Arsenic Binding and Transfer by the ArsD As(III) Metallochaperone
- 2 April 2010
- journal article
- research article
- Published by American Chemical Society (ACS) in Biochemistry
- Vol. 49 (17), 3658-3666
- https://doi.org/10.1021/bi100026a
Abstract
ArsD is a metallochaperone that delivers trivalent metalloids [As(III) or Sb(III)] to the ArsA ATPase, the catalytic subunit of the ArsAB pump encoded by the arsRDABC operon of Escherichia coli plasmid R773. Interaction with ArsD increases the affinity of ArsA for As(III), conferring resistance to environmental concentrations of arsenic. Previous genetic analysis suggested that ArsD residues Cys12, Cys13, and Cys18 are involved in the transfer of As(III) to ArsA. Here X-ray absorption spectroscopy was used to show that As(III) is coordinated with three sulfur atoms, consistent with the three cysteine residues forming the As(III) binding site. Two single-tryptophan derivatives of ArsD exhibited quenching of intrinsic protein fluorescence upon binding of As(III) or Sb(III), which allowed estimation of the rates of binding and affinities for metalloids. Substitution of Cys12, Cys13, or Cys18 decreased the affinity for As(III) more than 10-fold. Reduced glutathione greatly increased the rate of binding of As(III) to ArsD but did not affect binding of As(III) to ArsA. This suggests that in vivo cytosolic As(III) might be initially bound to GSH and transferred to ArsD and then to ArsAB, which pumps the metalloid out of the cell. The As(III) chelator dimercaptosuccinic acid did not block the transfer from ArsD to ArsA, consistent with channeling of the metalloid from one protein to the other, as opposed to release and rebinding of the metalloid. Finally, transfer of As(III) from ArsD to ArsA occurred in the presence of MgATP at 23 °C but not at 4 °C. Neither MgADP nor MgATP-γ-S could replace MgATP. These results suggest that transfer occurs with a conformation of ArsA that transiently forms during the catalytic cycle.Keywords
This publication has 41 references indexed in Scilit:
- Speciation, formation, stability and analytical challenges of human arsenic metabolitesJournal of Analytical Atomic Spectrometry, 2009
- Structural Biology of Copper TraffickingChemical Reviews, 2009
- Evolution of Metal(loid) Binding Sites in Transcriptional RegulatorsPublished by Elsevier BV ,2008
- Convergent Evolution of a New Arsenic Binding Site in the ArsR/SmtB Family of MetalloregulatorsPublished by Elsevier BV ,2007
- An arsenic metallochaperone for an arsenic detoxification pumpProceedings of the National Academy of Sciences of the United States of America, 2006
- Characterization of the Particulate Methane Monooxygenase Metal Centers in Multiple Redox States by X-ray Absorption SpectroscopyInorganic Chemistry, 2006
- Thermodynamics of the As(III)−Thiol Interaction: Arsenite and Monomethylarsenite Complexes with Glutathione, Dihydrolipoic Acid, and Other Thiol LigandsInorganic Chemistry, 2005
- Unisite and Multisite Catalysis in the ArsA ATPasePublished by Elsevier BV ,2002
- A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye bindingAnalytical Biochemistry, 1976
- Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4Nature, 1970