant, nucleotides had no effect. In contrast, in an A1 nucleotide-binding mutant, nucleotides still increased fluorescence, but there was no quenching with Mg(2+) and Sb(III). This suggests that the A2 site hydrolyzes ATP only when Sb(III) or As(III) is present and when the A1 nucleotide-binding domain is functional. These results support previous hypotheses in which only the A1 nucleotide-binding domain hydrolyzes ATP in the absence of activator (unisite catalysis), and both the A1 and A2 sites hydrolyze ATP when activated (multisite catalysis).     

Caenorhabditis elegans expresses a functional ArsA

Because arsenic is the most prevalent environmental toxin, it is imperative that we understand the mechanisms of metalloid detoxification. In prokaryotes, arsenic detoxification is accomplished by chromosomal and plasmid-borne operon-encoded efflux systems. Bacterial ArsA ATPase is the catalytic component of an oxyanion pump that is responsible for resistance to arsenite (As(III)) and antimonite (Sb(III)). Here, we describe the identification of a Caenorhabditis elegans homolog (asna-1) that encodes the ATPase component of the Escherichia coli As(III) and Sb(III) transporter. We evaluated the responses of wild-type and asna-1-mutant nematodes to various metal ions and found that asna-1-mutant nematodes are more sensitive to As(III) and Sb(III) toxicity than are wild-type animals. These results provide evidence that ASNA-1 is required for C. elegans' defense against As(III) and Sb(III) toxicity. A purified maltose-binding protein (MBP)-ASNA-1 fusion protein was biochemically characterized, and its properties compared with those of ArsAs. The ATPase activity of the ASNA-1 protein was dependent on the presence of As(III) or Sb(III). As(III) stimulated ATPase activity by 2 +/- 0.2-fold, whereas Sb(III) stimulated it by 4.6 +/- 0.15-fold. The results indicate that As(III)- and Sb(III)-stimulated ArsA ATPase activities are not restricted to bacteria, but extend to animals, by demonstrating that the asna-1 gene from the nematode, C. elegans, encodes a functional ArsA ATPase whose activity is stimulated by As(III) and Sb(III) and which is critical for As(III) and Sb(III) tolerance in the intact organism.     

Molecular characterization of an anion pump. The arsA gene product is an arsenite(antimonate)-stimulated ATPase

The products of the arsenical resistance operon of resistance plasmid R733 form an efflux system for arsenicals. Detoxification results from active efflux of the oxyanions, preventing their concentration from reaching toxic levels. The largest polypeptide encoded by the ars operon was purified. From N-terminal sequencing the purified protein, termed the ArsA protein, was shown to correspond to the product of the arsA gene. The purified protein was demonstrated to bind ATP by two methods. First, a photoadduct of the protein with [alpha-32P]ATP was formed by irradiation at 254 nm. Second, the purified protein bound a fluorescent ATP analogue, 2',3'-o-(2,4,6)trinitrophenyl ATP, with a half-maximal affinity of 2 microM. By both assays competition was observed with ATP or ADP, but not with AMP, GTP, CTP, or UTP. In both nucleotide binding assays, Mg2+ was required, but neither arsenite nor antimonate had any affect. In contrast, the ArsA protein exhibited an ATPase activity which was dependent on the presence of arsenite or antimonate. The results suggest that the ArsA protein is the catalytic subunit of an oxyanion-translocating ATPase.     

Ligand interactions in the ArsA protein, the catalytic component of an anion-translocating adenosinetriphosphatase.

The ars operon of the conjugative R-factor R773 produces resistance to arsenicals in cells of Escherichia coli. The operon encodes an oxyanion pump which is composed of a membrane subunit, the 45.5-kDa ArsB protein, and a catalytic subunit, the 63-kDa ArsA protein. Purified ArsA protein is an arsenite(antimonite)-stimulated ATPase. From its amino acid sequence, as deduced from the nucleotide sequence, the ArsA protein has four tryptophanyl residues which could serve as intrinsic fluorescent probes for the study of substrate-induced conformational changes. Both static and dynamic measurements of tryptophan fluorescence were performed with the ArsA protein. Results from static anisotropy measurements indicated differences in molecular motion with addition of ATP, SbO2-, or Mg2+. These results were supported by time decay measurements of fluorescence anisotropy. The results of time decay measurements indicated a shorter correlation time, reflecting localized motion in the vicinity of the probe, and a longer correlation time, which could have arisen from rotation of the major portion of the molecule. The longer correlation time changed with addition of the various effectors, especially MgCl2, suggesting that binding of Mg2+ decreases probe mobility.     

