The Inhibitory Effects of Au(III) and Ni(II) on Thioredoxin Reductase

Abstract

The selenoenzyme thioredoxin reductase (TrxR) keeps redox balance of the protein thioredoxin (Trx) skewed to the reduced state. Thioredoxin redox balance is critical to cellular processes such as DNA synthesis, gene expression, and cellular redox management. Thus, the activity of TrxR plays a major role in regulating these processes, and TrxR inhibitors may serve as important therapeutic modulators of these processes. The catalytic activity of TrxR relies on highly conserved cysteine and selenocysteine residues at its catalytic sites. Because Au(III) and Ni(II) have markedly different affinities for sulfur or selenium, the current study tested the hypothesis that these tons would inhibit TrxR activity to different degrees. If true, this hypothesis would support S/Se binding as a primary mechanism of TrxR inhibition by metal ions. TrxR activity was assessed in vitro by measuring the reduction of the surrogate substrate 5,5'-dithiobis- (2-nitrobenzoic acid) (DTNB) to its colored product 4-thio,2-nitrobenzoic acid (TNB). Au(III) or Ni(II) solutions were added at a range of concentrations to determine inhibitory doses. To further test the role of S/Se binding in inhibition, excess cysteine or selenocysteine was added as a competitive target for the metals. The results demonstrated a clear difference in inhibitory potency of the metal ions; Au(III) inhibited TrxR activity at concentrations above 5 nM, whereas Ni(ff) a. concentrations up to 50 pM (50,000 nM) were not inhibitory. However, cysteine and selenocysteine did not mitigate Au(III) inhibition, suggesting that simple distinctive binding competition was not occurring. Higher concentrations of these amino acids appeared to directly reduce the DTNB substrate. Collectively, these results support the hypothesis thai some transition metals inhibit TrxR according to their selenium or sulfur affinity, bu, .hat direct binding of these metals to selenium or sulfur may not account completely for their inhibitory behavior.