In Vitro and in Vivo Redox States of the Escherichia coli Periplasmic Oxidoreductases DsbA and DsbC

Abstract

DsbC is a periplasmic protein of Escherichia coli that was previously identified by a genetic selection that rescued sensitivity to dithiothreitol in Tn10 mutagenized cells. The Erwinia chrysanthemi dsbC gene was identified in a previous genetic screen to restore motility in a dsbA null strain. In order to analyze the biochemical role of E. coli DsbC, the protein was overexpressed, purified, and compared with DsbA in terms of disulfide isomerization, thiol oxidation, and in vivo redox state. In vitro, DsbC and DsbA have an equivalent kcat for disulfide isomerization with the model substrate, misfolded insulin-like growth factor-1. However, DsbA is a more effective oxidant than DsbC of protein dithiols. In vivo, DsbA is found exclusively in the oxidized state in wild-type strains grown in rich media. On the other hand, in vivo DsbC has one pair of cysteines oxidized and one pair reduced. DsbD is required to maintain this reduced pair of cysteines, confirming previous genetic results. A dsbC deletion strain showed decreases in the production of some, but not all, heterologous proteins containing multiple disulfide bonds. Notably, those proteins affected by the dsbC deletion do not have the cysteines paired consecutively.

Yeast Cox17 Solution Structure and Copper(I) Binding

“…If SDS is not included in the modification reaction, only three of seven cysteines are modified regardless of the reductant and conditions used, implying that the other four cysteines are not easily accessible. Notably, quantitation by absorption spectroscopy using both Ellman's reagent and a thiol detection kit led to an underestimated thiol content, a phenomenon also observed for the E. coli oxidoreductase DsbC (36).…”

Role and location of the unusual redox-active cysteines in the hydrophobic domain of the transmembrane electron transporter DsbD

“…Although cleavage of ␤ takes place efficiently, no band corresponding to that of alkylated cleaved ␤ was observed (Fig. 5, compare lane 4 with lanes [6][7][8][9][10][11][12][13][14][15][16][17][18][19][20]. This result indicates that ␣␤, exhibiting its periplasmic face outside on in right-side-out vesicles, is subject to cleavage by TEV protease but does not have either cysteine exposed on this face of the membrane.…”

“…It transfers reducing potential from cytoplasmic thioredoxin to a variety of periplasmic acceptors, such as the disulfide bond isomerase DsbC, responsible for shuffling wrongly formed disulfide bonds in proteins, and to a member of the cytochrome c maturation pathway, CcmG (8)(9)(10). In dsbD null mutants, wrongly formed disulfide bonds accumulate in periplasmic proteins with more than two cysteines, because of the absence of reduced (active) DsbC (9,11). In addition, these cells are depleted of c-type cytochromes because of disruption of the cytochrome c maturation pathway (6).…”

Nonconsecutive disulfide bond formation in an essential integral outer membrane protein

“…Introduction of the wrong disulfide bond by DsbA is particularly problematic for proteins that require a disulfide bond between nonconsecutive Cys (7)(8)(9)(10). When such proteins are oxidized incorrectly, the periplasmic disulfide bond isomerase DsbC rearranges their disulfide bonds to produce the final nonconsecutive configuration (11)(12)(13).…”