Contribution of selenocysteine to the peroxidase activity of selenoprotein S

Abstract

Selenoprotein S (SelS, VIMP) is an intrinsically disordered enzyme that utilizes selenocysteine to catalyze the reduction of disulfide bonds and peroxides. Here it is demonstrated that selenocysteine is the residue oxidized by the peroxide substrate. It is possible to trap the reaction intermediate selenenic acid when the resolving cysteine is mutated. The selenocysteine allows SelS to rapidly re-form its selenenylsulfide bond following its reduction, and to resist inactivation by H2O2. We propose that SelS's peroxidase mechanism is similar to that of atypical 2-Cys peroxiredoxin and that selenocysteine allows SelS to sustain activity under oxidative stress.

Figure 1

(A) Identification of the peroxidatic and resolving residues in SelS using peroxidase assays. NADPH consumption is monitored in the presence of 8 nM hTrxR, 5 μM hTrx and 200 μM H₂O₂ and with and without cSelS, with and without thioredoxin, and with C174S, U188S, and U188Δ mutants. (B) Trapping of the reaction intermediate, selenenic acid, in cSelS C174S. Mass spectrum showing cSelS C174S (15284 Da) and its dimedone adduct (15423 Da).

Figure 2

Electrospray ionization mass spectrometry-based detection of the rate of formation of selenenylsulfide bond in cSelS and the disulfide bond in cSelS U188C. (A) After 60 min the majority of cSelS U188C was still reduced, allowing for full alkylation by NEM. Molecular weights: oxidized cSelS U188C 15253 Da and alkylated cSelS U188C 15505 Da. (B) After 60 min the majority of cSelS was oxidized. Molecular weights: alkylated cSelS U188Δ 15219 Da (a truncated, inactive form that arise during expression), oxidized cSelS 15300 Da, and alkylated cSelS 15550 Da. R denotes percent of oxidized protein.

Figure 3

cSelS resistance to inactivation by H₂O₂. The ability of cSelS to reduce insulin’s intermolecular disulfide bond following incubations with H₂O₂ is monitored by recording the increasing turbidity caused by insulin’s chain B aggregation. (A) cSelS reductase activity assay after incubations with H₂O₂. (B) Percentage of remaining activity of wild type cSelS after treatment with H₂O₂. (C) cSelS C174S reductase activity assay after incubations with H₂O₂. (D) Percentage of remaining activity of cSelS C174S after treatment with H₂O₂. The error bars represent the range of measurements (that is, highest and lowest values) among two repetitions, using two independent protein preparations.

Scheme 1

Proposed reaction mechanism of SelS.