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 individual members of the family differ strongly in their intrinsic redox potentials. However, the role of the different redox potentials for the in vivo function of these enzymes is essentially unknown. To address the question of in vivo importance of redox potential for the most reducing member of the enzyme family, thioredoxin, we have employed a set of active site variants of thioredoxin with increased redox potentials (-270 to -195 mV) for functional studies in the cytoplasm of Escherichia coli. The variants proved to be efficient substrates of thioredoxin reductase, providing a basis for an in vivo characterization of NADPH-dependent reductive processes catalyzed by the thioredoxin variants. The reduction of sulfate and methionine sulfoxide, as well as the isomerization of periplasmic disulfide bonds by DsbC, which all depend on thioredoxin as catalyst in the E. coli cytoplasm, proved to correlate well with the intrinsic redox potentials of the variants in complementation assays. The same correlation could be established in vitro by using the thioredoxin-catalyzed reduction of lipoic acid by NADPH as a model reaction. We propose that the rate of direct reduction of substrates by thioredoxin, which largely depends on the redox potential of thioredoxin, is 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.
Mössner
E
E
Institut für Molekularbiologie und Biophysik, Eidgenössische Technische Hochschule Hönggerberg, CH-8093 Zürich, Switzerland.
Huber-Wunderlich
M
M
Rietsch
A
A
Beckwith
J
J
Glockshuber
R
R
Aslund
F
F
D003593:Cytoplasm / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism*; D009602:Nitrosamines / Q000378:metabolism; D010084:Oxidation-Reduction; D013879:Thioredoxins / Q000378:metabolism*
D009602:Nitrosamines; D013879:Thioredoxins; C041199:N-nitrosoallyl-2,3-dihydroxypropylamine