A BACTERIAL THIOREDOXIN-LIKE PROTEIN THAT IS EXPOSED TO THE PERIPLASMHAS REDOX PROPERTIES COMPARABLE WITH THOSE OF CYTOPLASMIC THIOREDOXINS

The membrane-anchored thioredoxin-like protein (TlpA) from the Gram-ne gative soil bacterium Bradyrhizobium japonicum was initially discovere d due to its essential role in the maturation of cytochrome aa,, A sol uble form of TlpA lacking the N-terminal membrane anchor acts as a pro tein thiol:disulfide oxidoreductase. TlpA possesses an active-site dis ulfide bond common to all members of the thiol:disulfide oxidoreductas e 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) fur. 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 di thiothreitol and show that the non-active-site disulfide bond is not r equired for the integrity of TlpA's native conformation In contrast to dithiothreitol, reduced glutathione (GSH) selectively reduces the act ive-site disulfide and leaves the non active-site disulfide bond intac t, even at high molar excess over TlpA The selective reduction of the active-site disulfide bond leads to a 10-fold increase of the intrinsi c tryptophan fluorescence of TlpA at 555 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 red ox state, a value of 1.9 +/- 0.2 FA was determined for the TlpA:glutat hione equilibrium constant at pH 7.0, demonstrating that TlpA is a red uctant, like cytoplasmic thioredoxins. The DsbA protein, which acts as the final oxidant of periplasmic secretory proteins in Escherichia co li, 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 ce rtain proteins reduced and that TIpA's active, reduced state may be ma intained owing to its kinetically restricted oxidation by other peripl asmic disulfide oxidoreductases such as DsbA.