Characterization of the metalloactivation domain of an arsenite/antimonite resistance pump

The ArsAB extrusion pump encoded by the ars operon of Escherichia coli plasmid R773 confers resistance to the toxic trivalent metalloids arsenite [As(III)] and antimonite [Sb(III)]. The ArsA ATPase, the catalytic subunit of the pump, has two homologous halves, A1 and A2. At the interface of these two halves are two nucleotide-binding domains and a metalloid-binding domain. Cys-113 and Cys-422 have been shown to form a high-affinity metalloid binding site. The crystal structure of ArsA shows two other bound metalloid atoms, one liganded to Cys-172 and His-453, and the other liganded to His-148 and Ser-420. The contribution of those putative metalloid sites was examined. There was little effect of mutagenesis of residues His-148 and Ser-420 on metalloid binding. However, a C172A ArsA mutant and C172A/H453A double mutant exhibited significantly decreased affinity for Sb(III). These results suggest first that there is only a single high-affinity metalloid binding site in ArsA, and second that Cys-172 controls the affinity of this site for metalloid and hence the efficiency of metalloactivation of the ArsAB efflux pump.     

A 3D localized surface plasmon resonance biosensor for the study of trivalent arsenic binding to the ArsA ATPase

A self-assembled 3D hydrogel-nanoparticle composite integrated surface plasmon resonance (SPR) sensor is reported here. The novel assembled substrate was developed by means of a surface mediated radical co-polymerization process to obtain a highly sensitive hydrogel-based thin film that possesses specific binding sites for target analytes. Initially, amino group modified gold nanoparticles (AuNPs) were covalently linked to acrylic acid monomer. Following this, N-isopropylacrylamide (NIPAAm) and AuNPs linked acrylic acid (AAc) monomers were randomly co-polymerized by the "grafting from" method in the presence of initiator and crosslinker onto the sensing surface. Surface characterization techniques were utilized to evaluate the thickness and composition of the hydrogel-nanoparticle film. The sensing platform was employed to study the binding kinetics and conformational changes of the ArsA ATPase as a consequence of binding trivalent arsenicals under a variety of conditions. ArsA, the catalytic subunit of the ArsAB arsenite (As(III)) translocating ATPase, is one of the five proteins encoded by the arsenical resistance (ars) operon of plasmid R773 in cells of Escherichia coli, that confers resistance to trivalent and pentavalent salts of the metalloid arsenic. SPR measurements indicate that the 3D hydrogel-nanoparticle coated sensors exhibited a higher sensitivity than that of the 2D AuNPs decorated sensors. Binding of As(III) to ArsA is greatly facilitated by the presence of magnesium ion and ATP.     

Original Research Report: Structure-Function Analysis of the ArsA ATPase: Contribution of Histidine Residues

The ArsA ATPase is the catalytic subunit of the ArsAB oxyanion pump in Escherichia coli that is responsible for extruding arsenite or antimonite from inside the cell, thereby conferring resistance. Either antimonite or arsenite stimulates ArsA ATPase activity. In this study, the role of histidine residues in ArsA activity was investigated. Treatment of ArsA with diethyl pyrocarbonate (DEPC) resulted in complete loss of catalytic activity. The inactivation could be reversed upon subsequent incubation with hydroxylamine, suggesting specific modification of histidine residues. ATP and oxyanions afforded significant protection against DEPC inactivation, indicating that the histidines are located at the active site. ArsA has 13 histidine residues located at position 138, 148, 219, 327, 359, 368, 388, 397, 453, 465, 477, 520, and 558. Each histidine was individually altered to alanine by site-directed mutagenesis. Cells expressing the altered ArsA proteins were resistant to both arsenite and antimonite. The results indicate that no single histidine residue plays a direct role in catalysis, and the inhibition by DEPC may be caused by steric hindrance from the carbethoxy group.     

