Abstract
In Escherichia coli, the periplasmic disulfide oxidoreductase DsbA is thought to be a powerful but nonspecific oxidant, joining cysteines together the moment they enter the periplasm. DsbC, the primary disulfide isomerase, likely resolves incorrect disulfides. Given the reliance of protein function on correct disulfide bonds, it is surprising that no phenotype has been established for null mutations in dsbC. Here we demonstrate that mutations in the entire DsbC disulfide isomerization pathway cause an increased sensitivity to the redox-active metal copper. We find that copper catalyzes periplasmic disulfide bond formation under aerobic conditions and that copper catalyzes the formation of disulfide-bonded oligomers in vitro, which DsbC can resolve. Our data suggest that the copper sensitivity of dsbC- strains arises from the inability of the cell to rearrange copper-catalyzed non-native disulfides in the absence of functional DsbC. Absence of functional DsbA augments the deleterious effects of copper on a dsbC- strain, even though the dsbA- single mutant is unaffected by copper. This may indicate that DsbA successfully competes with copper and forms disulfide bonds more accurately than copper does. These findings lead us to a model in which DsbA may be significantly more accurate in disulfide oxidation than previously thought, and in which the primary role of DsbC may be to rearrange incorrect disulfide bonds that are formed during certain oxidative stresses.
Highlights
DsbC, a second periplasmic thiol-disulfide oxidoreductase, appears to function as a disulfide isomerase both in vitro and in vivo
The DsbC Disulfide Isomerization Pathway Is Involved in Copper Resistance—Given the importance of proper protein folding to the well being of the cell, it is surprising that no clear phenotype has yet been found for null mutations in the E. coli principal disulfide isomerase, DsbC
It seemed possible that the entire disulfide isomerization pathway might be involved in copper resistance and that dsbCϪ strains would be copper-sensitive, despite previous reports that did not observe dsbCϪ copper sensitivity [13]
Summary
DsbC, a second periplasmic thiol-disulfide oxidoreductase, appears to function as a disulfide isomerase both in vitro and in vivo. Berkmen et al [3] recently showed that the folding of Agp (three consecutive disulfides) becomes DsbC-dependent with the introduction of a non-consecutive disulfide bond These results suggest that the principal role of DsbC under nonstress conditions is to rearrange disulfide bonds that DsbA forms incorrectly. Whereas a dsbAϪ strain shows pleiotropic phenotypes, no consistent phenotype has yet been found for a dsbCϪ or dsbGϪ strain This suggests that disulfide isomerization may be less important under non-stress conditions than previously believed. Our data suggest that the copper sensitivity of dsbCϪ strains arises from the inability of the cell to rearrange copper-catalyzed nonnative disulfide bonds in the absence of functional DsbC. This may indicate a role for DsbC in combating periplasmic oxidative stress
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