The CXXC motif is more than a redox rheostat.

The CXXC active-site motif of thiol-disulfide oxidoreductases is thought to act as a redox rheostat, the sequence of which determines its reduction potential and functional properties. We tested this idea by selecting for mutants of the CXXC motif in a reducing oxidoreductase (thioredoxin) that complement null mutants of a very oxidizing oxidoreductase, DsbA. We found that altering the CXXC motif affected not only the reduction potential of the protein, but also its ability to function as a disulfide isomerase and also impacted its interaction with folding protein substrates and reoxidants. It is surprising that nearly all of our thioredoxin mutants had increased activity in disulfide isomerization in vitro and in vivo. Our results indicate that the CXXC motif has the remarkable ability to confer a large number of very specific properties on thioredoxin-related proteins.