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

Abstract

Selenophosphate is the active selenium-donor compound required by bacteria and mammals for the specific synthesis of Secys-tRNA, the precursor of selenocysteine in selenoenzymes. Although free selenide can be used in vitro for the synthesis of selenophosphate, the actual physiological selenium substrate has not been identified. Rhodanese (EC ) normally occurs as a persulfide of a critical cysteine residue and is believed to function as a sulfur-delivery protein. Also, it has been demonstrated that a selenium-substituted rhodanese (E-Se form) can exist in vitro. In this study, we have prepared and characterized an E-Se rhodanese. Persulfide-free bovine-liver rhodanese (E form) did not react with SeO(3)(2-) directly, but in the presence of reduced glutathione (GSH) and SeO(3)(2-) E-Se rhodanese was generated. These results indicate that the intermediates produced from the reaction of GSH with SeO(3)(2-) are required for the formation of a selenium-substituted rhodanese. E-Se rhodanese was stable in the presence of excess GSH at neutral pH at 37 degrees C. E-Se rhodanese could effectively replace the high concentrations of selenide normally used in the selenophosphate synthetase in vitro assay in which the selenium-dependent hydrolysis of ATP is measured. These results show that a selenium-bound rhodanese could be used as the selenium donor in the in vitro selenophosphate synthetase assay.

Figure 1

Profile of the products from the reaction of the E form of rhodanese with selenium after elution on a desalting column. A reaction mixture (100 μl) containing PBS (pH 7.4), 20 μM E form rhodanese and 100 μM selenite was incubated with (A) or without (B) 400 μM GSH. After a 10-min incubation at 25°C, the reaction mixture was applied to a gel filtration column (1.0× 10 cm) and eluted with PBS at pH 7.4. Aliquots of each fraction were assayed to determine selenium (○) and protein (▴) as described in Materials and Methods.

Figure 2

Stability of selenium bound to rhodanese and GSSeSG. Reaction mixtures (100 μl) containing PBS at pH 7.4, 100 μM selenite, and 400 μM GSH were incubated with 20 μM E form of rhodanese at 37°C for 120 min (○) and without rhodanese at 25°C for 10 min (□) and for 120 min (▴). Gel filtration profile of a desalting column (1.0× 10 cm) eluted with PBS at pH 7.4. Aliquots of each fraction were assayed to determine the concentration of selenium.

Figure 3

Effect of modification of thiol residues by iodoacetamide on the reaction of E form of rhodanese plus selenite in the presence of GSH. E form of rhodanese was treated with iodoacetamide as described in Materials and Methods. Reaction mixtures (100 μl) containing iodoacetamide-treated 20 μM E form rhodanese, 100 μM selenite, and 400 μM GSH were incubated at 25°C. After a 10-min incubation, the reaction mixture was applied to a gel filtration column (1.0× 10 cm) and eluted with PBS at pH 7.4. Aliquots of each fraction were assayed to determine selenium (○) and protein (▴) as described in Materials and Methods.

Figure 4

Effect of the ratio of GSH and selenite on the formation of E-Se rhodanese. Reaction mixtures (100 μl) containing PBS at pH 7.4 and 20 μM E form rhodanese were incubated in the presence of the indicated ratio of GSH and selenite. After a 10-min incubation at 25°C, the reaction mixture was applied to a gel filtration column. Quantitation of bound selenium to rhodanese was determined.

Figure 5

Effect of the GSSeSG concentration on the formation of E-Se rhodanese. Reaction mixtures (100 μl) containing PBS at pH 7.4 and 20 μM E form rhodanese were incubated in the presence of the indicated concentrations of GSSeSG, theoretically produced by the reaction of GSH with selenite in a molar ratio of 4:1. After a 10-min incubation at 25°C, the reaction mixture was applied to the gel filtration column. The amount of selenium bound per mole of rhodanese was determined by the measurement of selenium and protein contents of peak enzyme fractions from a desalting column.

Figure 6

SPS assay with E-Se rhodanese. Assays were performed anaerobically at 37°C in 50 mM Tricine/KOH, pH 8.0/25 mM DTT/8 mM MgCl₂/50 mM KCl/0.1 mM Mg triplex/2 mM ATP/0.2 μCi [¹⁴C]ATP/10 μM SPS. The standard assay, performed in the absence of rhodanese, contained 1.5 mM selenide. E-Se rhodanese at the indicated concentrations was added in the absence of selenide. After a 30-min incubation, reactions were terminated, and the reaction products were separated chromatographically as described in Materials and Methods. AMP was quantitated by liquid scintillation spectroscopy.

Figure 7

Proposed reactions for the formation of protein perselenide and pathways for selenophosphate formation. Cysteine-free rhodanese-type enzyme is shown as a protein (S⁻). Se*, an unidentified transfer form of selenium.