The mammalian selenoproteinthioredoxinreductase 1(TrxR1)is one of the most importantenzymes in redox regulation,antioxidant defense,and cellular growth.TrxR1 can catalyze efficient reduction of juglone(5-hydroxy-1,4-naphthoquinone;walnut toxin)in a reaction which,in contrast to reduction of most other substrates of the enzyme,is not selenocysteine(Sec,U)dependent.Using site-directed mutagenesis,we here found that a sole Cys residue at the C-terminus of TrxR1 is highly required for high-efficiency juglone-coupled NADPH oxidase activity of Sec-deficient enzyme,occurring with mixed one-and two-electron reactions producing superoxide.The activity also utilizes the noncovalently bound FAD and the N-terminal redox active disulfide/dithiol motif of TrxR1.If a sole Cys residue at the C-terminal tail of TrxR1,in the absence of Sec,was moved further towards the C-terminal end of the protein compared to its natural position as residue 497,juglone reduction was,surprisingly,further increased.Four residues of Sec-deficient TrxR1 were found to be easily arylated by juglone,including the Cys residue at position 497.Based upon our observations we suggest a model for involvement of the juglone-arylated C-terminal motif of TrxR1 to explain its high activity with juglone.This study thus providesnovel insights into the catalytic mechanisms of TrxR1.One-electron juglone reduction by TrxR1 producing superoxide should furthermore contribute to the well-known prooxidant cytotoxicity of juglone.