Mammalian thioredoxin reductase (TR) contains the rare amino acid selenocysteine (Sec), which is essential for the enzyme’s catalytic activity. Substitution of the catalytic Sec residue for a cysteine (Cys) residue, results in a drop in kcat of 100- fold. Homologous high molecular weight TRs from other eukaryotes such as D. melanogaster and C. elegans, have naturally evolved a Sec to Cys substitution in their active sites and these enzymes function with high catalytic activity without the need for a Sec residue. Thus, various TRs can catalyze an identical reaction with either a Cys or Sec residue. A natural assumption in the field has always been that the lower nucleophilicity of a Cys thiol, relative to the selenol of Sec, is the reason for the much lower activity of the mammalian Cys-containing mutant. However, here I provide an alternative explanation. High Mr TRs contain either a Cys-Cys or Cys-Sec dyad that forms an eight-membered ring in the oxidized state during the redox cycle of the enzyme. These eight-membered ring structures are rare in protein structures, presumably due to the strain induced in the intervening peptide bond between the Cys residues. Here I take a “chemical approach” to studying the enzyme mechanism of TR by breaking it into two pieces. This approach is possible because of TR’s structural and mechanistic similarity to glutathione reductase (GR). In comparison to GR, TR contains an additional thiol-disulfide exchange step resulting from the presence of a sixteen amino acid C-terminal extension containing either a vicinal disulfide bond or vicinal selenylsulfide bond. This additional thiol-disulfide exchange step is in the form of the reduction and opening of the eight-membered ring motif. I have constructed a truncated version of the enzyme lacking the amino acid sequence possessing the ring motif so that I could isolate this ring-opening step from the rest of the catalytic cycle by using peptide disulfides/selenylsulfides as substrates. The results of this study using peptide substrates show that the ring opening step is the step of the catalytic cycle that is most effected by Sec to Cys substitution because the higher pKa of the Cys thiolate in comparison to the Sec selenolate means that the Cys residue must be protonated in this step.