Biochemical and biophysical characterization of the Selenium binding and reducing site in Arabidopsis thaliana homologue to mammals Selenium Binding Protein 1 [Plant Biology]

October 1st, 2014 by Schild, F., Kieffer-Jaquinod, S., Palencia, A., Cobessi, D., Sarret, G., Zubieta, C., Jourdain, A., Dumas, R., Forge, V., Testemale, D., Bourguignon, J., Hugouvieux, V.

The function of Selenium Binding Protein 1 (SBP1), present in almost all organisms, has not yet been established. In mammals, SBP1 is known to bind the essential element, Selenium but the binding site has not been identified. In addition, the SBP family has numerous potential metal binding sites that may play a role in detoxification pathways in plant. In Arabidopsis thaliana , SBP1 over−expression increases tolerance to two toxic compounds for plants, selenium and cadmium, often found as soil pollutants. For a better understanding of AtSBP1 function in detoxification mechanisms, we investigated the chelating properties of AtSBP1 towards different ligands with a focus on selenium using biochemical and biophysical techniques. Thermal − shift assay together with inductively coupled plasma mass spectrometry revealed that AtSBP1 binds selenium after incubation with selenite (SeO32) with a ligand to protein molar ratio of 1:1. Isothermal titration calorimetry confirmed the stoichiometry 1:1 and revealed an unexpectedly large value of binding enthalpy suggesting a covalent bond between selenium and AtSBP1. Titration of reduced Cys residues and comparative mass spectrometry on AtSBP1 and the purified selenium−AtSBP1 complex, identified Cys21 and Cys22 as being responsible for the binding of one selenium. These results were validated by site directed mutagenesis. Selenium K−edge X ray absorption near edge structure spectroscopy performed on the Selenium −AtSBP1 complex demonstrated that AtSBP1 reduced SeO3 2− to form a R−S−Se−S−R−type complex. The capacity of AtSBP1 to bind different metals and selenium is discussed with respect to the potential function of AtSBP1 in detoxification mechanisms and selenium metabolism.
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