Abstract
Isomerization of disulfide bonds is vital for the proper folding of proteins that possess multiple disulfides. In prokaryotes, the catalytic pathway responsible for disulfide isomerization involves thioredoxin, thioredoxin reductase, and the DsbC, DsbG, and DsbD proteins. To be active as isomerases, DsbC and DsbG must be kept reduced. This task is performed by the cytoplasmic membrane protein DsbD. DsbD in turn is reduced by the cytoplasmic thioredoxin and is composed of three domains. The beta domain is membrane-embedded, whereas the alpha and gamma domains are localized to the periplasm. It had been proposed that electrons are transferred within DsbD by a succession of disulfide exchange reactions between the three domains. To test this model using biochemical methods, we purified to homogeneity different polypeptides corresponding to the alpha, beta, gamma, and betagamma domains. Using these domains, we could reconstitute a DsbD activity and, for the first time, reconstitute in vitro the electron transport pathway from NADPH and thioredoxin to DsbC and DsbG. We showed that electrons are transferred from thioredoxin to the beta domain then successively to the gamma domain, the alpha domain, and finally on to DsbC or DsbG. We also determined the redox potential of the gamma domain to be -241 mV, and that of the alpha domain was found to be -229 mV. This shows that the direction of electron flow within DsbD is thermodynamically driven.
Highlights
A critical step in the folding of newly synthesized proteins is the formation of native disulfide bonds between the thiol groups of two cysteine residues
This disulfide formation occurs in the periplasm and is catalyzed by a protein called DsbA1 (DsbA stands for disulfide bond) [2]
A model has been proposed for the direction of electron flow within DsbD. This model relies on two types of in vivo experiments: first, the identification of mixed disulfide bonds between thioredoxin and the  domain and between the ␣ domain and DsbC [11, 12] and second, the demonstration that, when the ␥ domain is removed from the system, the  domain is found reduced and the ␣ domain is found oxidized [12]
Summary
The first cysteine of the CXXC motif, present in the reduced form, is thought to attack a non-native disulfide in a misfolded protein This results in the formation of a mixeddisulfide between the isomerase and the target protein. A model has been proposed for the direction of electron flow within DsbD This model relies on two types of in vivo experiments: first, the identification of mixed disulfide bonds between thioredoxin and the  domain and between the ␣ domain and DsbC [11, 12] and second, the demonstration that, when the ␥ domain is removed from the system, the  domain is found reduced and the ␣ domain is found oxidized [12]. This result helps explain how the DsbA-dependent disulfide oxidation system fails to oxidize and inactivate the DsbD-dependent isomerization system
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