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J. Biol. Chem., Vol. 281, Issue 15, 9925-9934, April 14, 2006

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Cys-113 and Cys-422 Form a High Affinity Metalloid Binding Site in the ArsA ATPase^*

From the Department of Biochemistry and Molecular Biology, Wayne State University, School of Medicine, Detroit, Michigan 48201

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 Mg²⁺-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/C422AB 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/C422AB growing in subtoxic concentrations of arsenite, cells bearing wild type arsAB replaced cells with mutant arsA_C113A/C422AB in less than 1 week, showing that the metalloid binding site confers an evolutionary advantage.

Received for publication, January 5, 2006 , and in revised form, February 1, 2006.

^* This work was supported by United States Public Health Service Grant GM55425. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed. Tel.: 313-577-1512; Fax: 313-577-2765; E-mail: brosen{at}med.wayne.edu.

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