The ATPase mechanism of ArsA, the catalytic subunit of the arsenite pump.

The ArsA ATPase is the catalytic subunit of a novel arsenite pump, with two nucleotide-binding consensus sequences in the N- and C-terminal halves of the protein. The single tryptophan-containing Trp159 ArsA was used to elucidate the elementary steps of the ATPase mechanism by fluorescence stopped-flow experiments. The binding and hydrolysis of MgATP is a multistep process with a minimal kinetic mechanism (Mechanism 1). A notable feature of the reaction is that MgATP binding induces a slow transient increase in fluorescence of ArsA, which is independent of the ATP concentration, indicative of the build-up of a pre-steady state intermediate. This finding, coupled with a phosphate burst, implies that the steady-state intermediate builds up subsequent to product release. We propose that the rate-limiting step is an isomerization between different conformational forms of ArsA. kcat is faster than the phosphate burst, indicating that both nucleotide binding sites of ArsA are catalytic. Consistent with this interpretation, approximately 2 mol of phosphate are released per mole of ArsA during the phosphate burst.     

Nonequivalence of the nucleotide binding domains of the ArsA ATPase

The arsRDABC operon of Escherichia coli plasmid R773 encodes the ArsAB pump that catalyzes extrusion of the metalloids As(III) and Sb(III), conferring metalloid resistance. The catalytic subunit, ArsA, is an ATPase with two homologous halves, A1 and A2, connected by a short linker. Each half contains a nucleotide binding domain. The overall rate of ATP hydrolysis is slow in the absence of metalloid and is accelerated by metalloid binding. The results of photolabeling of ArsA with the ATP analogue 8-azidoadenosine 5'-[alpha-(32)P]-triphosphate at 4 degrees C indicate that metalloid stimulation correlates with a >10-fold increase in affinity for nucleotide. To investigate the relative contributions of the two nucleotide binding domains to catalysis, a thrombin site was introduced in the linker. This allowed discrimination between incorporation of labeled nucleotides into the two halves of ArsA. The results indicate that both the A1 and A2 nucleotide binding domains bind and hydrolyze trinucleotide, even in the absence of metalloid. Sb(III) increases the affinity of the A1 nucleotide binding domain to a greater extent than the A2 nucleotide binding domain. The ATP analogue labeled with (32)P at the gamma position was used to measure hydrolysis of trinucleotide at 37 degrees C. Under these catalytic conditions, both nucleotide binding domains hydrolyze ATP, but hydrolysis in A1 is stimulated to a greater degree by Sb(III) than A2. These results suggest that the two homologous halves of the ArsA may be functionally nonequivalent.     

Involvement of the 50-kDa peptide of myosin heads in the ATPase activity revealed by fluorescent modification with 4-fluoro-7-nitrobenz-2-oxa-1,3-diazole

The fluorescent reagent 4-fluoro-7-nitrobenz-2-oxa-1,3-diazole (NBD-F) reacted specifically with 1.9 lysyl residues/mol of the myosin subfragment-1 (S-1) ATPase. When 1.9 lysyl residues were modified, the K+- and Ca2+-ATPase activities were almost completely inhibited, whereas the Mg2+-ATPase activity was increased to 180% of original activity. The actin-activated Mg2+-ATPase activity was decreased to 30% of original activity by this modification. However, affinity of S-1 for actin in the presence of ATP was unchanged. The NBD fluorescence of the modified S-1 was quenched on addition of ATP, suggesting that ATP induced conformational changes around the NBD groups attached to S-1. Tryptic digestion of the modified S-1 revealed that the NBD groups are attached mainly to the 50-kDa peptide of S-1, more precisely the 45-kDa peptide. These results confirm the recent reports that the 50-kDa peptide of S-1 is involved in the myosin ATPase reaction (K?rner, M., Thiem, N. V., Cardinaud, R., and Lacombe, G. (1983) Biochemistry 22, 5843-5847; Hiratsuka, T. (1986) Biochemistry 25, in press).     

