Reactivity of thioredoxin as a protein thiol-disulfide oxidoreductase

Thiol-disulfide reactions are crucial for redox homeostasis in the cell.1-2 As a disulfide oxidoreductase, thioredoxin (Trx, ∼12 kDa) regulates a wide variety of biological molecules in both eukaryotic and prokaryotic species, including ribonucleotide reductase, peroxiredoxin, methionine sulfoxide reductase, phosphatase and tensin homolog (PTEN), transcription factors such as nuclear factor-κB (NF-κB), redox factor-1 (Ref-1), and activator protein-1 (AP-1).3 Thus, Trx has been implicated in such diverse processes as antioxidant defense, DNA repair and synthesis, redox regulation, and apoptosis.4-9 In the evolution of Trx catalysis important physical factors (e.g., a hydrophobic binding groove) have appeared to make Trx bind the disulfide substrate in a specific fashion and generate stabilizing interactions.10-11 The interactions and substrate binding were shown to regulate the geometry and orientation of the target disulfide in the catalytic site of the enzyme, and account for Michaelis-Menten–type kinetics of disulfide reduction by Trx.10-11 Trx molecules contain two cysteines in the active site in a CXXC motif. The chemistry of Trx-catalyzed protein disulfide reduction following substrate-binding includes a nucleophilic attack on the disulfide of target proteins by the N-terminal cysteine thiolate of Trx (which is Cys32 in the E. coli numbering) to form an intermolecular mixed disulfide, and a subsequent attack on the disulfide intermediate by the thiolate of the C-terminal cysteine of Trx (Cys35), producing the reduced target protein and oxidized Trx (Figure 1).12-13 To date a number of factors, including the identity of the amino acid residues spanning the CXXC motif, the pKa of the redox active thiols, the redox potential of the disulfide/dithiol couple, the acid/base catalysts, the molecular interactions and the local conformational changes after substrate binding, have been implicated in the regulation of Trx activity as a protein disulfide oxidoreductase, albeit with controversies and debates. In this work, we review the emerging evidence that support or dispute the regulating roles of these factors, and propose the likely prime determinants of Trx activity that deserve more attention in future research, with the hope to promote a better understanding of the mechanistic factors of Trx reactivity. Figure 1 A schematic cartoon view of a Trx-catalyzed disulfide reduction. A number of factors are involved in the regulation of Trx activity, including the amino acid residues spanning the redox active CXXC motif, molecular interaction (e.g., electrostatic force), ... The 3-D structures of Trx proteins are highly conserved, with the five central β-strands surrounded by four α-helices (Figure 2, A and B). Part of the redox active center (-CGPC- motif) protrudes at the surface of the molecule at the very N-terminus of helix α2, with Cys35 largely covered by the N-terminal portion of the helix α2. Only one side of the Cys32-SH is accessible to solvent for the transfer of reducing equivalents in Trxox but the side chain of Cys32 turns into the solvent upon reduction.14-16 The Trx fold is also found in several other classes of enzymes that interact with substrates containing either disulfides or dithiols. Protein families that have a Trx-fold include the thioredoxin, Dsb (disulfide bond formation protein) proteins, glutaredoxin (Grx), glutathione S-transferase, and protein disulfide isomerase (PDI) families.2,16-17 We performed a structural bioinformatics study on 515 sequences of the Trx family, 495 sequences of the DsbA family, and 382 sequences of the PDI family (from the Conserved Domain Database (CDD),18 http://www.ncbi.nlm.nih.gov/sites/entrez?db=cdd). This search confirmed a recent observation that the two components that are most conserved across these 3 families of proteins include an active site CXXC motif, and a conserved proline that is distant in sequence from this active site but immediately adjacent in 3 dimensional space.19 In the Trx family, the CXXC motif has the sequence CGPC and is present at position 32–35, while the proline residue is at position 76 in its best-studied member, E. coli thioredoxin 1 (EcTrx; PDB: 2TRX_A) (Figure 2C). The highly conserved proline is at position 151 in E. coli DsbA (PDB: 1DSB_A) and at position 83 in human PDI (PDB: 1MEK) (Figure 2, D and E). In EcTrx, the distances between the N-atom of Pro76 and the two sulfur atoms of the cysteine pair are 4.07 A (Cys32) and 3.62A (Cys35) (Figure 2F). The next closest residue is Asp26, and the shortest distance between an O-atom of Asp26 and the sulfur atom of Cys35 is 5.59 A (Figure 2G). Pro76 and Asp26 have been shown to play important roles in the redox reactions due to their close contact with the Cys pair.20-21 Figure 2 The structure of EcTrx and conserved amino acid residues in the Trx family. (A) and (B) are the ribbon diagrams of oxidized and reduced EcTrx, respectively. They show the spatial distances between Trp28 and Cys32, and between Trp28 and Asp26. The color ... Conserved structural features such as the Trx-fold and secondary structure in Trx family members, never-the-less, allow diverse reactivities in catalyzing protein disulfide interchange reactions. Quantum mechanical calculations suggest that the relative stability of thiolates in the CXXC motif determines whether these enzymes catalyze oxidation, reduction, or isomerization.2,22-23 Because the cysteine thiolates in Trx are both poorly stabilized, Trx is a good reducing agent. In contrast, DsbA stabilizes both cysteine thiolates and thus is a good oxidizing agent; isomerases such as PDI have one thiolate relatively solvent exposed and poorly stabilized while the other (relatively buried) thiolate is highly stabilized.22-23 Static calculations, such as the quantum mechanical calculations, provide insights into the thermodynamics of thiol-disulfide reactions, but as previously noted and as recently further investigated,1,24 the reactivity in an actual reaction is more complex due to dynamics and conformational changes that occur during substrate binding. For instance, studies with site-specific mutagenesis indicate that the relative stability of the oxidized versus the reduced form determines the difference in the redox potentials of Trx from Staphylococcus aureus (SaTrx).25-27 Replacement of the conserved proline in the CXXC motif by threonine or serine greatly reduced the relative stability value and made SaTrx less reducing.25 Moreover, the activation energy barrier for forming a transition state complex must be overcome by dynamics or other properties to accomplish a thermodynamically favorable thiol-disulfide reaction.1 However, DsbA from Staphylococcus aureus shows identical stabilities in oxidized and reduced forms, suggesting that alternative mechanisms beyond thermodynamic stability underlie the activity of SaDsbA in thiol-disulfide reactions.28-29 In this work, we review recent studies on the reactivity of thioredoxins as a protein disulfide oxidoreductase. Our goal is to provide an updated view and a better understanding of the determinants of Trx reactivity and their critical roles in redox homeostasis.