Thioredoxin Reductase
Created by Andaleeb Rahman
Thioredoxin reductase (pdb id: 1F6M) is an enzyme that belongs to the pyridine nucleotide-disulfide oxidoreductase family of flavoenzymes. This enzyme is responsible for catalyzing the reduction of thioredoxin, an important antioxidant found in all organisms (1). The general reaction as well as the reaction mechanism can be seen in Figures 1 and 2, respectively, under the Reaction Scheme tab. The reaction catalyzed by thioredoxin reductase is critical to maintaining proteins in their reduced state, otherwise the biological function of proteins can become altered if they are subject to oxidation. In higher, more complex organisms thioredoxin reductase develops broader substrate specificity as well as an additional redox active motif. However, the fundamental role of thioredoxin is disulfide reductase activity, which generally depends on NADPH and thioredoxin reductase activity (2).
The crystallized structure of the thioredoxin reductase - thioredoxin complex is made up of eight chains where chains A, B, E, and F are four identical chains of the enzyme thioredoxin reductase, seen in red, and chains C, D, G, and H are four identical chains of the substrate thioredoxin, seen in blue (3). Thioredoxin reductase is a homodimeric flavoprotein responsible for catalyzing the reaction that transfers electrons from NADPH to FAD, from reduced FAD to the active-site disulfide, and from the active-site dithiol to the small protein thioredoxin (4). Each monomer subunit of thioredoxin reductase in Escherichia coli consists of two domains: the FAD binding and AADP binding domains (5). Both of these subunits contain one FAD molecule and one redox active disulfide (6).
Thioredoxin reductase is a unique member of the pyridine nucleotide-disulfide oxidoreductase family of flavoenzymes because its active site disulfide exists in the same domain as the AADP binding site instead of the FAD binding domain (6). Furthermore, each thioredoxin reductase monomeric subunit contains a total of 10 cavities that are completely closed off from binding to the substrate or other molecules.
The secondary structure of thioredoxin reductase is composed of 11 alpha helices, 23 beta sheets, and various random coils (3). The random coils include beta alpha beta units, beta hairpins, beta bulges, beta turns, and gamma turns (3). The polarity of thioredoxin reductase demonstrates how nonpolar residues are greatly outnumbered by polar, acidic, and basic residues that can be seen as yellow, red, and blue, respectively. Furthermore, a double-stranded beta sheet is responsible for connecting the two globular domains of each monomer (4). These antiparallel beta strands are made up the following residues: 113-Gly-Ala-Ser-Ala-Arg-Tyr-Leu-119 and 244-Gly-His-Ser-Pro-Asn-248 (5).
The redox active disulfide in thioredoxin reductase is formed by residues Cys-135 and Cys-138, while the corresponding redox active disulfide of thioredoxin is found at residues Cys-32 and Cys-35 (1, 7). The Asp-139 residue is suggested to be the active site acid-base catalyst involved in the protonation of the nascent thiolate ion of reduced thioredoxin (1). Arg-130 and nearby residues in the NADPH domain of thioredoxin reductase interact with the protruding Arg-73 residue of thioredoxin through hydrogen bonding (1). Also, Phe-141 and Phe-142 from thioredoxin reductase fit into the hydrophobic pocket on thioredoxin composed of the residue Trp-31, Ile-60, Gly-74, and Ile-75 (3).
In order to catalyze the reduction of thioredoxin, the enzyme thioredoxin reductase has both FAD and AADP binding domains in addition to its catalytic sites for binding the substrate (5). The FAD binding domain includes residues from Gly-1 to Tyr-118 and His-245 to Lys-320 while the AADP binding domain includes residues from Lys-119 to Gly-244 (3). The prosthetic groups AADP and FAD are required as sources of reducing equivalents that can be transferred to the active site disulfide of thioredoxin (8).
Thioredoxin reductase has two conformations ( FO and FR) that arise from the unimpeded rotation of the AADP binding domain by 66° with respect to the FAD binding domain (1). The FO conformation allows the enzyme disulfide to oxidize the flavin (6). In this conformation the active site disulfide is unavailable for reaction with thioredoxin because it is buried in the structure (6). Thus, the AADP binding domain must be rotated into a position more favorable for hydride transfer to the flavin, which frees the active site cysteines to move to the surface of the enzyme so they can react with the substrate (6). This conformation is called the FR conformation because it allows flavin reduction by AADP (6). Rearrangement from the FO conformation to the FR conformation requires exchanges of the smooth hydrophilic interfaces of the FAD and AADP binding domains, like ball-and-socket rotation (3). Mostly residues in the AADP binding domain change their position relative to the flavin ring after rotation, while Cys-135, Cys-138, and Asp-139 move only slightly (1).