Protein disulfide isomerases are ubiquitous proteins that catalyze the proper formation of disulfide bonds during oxidative protein folding. The bacterial protein disulfide isomerase DsbC is a homodimeric V-shaped enzyme that consists of a dimerization domain, two α-helical linkers and two opposing thioredoxin fold catalytic domains. The functional significance of the two opposing catalytic sites is not well understood yet. To address this problem we fused the two polypeptides that compose the DsbC homodimer into a single protein. The resulting protein, mDsbC, was shown to exhibit high catalytic activity. Mutants containing only one active site or catalytic domain were constructed and their catalytic properties, including their ability to serve as a thiol oxidant and as a catalyst of disulfide bond rearrangement, were studied in detail and will be discussed. Apart from helping understand the catalytic cycle of disulfide isomerization, this study also provides insights onto the evolution of the eukaryotic enzymes (such as PDI) relative to their bacterial counterparts.