Abstract.
Staphylococcus aureus pI258 CadC is a metal sensor protein that regulates the expression of the cad operon which encodes metal ion resistance proteins involved in the efficient efflux of Cd(II), Pb(II), Zn(II) and, according to one report , Bi(III) ions. In this paper, direct evidence is presented that Bi(III) binds to CadC and negatively regulates cad operator/promoter (O/P) binding. Optical absorption spectroscopy reveals that dimeric CadC binds ∼0.8 mol equivalents of Bi(III) per CadC monomer to form a coordination complex characterized by three S–→Bi(III) ligand-to-metal charge transfer transitions, with the longest wavelength absorption band centered at 415 nm (ε415=4000 MBi –1 cm–1). UV-Vis absorption spectra of wild-type and mutant Cys→Gly (Ser) substitution CadC mutants compared to [Bi(DTT)2], [Bi(GSH)3] and [Bi(NAC)]3 model complexes reveal that Cys7, Cys11, Cys60 and Cys58 directly coordinate Bi(III) in a tetrathiolate coordination complex. The apparent affinity derived from a Bi(III)-displacement optical titration with Cd(II) is estimated to be K Bi≤1012 M–1. Apo-CadC binds with high affinity [K a=1.1(±0.3)×109 M–1; 0.40 M NaCl, pH 7.0, 25 °C] to a 5′-fluorescein-labeled cad O/P oligonucleotide ,while the binding of one molar equivalent of Bi(III) per CadC monomer (Bi1-CadC) reduces the affinity by ∼170-fold. Strikingly, Bi(III)-responsive negative regulation of cad O/P binding is abrogated for Bi1-C60G CadC and severely disrupted in Bi1-C7G CadC, whose relative affinity is reduced only 10-fold. The mechanism of Bi(III)-responsive metalloregulation is discussed, based on the findings presented here. Electronic supplementary material to this paper can be obtained by using the Springer Link server located at http://dx.doi.org/10.1007/s00775-001-0336-9.
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Busenlehner, L.S., Apuy, J.L. & Giedroc, D.P. Characterization of a metalloregulatory bismuth(III) site in Staphylococcus aureus pI258 CadC repressor. J Biol Inorg Chem 7, 551–559 (2002). https://doi.org/10.1007/s00775-001-0336-9
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DOI: https://doi.org/10.1007/s00775-001-0336-9