Formation of a selenium-substituted rhodanese by reaction with selenite and glutathione: Possible role of a protein perselenide in a selenium delivery system

Selenophosphate is the active selenium-donor compound required by bacteria and mammals for the specific synthesis of Secys-tRNA, the precursor of sele nocysteine in selenoenzymes. Although free selenide can be used in vitro fo r the synthesis of selenophosphate, the actual physiological selenium subst rate has not been identified. Rhodanese (EC 2.3.1.1) normally occurs as a p ersulfide of a critical cysteine residue and is believed to function as a s ulfur-delivery protein. Also, it has been demonstrated that a selenium-subs tituted rhodanese (E-Se form) can exist in vitro. In this study, we have pr epared and characterized an E-Se rhodanese. Persulfide-free bovine-liver rh odanese (E form) did not react with SeO32- directly, but in the presence of reduced glutathione (GSH) and SeO32- E-Se rhodanese was generated. These r esults indicate that the intermediates produced from the reaction of GSH wi th SeO32- are required for the formation of a selenium-substituted rhodanes e. E-Se rhodanese was stable in the presence of excess GSH at neutral pH at 37 degreesC. E-Se rhodanese could effectively replace the high concentrati ons of selenide normally used in the selenophosphate synthetase in vitro as say in which the selenium-dependent hydrolysis of ATP is measured. These re sults show that a selenium-bound rhodanese could be used as the selenium do nor in the in vitro selenophosphate synthetase assay.