Methanococcus vannielii selenium-binding protein (SeBP): chemical reactivity of recombinant SeBP produced in Escherichia coli.
A selenium-binding protein (SeBP) from Methanococcus vannielii was recently identified, and its gene was isolated and overexpressed in Escherichia coli [Self, W. T., Pierce, R. & Stadtman, T. C. (2004) IUBMB Life 56, 501-507]. SeBP and recombinant SeBP (rSeBP) migrated as approximately 42-kDa species on native gels and as approximately 33-kDa species on SDS gels. rSeBP consists of identical 8.8-kDa subunits, each containing a single cysteine residue. rSeBP isolated in the absence of reducing agents contained oxidized cysteine (89%) and very little bound selenium (0.05 eq or less per subunit). Complete reduction of the oxidized cysteine residues in rSeBP with Tris(2-carboxyethyl)phosphine required addition of a denaturant, such as 1 M guanidine-hydrochloride. With selenite as the selenium source and the isolated reduced protein as sole reductant, binding of one selenium per tetramer under anaerobic conditions required four cysteine thiol groups, one on each subunit. In the corresponding reaction, with reduced glutathione (GSH), equimolar amounts of selenodiglutathione (GSSeSG) and glutathione disulfide are formed from selenite and 4 GSH. At GSH-to-selenite ratios >4:1, conversion of GSSeSG to a perselenide derivative, GSSe(-), occurs. However, with the reduced rSeBP as sole electron donor in the reaction with selenite, further conversion of the R-SSeS-R product apparently did not occur. Prior alkylation of the cysteine thiol groups in reduced rSeBP prevented selenite reduction and selenium binding under comparable conditions.
Patteson
Kemberly G
KG
Laboratory of Biochemistry, National Heart, Lung, and Blood Institute, National Institutes of Health, Building 50 Room 2120, 50 South Drive, Bethesda, MD 20892-8012, USA.
Trivedi
Neel
N
Stadtman
Thressa C
TC
D002152:Calorimetry, Differential Scanning; D003545:Cysteine / Q000378:metabolism; D004586:Electrophoresis; D004926:Escherichia coli; D005978:Glutathione / Q000378:metabolism; D019791:Guanidine; D006863:Hydrogen-Ion Concentration; D017017:Methanococcus / Q000378:metabolism*; D010720:Phosphines; D011485:Protein Binding; D021122:Protein Subunits; D011994:Recombinant Proteins / Q000378:metabolism*; D051137:Selenium-Binding Proteins / Q000378:metabolism*; D018038:Sodium Selenite / Q000378:metabolism; D013054:Spectrophotometry, Atomic
D003545:Cysteine; D005978:Glutathione; D019791:Guanidine; D010720:Phosphines; D021122:Protein Subunits; D011994:Recombinant Proteins; D051137:Selenium-Binding Proteins; D018038:Sodium Selenite; C080938:tris(2-carboxyethyl)phosphine