A Mechanistic Investigation of the C‑Terminal Redox Motif of Thioredoxin Reductase from Plasmodium falciparum

High-molecular mass thioredoxin reductases (TRs) are pyridine nucleotide disulfide oxidoreductases that catalyze the reduction of the disulfide bond of thioredoxin (Trx). Trx is responsible for reducing multiple protein disulfide targets in the cell. TRs utilize reduced β-nicotinamide adenine dinucleotide phosphate to reduce a bound flavin prosthetic group, which in turn reduces an N-terminal redox center that has the conserved sequence CICVNVGCCT, where CIC is denoted as the interchange thiol while the thiol involved in charge-transfer complexation is denoted as CCT. The reduced N-terminal redox center reduces a C-terminal redox center on the opposite subunit of the head-to-tail homodimer, the C-terminal redox center that catalyzes the reduction of the Trx-disulfide. Variations in the amino acid sequence of the C-terminal redox center differentiate high-molecular mass TRs into different types. Type Ia TRs have tetrapeptide C-terminal redox centers of with a GCUG sequence, where U is the rare amino acid selenocysteine (Sec), while the tetrapeptide sequence in type Ib TRs has its Sec residue replaced with a conventional cysteine (Cys) residue and can use small polar amino acids such as serine and threonine in place of the flanking glycine residues. The TR from Plasmodium falciparum (PfTR) is similar in structure and mechanism to type Ia and type Ib TRs except that the C-terminal redox center is different in its amino acid sequence. The C-terminal redox center of PfTR has the sequence G534CGGGKCG541, and we classify it as a type II high-molecular mass TR. The oxidized type II redox motif will form a 20-membered disulfide ring, whereas the absence of spacer amino acids in the type I motif results in the formation of a rare eight-membered ring. We used site-directed mutagenesis and protein semisynthesis to investigate features of the distinctive type II C-terminal redox motif that help it perform catalysis. Deletion of Gly541 reduces thioredoxin reductase activity by ∼50-fold, most likely because of disruption of an important hydrogen bond between the amide NH group of Gly541 and the carbonyl of Gly534 that helps to stabilize the β–turn−β motif. Alterations of the 20-membered disulfide ring either by amino acid deletion or by substitution resulted in impaired catalytic activity. Subtle changes in the ring structure and size caused by using semisynthesis to substitute homocysteine for cysteine also caused significant reductions in catalytic activity, demonstrat