Kinetics of the intramolecular disulfide exchange between the periplasmic domains of DsbD.
DsbD from Escherichia coli catalyzes the transport of electrons from cytoplasmic thioredoxin to the periplasmic substrate proteins DsbC, DsbG and CcmG. DsbD consists of a periplasmic, N-terminal domain (nDsbD), a central transmembrane domain and a periplasmic, C-terminal domain (cDsbD). Each of these domains contains two essential cysteine residues that are required for intermolecular disulfide exchange between DsbD and substrates, and intramolecular disulfide exchange between the three DsbD domains. In order to determine the rate of intramolecular electron transfer from cDsbD to nDsbD, we constructed a redox-sensitive tryptophan variant of cDsbD (cDsbD(W)) that shows an approximately threefold increase in fluorescence upon reduction and has the same redox potential and reactivity as wild-type cDsbD. cDsbD(W) was then used for the construction of fusion proteins with nDsbD and cDsbD(W), connected via flexible linkers of different length. Using the DsbD substrate DsbC, which can only be reduced by nDsbD and does not react with cDsbD, we could directly measure the intramolecular electron transfer from cDsnD(W) to nDsbB in the fusion proteins. We show that the intramolecular disulfide exchange is significantly faster than the reaction between isolated nDsbD and cDsbD. Nevertheless, the effective concentration of 0.2 mM of the domains in the fusions is comaparably low. The rate of 23 s(-1) for the intramolecular disulfide exchange in the fusions was independent of the linker length and may represent the upper limit for the substrate turnover of full-length DsbD.
Rozhkova
Anna
A
Institute of Molecular Biology and Biophysics, ETH Zurich 8093, Switzerland.
Glockshuber
Rudi
R
D004220:Disulfides / Q000737:chemistry* / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D029968:Escherichia coli Proteins / Q000737:chemistry*; D007700:Kinetics; D008954:Models, Biological; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000737:chemistry*; D019897:Periplasm / Q000737:chemistry; D017434:Protein Structure, Tertiary
D004220:Disulfides; D029968:Escherichia coli Proteins; D010088:Oxidoreductases; C094848:DsbD electron transport protein, E coli