Date of Award


Document Type


Degree Name

Master of Science (MS)



First Advisor

Robert J. Hondal


Selenocysteine (Sec) is referred to as the 21st proteogenic amino acid and is found in place of the redox-sensitive amino acid cysteine (Cys) in a small number of proteins. Sec and Cys carry out similar chemistry and are structural isomers save for a single atom difference; the former contains selenium (Se), while the latter contains sulfur (S) in the identical position. Sec poses a high bioenergetic cost for its synthesis and subsequent incorporation into protein not shared by Cys. Since Sec’s discovery in 1976, scientists have debated why certain proteins express Sec while others express Cys. In recent years, it has been shown that redox-active enzymes expressing Sec exhibit a distinct biological advantage over their Cys-containing counterparts. Sec-containing enzymes retain their catalytic activities when exposed to highly oxidizing conditions, while analogous Cys-containing enzymes become significantly inactivated.

This thesis examines the enzyme thioredoxin reductase (TrxR), an essential regulator of cellular redox homeostasis. TrxR is expressed in higher-order organisms such as humans and other mammals with a penultimate Sec residue in its C-terminal redox center, while lower-order organisms such as Drosophila melanogaster (D. melanogaster) express TrxR with Cys. Using spectrophotometric activity assays, we show that the presence of Sec preserves thioredoxin reductase’s ability to reduce its canonical substrate, thioredoxin (Trx), in the presence of hydrogen peroxide. Herein, we use mass spectrometry analysis to provide a biophysical basis for this phenomenon through the characterization and quantitation of the TrxR’s two redox centers that drive its mechanism of action. Our findings show that Sec confers superior resistance to oxidation-induced inactivation not because Sec better resists irreversible hyperoxidation but instead TrxR’s redox centers to better retain their reductive abilities. In D. melanogaster’s Cys-containing TrxR, we show that a significant loss of its redox center’s reductive ability coincides with the loss of its enzymatic activity.



Number of Pages

103 p.