A bacterial thioredoxin-like protein that is exposed to the periplasm has redox properties comparable with those of cytoplasmic thioredoxins.
The membrane-anchored thioredoxin-like protein (TlpA) from the Gram-negative soil bacterium Bradyrhizobium japonicum was initially discovered due to its essential role in the maturation of cytochrome aa3. A soluble form of TlpA lacking the N-terminal membrane anchor acts as a protein thiol:disulfide oxidoreductase. TlpA possesses an active-site disulfide bond common to all members of the thiol:disulfide oxidoreductase family. In addition, it contains two non-active-site cysteines that form a structural disulfide bond (Loferer, H., Bott, M., and Hennecke, H. (1993) EMBO J. 12, 3373-3383; Loferer, H., and Hennecke, H. (1994) Eur. J. Biochem. 223, 339-344). Here, we compare the far- and near-UV CD spectra of TlpA before and after reduction of both disulfides by dithiothreitol and show that the non-active-site disulfide bond is not required for the integrity of TlpA's native conformation. In contrast to dithiothreitol, reduced glutathione (GSH) selectively reduces the active-site disulfide and leaves the non-active-site disulfide bond intact, even at high molar excess over TlpA. The selective reduction of the active-site disulfide bond leads to a 10-fold increase of the intrinsic tryptophan fluorescence of TlpA at 355 nm, which may be interpreted as a quenching of tryptophan fluorescence by the active-site disulfide bond. Using the specific fluorescence of TlpA as a measure of its redox state, a value of 1.9 +/- 0.2 M was determined for the TlpA:glutathione equilibrium constant at pH 7.0, demonstrating that TlpA is a reductant, like cytoplasmic thioredoxins. The DsbA protein, which acts as the final oxidant of periplasmic secretory proteins in Escherichia coli, is not capable of oxidizing the active-site cysteines of TlpA. This suggests that TlpA's primary role in vivo is keeping the thiols of certain proteins reduced and that TlpA's active, reduced state may be maintained owing to its kinetically restricted oxidation by other periplasmic disulfide oxidoreductases such as DsbA.
Loferer
H
H
Mikrobiologisches Institut, Eidgenössische Technische Hochschule, Zürich, Switzerland.
Wunderlich
M
M
Hennecke
H
H
Glockshuber
R
R
D001426:Bacterial Proteins / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D002942:Circular Dichroism; D003001:Cloning, Molecular; D003545:Cysteine; D017931:DNA Primers; D004229:Dithiothreitol / Q000494:pharmacology; D004926:Escherichia coli; D005978:Glutathione / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology; D019803:Glutathione Disulfide; D007700:Kinetics; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D015183:Restriction Mapping; D012230:Rhizobiaceae / Q000378:metabolism*; D013879:Thioredoxins / Q000378:metabolism*
D001426:Bacterial Proteins; D017931:DNA Primers; D011993:Recombinant Fusion Proteins; C075138:TlpA protein, Salmonella; D013879:Thioredoxins; D005978:Glutathione; D003545:Cysteine; D004229:Dithiothreitol; D019803:Glutathione Disulfide