Energy transduction in Escherichia coli. The role of the Mg2+ATPase.

Inverted membrane vesicles from strain 7, a wild type Escherichia coli K12 strain, actively transport calcium with energy supplied either by respiration or by ATP. These vesicles also have energy-linked quenching of quinacrine fluorescence. Membranes of strain 7, depleted of Mg2+ATPase by EDTA treatment, lack both activities. Membrane vesicles from strain NR70, a mutant lacking the Mg2+ATPase, show neither calcium transport nor energy-linked fluorescence quenching. Neither EDTA treatment nor genetic loss of the Mg2+atpase causes a reduction in respiration. Purified Mg2+ATPase from strain 7 can bind to EDTA-treated membrane vesicles from either strain 7 or NR70. This binding restored both calcium transport and fluorescence quenching, driven either by respiration or by ATP. Dicyclohexylcarbodiimide treatment mimics the effect of the Mg2+ATPase in the case of respiration-driven reactions. Treatment with EDTA, while not essential for the binding of the Mg2+ATPase to membrane vesicles of NR70, produced better restoration of both activities. The rate of restoration of fluorescence quenching showed a time lag which may indicate that binding of the Mg2+ATPase is a relatively slow process. Antiserum prepared against the Mg2+ATPase inhibited the quenching of quinacrine fluorescence when driven by ATP but not when driven by respiration. Addition of antiserum prior to addition of Mg2+ATPase prevented the restoration of fluorescence quenching, whether driven by respiration or ATP. These results clearly show that MG2+ATPase has an important role not only in catalyzing ATP synthesis and hydrolysis but also in maintaining the energized membrane state.     

Trinitrophenyl-ATP binding to the ArsA protein: the catalytic subunit of an anion pump.

The ars operon of the conjugative R-factor R773 confers resistance to arsenicals by coding for an anion pump for extrusion of arsenicals from cells of Escherichia coli. Extrusion of arsenite requires only two polypeptides, the ArsA and ArsB proteins. Purified ArsA protein exhibits oxyanion-stimulated ATPase activity and has been shown to bind ATP by photoaffinity labeling with [alpha-32P]ATP. From sequence analysis the ArsA protein is predicted to have two nucleotide binding folds, one in the N-terminal half and one in the C-terminal half of the protein. Purified ArsA protein bound a fluorescent ATP analogue, 2',3'-O-(2,4,6-trinitrophenylcyclohexadienylidene)adenosine- 5'-triphosphate, with an apparent stoichiometry of 2 mol of nucleotide per mole of ArsA. Strains expressing plasmids with mutations in the N-terminal consensus nucleotide sequence bound only 1 mol of nucleotide per mole of protein.     

Conformational states of (K+ + H+)-ATPase studied using tryptic digestion as a tool

The (K+ + H+)-ATPase from gastric mucosa has been treated by limited proteolytic digestion with trypsin to study the conformational states of the enzyme. The existence of a K+- and an ATP-form of the enzyme follows from the kinetics of inactivation and from the specific cleavage products. In the presence of K+ the 95 kDa chain is cleaved into two fragments of 56 and 42 kDa, whereas in the presence of ATP fragments of 67 and 35 kDa are formed. When Mg2+ is present during tryptic digestion cleavage products which are specific for both the ATP- and the K+-form of the enzyme are yielded. In analogy to ATP, Mg2+ is able to convert the enzyme from a K+-conformation to a more protected form. Moreover Mg2+ supports the protecting effect of ATP against tryptic inactivation. The K0.5 for ATP is lowered from 1.6 mM (no Mg2+) to 0.2 mM in the presence of 10 mM Mg2+. Mg2+, which in previous studies has been shown to induce a specific conformation, apparently induces a conformation different from the K+-form of the enzyme and has ATP-like effects on the enzyme. In addition it has been found that in the initial rapid phase of the digestion process the K+-ATPase activity is interrupted at a step which is very likely the interconversion of the phosphoenzyme forms E1P and E2P, since neither the K+-stimulated p-nitrophenylphosphatase activity nor the phosphorylation of the enzyme are inhibited in this phase. During the tryptic digestion in the presence of K+ there is a good correlation between the residual ATPase activity and the amount of the catalytic subunit left, suggesting that the latter is homogeneous. After tryptic digestion in the presence of K+, phosphorylation only occurs in the 42 kDa and not in the 56 kDa band. The same experiments in the presence of ATP yield only phosphorylation in the 67 kDa band and not in the 35 kDa band. A provisional model for the structure of the catalytic subunit is given.     

