Reduction of L-methionine selenoxide to seleno-L-methionine by endogenous thiols, ascorbic acid, or methimazole

Abstract

Seleno-L-methionine (SeMet) can be oxidized to L-methionine selenoxide (MetSeO) by flavin-containing monooxygenase 3 (FMO3) and rat liver microsomes in the presence of NADPH. MetSeO can be reduced by GSH to yield SeMet and GSSG. In the present study, the potential reduction of MetSeO to SeMet by other cellular components and antioxidants was investigated. Besides GSH, other thiols (L-cysteine, or N-acetyl-L-cysteine) and antioxidants (ascorbic acid and methimazole) also reduced MetSeO to SeMet. This reduction is unique to MetSeO since methionine sulfoxide was not reduced to methionine under similar conditions. The MetSeO reduction by thiols was instaneous and much faster than the reduction by ascorbic acid or methimazole. However, only one molar equivalent of ascorbic acid or methimazole was needed to complete the reduction, as opposed to two molar equivalents of thiols. Whereas the disulfides produced by the reactions of MetSeO with thiols are chemically stable, methimazole disulfide readily decomposed at pH 7.4, 37 degrees C to yield methimazole, methimazole-sulfenic acid, methimazole sulfinic acid, methimazole S-sulfonate, 1-methylimidazole (MI) and sulfite anion. Collectively, the results demonstrate reduction of MetSeO to SeMet by multiple endogenous thiols, ascorbic acid, and methimazole. Thus, oxidation of SeMet to MetSeO may result in depletion of endogenous thiols and antioxidant molecules. Furthermore, the novel reduction of MetSeO by methimazole provides clear evidence that methimazole should not be used as an alternative FMO substrate when studying FMO-mediated oxidation of SeMet.

Figure 1

Loss of MetSeO and formation of SeMet by endogenous thiols (GSH, A, and L-cysteine, B) and antioxidants (MIZ, C, and ascorbic acid, D) after in vitro incubations of MetSeO with these compounds at different molar ratios at physiological conditions (pH 7.4, 37ºC). Solid lines indicate SeMet values and dashed lines indicate MetSeO values. Filled circles represent experiments conducted at a 1:1 molar ratio, open circles 1:2 molar ratio, and filled triangles 1:5 molar ratio. Values presented are means ± S.D. (n=3).

Figure 2

Typical HPLC chromatogram of the reaction of MetSeO with MIZ with UV detection at 220 nm after 0 min (A), 50 min (B), and 150 min (C) reaction time. MI = 1-methylimidazole.

Figure 3

A) LC/MS chromatogram of the reaction mixture of MetSeO and MIZ at pH 7.4, 37ºC. The identities of peaks I-IV, were confirmed by MS analyses as 1-methylimidazole, SeMet, MIZ-S-sulfonate and MIZ, respectively. The mass spectrum of the MIZ S-sulfonate peak is shown (B) as to our knowledge, the mass spectrum of this compound has not been previously reported.

Figure 4

Structures of MIZ and related compounds formed in its reaction with MetSeO and possible mechanisms of MIZ disulfide and MIZ S-sulfonate formation and/or breakdown. MI = 1-methylimidazole; MIZ-S-S-MIZ = MIZ disulfide.

Figure 5

Oxidative metabolism of SeMet by FMO3 and the reduction of MetSeO by GSH, MIZ, or ascorbic acid to regenerate SeMet.