Importance of redox potential for the in vivo function of the cytoplasmic disulfide reductant thioredoxin from Escherichia coli

The thioredoxin superfamily consists of enzymes that catalyze the reduction , formation, and isomerization of disulfide bonds and exert their activity through a redox active disulfide in a Cys-Xaa(1)-Xaa(2)-Cys motif, The indi vidual members of the family differ strongly in their intrinsic redox poten tials. However, the role of the different redox potentials for the in vivo function of these enzymes is essentially unknown. To address the question o f in vivo importance of redox potential for the most reducing member of the enzyme family, thioredoxin, me have employed a set of active site variants of thioredoxin with increased redox potentials (-270 to -195 mV) for funct ional studies in the cytoplasm of Escherichia coil, The variants proved to be efficient substrates of thioredoxin reductase, providing a basis for an in vivo characterization of NADPH-dependent reductive processes catalyzed b y the thioredoxin variants. The reduction of sulfate and methionine sulfoxi de, as well as the isomerization of periplasmic disulfide bonds by DsbC, wh ich all depend on thioredoxin as catalyst in the E. coil cytoplasm, proved to correlate well with the intrinsic redox potentials of the variants in co mplementation assays. The same correlation could be established in vitro by using the thioredoxin-catalyzed reduction of lipoic acid by NADPH as a mod el reaction. We propose that the rate of direct reduction of substrates by thioredoxin, which largely depends on the redox potential of thioredoxin, i s the most important parameter for the in vivo function of thioredoxin, as recycling of reduced thioredoxin through NADPH and thioredoxin reductase is not rate-limiting for its catalytic cycle.