Thioredoxin was purified to homogeneity from the Escherichia coli mutant tsnC 7007 that is defective in phage T7 DNA replication and previously shown to contain a missense thioredoxin. Tryptic peptide maps of reduced and carboxymethylated 7007 thioredoxin combined with amino acid sequence analysis revealed one amino acid substitution; Gly-92 in thioredoxin is exchanged to an aspartic acid residue in the 7007 protein. The missense thioredoxin gave no activity with the gene 5 protein of phage T7 in the complementation to active T7 DNA polymerase. It competitively inhibited the complementation of wild type thioredoxin and gene 5 protein and formed a complex with the gene 5 protein that was retained by antithioredoxin Sepharose. The 7007 thioredoxin has reduced catalytic activity with thioredoxin reductase, ribonucleotide reductase, or as a protein disulfide reductase. The apparent Km value of 7007 thioredoxin as a substrate for thioredoxin reductase was increased 3-fold relative to normal thioredoxin, and the Vmax value was decreased 7-fold. The position of GLy-92 in the known three-dimensional structure of thioredoxin-S2 is correlated with the changed functional properties of the substituted mutant protein.