The ubiquitous thioredoxin fold proteins catalyze oxidation, reduction, or disulfide exchange reactions depending on their redox properties. They also play vital roles in protein folding, redox control, and disease. Here, we have shown that a single residue strongly modifies both the redox properties of thioredoxin fold proteins and their ability to interact with substrates. This residue is adjacent in three-dimensional space to the characteristic CXXC active site motif of thioredoxin fold proteins but distant in sequence. This residue is just N-terminal to the conservative cis-proline. It is isoleucine 75 in the case of thioredoxin. Our findings support the conclusion that a very small percentage of the amino acid residues of thioredoxin-related proteins are capable of dictating the functions of these proteins.The thioredoxin fold is the core scaffold of numerous proteins that control disulfide redox activity in the cell (1-3). These redox proteins share very little sequence homology, but all of them incorporate the four-stranded ␤-sheet, three flanking ␣-helices, and the redox-active CXXC motif of the TRX 5 fold (Fig. 1A). The archetype of the family is thioredoxin (4), a disulfide reductase that maintains a reducing cytosolic environment. Other TRX fold redox proteins include the Dsb proteins (1), which regulate the formation of disulfide bonds in prokaryotes, and protein-disulfide isomerase (5), which catalyzes the oxidation and shuffling of disulfides in the endoplasmic reticulum of eukaryotic cells.This wide range of redox activities of TRX fold proteins is thought to be a consequence of modifications to the common scaffold, which result in different redox properties. Thus, the redox potential of Escherichia coli thioredoxin is very reducing, at Ϫ271 mV (6, 7), whereas that of the oxidizing periplasmic protein E. coli DsbA is Ϫ120 mV (8). Thioredoxin fold proteins that participate in a wide range of thiol disulfide exchange reactions, such as the eukaryotic protein-disulfide isomerases, have intermediate redox potentials (around Ϫ160 mV (9)).Thioredoxin-related proteins provide an attractive model for the study of how protein function is dictated by sequence and three-dimensional structure; this is because their functions are, in part, determined by their redox properties, which in turn, are easy to quantify. For example, mutations in thioredoxin that make its redox potential more oxidative complement null mutations in the oxidase DsbA (10, 11). A detailed understanding of how thioredoxin fold sequence affects redox properties provides an excellent opportunity to relate sequence and function. Previous work has focused on the role of the CXXC "redox rheostat" active site in determining the properties of thioredoxin-related proteins (3,12,13). Experiments that exchange the X-X dipeptide of one thiol-disulfide oxidoreductase with that of another generally result in an oxidoreductase with a redox potential partially shifted in the direction of the oxidoreductase protein that served as the source of the dipepti...