ArsD: an As(III) metallochaperone for the ArsAB As(III)-translocating ATPase

The toxic metalloid arsenic is widely disseminated in the environment and causes a variety of health and environment problems. As an adaptation to arsenic-contaminated environments, organisms have developed resistance systems. Many ars operons contain only three genes, arsRBC. Five gene ars operons have two additional genes, arsD and arsA, and these two genes are usually adjacent to each other. ArsA from Escherichia coli plasmid R773 is an ATPase that is the catalytic subunit of the ArsAB As(III) extrusion pump. ArsD was recently identified as an arsenic chaperone to the ArsAB pump, transferring the trivalent metalloids As(III) and Sb(III) to the ArsA subunit of the pump. This increases the affinity of ArsA for As(III), resulting in increased rates if extrusion and resistance to environmentally relevant concentrations of arsenite. ArsD is a homodimer with three vicinal cysteine pairs, Cys12–Cys13, Cys112–Cys113 and Cys119–Cys120, in each subunit. Each vicinal pair binds one As(III) or Sb(III). ArsD mutants with alanines substituting for Cys112, Cys113, Cys119 or Cys120, individually or in pairs or truncations lacking the vicinal pairs, retained ability to interact with ArsA, to activate its ATPase activity. Cells expressing these mutants retained ArsD-enhanced As(III) efflux and resistance. In contrast, mutants with substitutions of conserved Cys12, Cys13 or Cys18, individually or in pairs, were unable to activate ArsA or to enhance the activity of the ArsAB pump. It is proposed that ArsD residues Cys12, Cys13 and Cys18, but not Cys112, Cys113, Cys119 or Cys120, are required for delivery of As(III) to and activation of the ArsAB pump.     

Mutagenesis of the C-terminal nucleotide-binding site of an anion-translocating ATPase.

An oxyanion-translocating ATPase encoded by a bacterial plasmid confers resistance to antiomonials and arsenicals in Escherichia coli by extrusion of the toxic oxyanions from the cytosol. The anion pump is composed of two polypeptides, the ArsA and ArsB proteins. Purified ArsA protein is an oxyanion-stimulated ATPase with two nucleotide-binding consensus sequences, one in the N-terminal half and one in the C-terminal half of the protein. The ArsA protein can be labeled with [alpha-32P]ATP by a UV-catalyzed reaction. Previously reported mutations in the N-terminal site abolish photoadduct formation. Using site-directed mutagenesis the glycine-rich region of the C-terminal putative nucleotide-binding sequence was altered. Three C-terminal site mutant proteins (GR337, KE340, KN340) were analyzed, as well as one additional N-terminal mutant protein (KE21). Strains bearing the mutated plasmids were arsenite sensitive to varying degrees. The purified ArsA protein from mutant KE340 retained approximately 20% of the wild type oxyanion-stimulated ATPase activity, while the purified proteins from the other mutants were catalytically inactive. The KE21 mutation in the N-terminal nucleotide-binding site eliminated photoadduct formation with [alpha-32P] ATP, while the purified proteins with mutations in the C-terminal site retained the ability to form a photoadduct. Each mutant protein was capable of forming a membrane-bound complex in arsB expressing strains. These results suggest first that both sites are required for resistance and ATPase activity, and second that the conserved lysyl residue in the glycine-rich loop of the C-terminal nucleotide-binding site is not essential for catalytic activity.